The half-life of plasma albumin and the possibility of its partial conversion into globulin as studied in the normal adult dog

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Content THE HAIF-LIFE OF PLASMA ALBUMIN AND THE POSSIBILITY OF ITS PARTIAL CONVERSION INTO GLOBUILN AS STUDIED IN THE NORMAL ADULT DOG by Samuel M. Mozerslsy A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry and Nutrition) June 1957 UMI Number: DP21574 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Di ssert at i on Publ i shi ng UMI DP21574 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 T his dissertation, w ritte n by Sam aelil«,tfQzersky......................................... u n d e r the d ire c tio n o f his G uidance C o m m itte e , a nd a p p ro v e d by a ll its m em bers, has been p r e sented to a n d accepted by the F a c u lty o f the G ra d u a te S cho o l, in p a r tia l f u lfillm e n t o f re quirem ents f o r the degree o f D O C T O R O F P H I L O S O P H Y G uidance C om m ittee . . . L C y y y J L S L C h a irm a n % r ^ GRATEFULLY DEDICATED TO MY PARENTS ACKNOWLEDGMENT The author wishes to express his appreciation to the Committee on Growth of the American Cancer Society and to the Araiy Air Force for financial support of this work, under the direction of Dr, Richard J. Winzler. I also wish to convey my sincere thanks to Dr, John W, Mehl, whose lectures on the Chemistry of Proteins inspired me to do research in this field. My most grateful acknowledgment to my wife Faye, without whom this dissertation may never have been completed. TABLE OF CONTENTS CHAPTER PACE I. INTRODUCTION ............................. 1 Preliminary Remarks • ••••••••••.• 1 Statement of the Problem ................ 2 Terminology ........................... 3 Graphic Representation of Protein Synthesis and Degradation........................ 6 II. HISTORY ...................... 11 General......... 11 Conservation of Amino Acids in the Form of Protein ........................... lit Plasma Proteins Outside the Bloodstream ... 16 Heart Fibroblasts in vitro................ 17 Hemoglobin ........................... 18 Proteins of the Mammaxy Gland............. 18 Plasma Proteins.......................... 19 III. MATERIALS AMD METHODS...................... 20 Electrophoresis in Supporting Media (Paper and Starch) ......... 20 Apparatus and Reagents .......... 20 The Electrophoresis "Cell” ........... 20 Electrodes .......................... 28 Buffers ........................... 33 Staining Reagent .................... 3k ill CHAPTER PAGE Procedure................................ 35 Preparation of the Paper * * ........... 35 Application of the Sample for Preparative Purposes (1 to 2 ml* Sample) .......... 36 Application of Electrical Field • • . * . 39 Location of Components ................. 1*0 Isolation of Components * ............... 1*2 Procedure for Analytical Purposes (0 to b00 microliters).................. 1*3 Reproducibility ...................... 1 * 1 * Composition of Isolated Fractions * • • • 1 * 1 * Fractionation of Crude Albumin............... 60 Reagents .............................. 60 Preparation of S-^-Labeled Albumin • • • • 62 Chemical Fractionation .............. * 62 Electrophoresis in Starch •••••*** 71 Specific Activity of Sulfur ................. 8b General ••••••• ..................... 8b Apparatus................................ 8$ Reagents •••••••«•••**•••• 88 Isolation of Protein from Solution • • • • 89 Oxidation................................ 90 Removal of Perchloric Acid.................. 91 Removal of Copper .............. 91 iV CHAPTER PAGE Isolation of Sulfur.................... 93 Determi nation of Radioactivity ......... 95 Determination of Sulfur.................. 100 Care and Treatment of Animals ............ 102 IV. RESULTS ................................... 10U V. DISCUSSION ................................... na Design of the Experiments ................ Utl Interpretation of Results ...................Iii3 H. SUMMARY AMD CONCLUSION.......................... 1$7 BIBLIOGRAPHY ............................... 160 APPENDICES .......................................... 166 V LIST OF FIGURES FIGURE PAGE 1* A Di&grsomatic Representation of the Structure of Insulin ....... 7 2. The Apparatus for Electrophoresis in Supporting Media. The Loner Section............ 21 3. The Apparatus for Electrophoresis in Supporting Media. The Middle Section ............... 23 U* The Apparatus for Electrophoresis in Supporting Media. The Upper Section .............. 25 5* Suspension of the Paper for Electrophoresis . • 27 6. The Starch Block and Supporting Frame........ 29 7* The Electrode Vessel .................... 31 8. Application of Grease to the Edges of the Paper 35 9* Appearance of the Paper Prior to Application of the Sample ......... ......... 36 10. Application of the Sample for Electrophoresis in Paper ........................... 37 11. Arrangement of Paper Sheets for Preparative Electrophoresis................ 38 12. Removal of Ends of Paper Sheets After Electrophoresis .......................... kO 13* The Arrangement for Elution of Protein from Paper Strips .......................... U2 Vi FIGURE PAGE 1) 4. Fractionation of S^-Labeled Plasma (Dog) in Cold Ethanol ........... 6k lf>. Fractionation of Unlabeled Plasma (Dog) in Cold Ethanol • 66 16. Fractionation of "Mixed Plasma" (Dog) in Cold Ethanol • •••••••••....••.• 68 17. Distribution of Protein After Electrophoresis of Crude -Labeled Albumin in a Starch Medium • • 82 18. The Oxidation Tube and Condenser for Wet Digestion of S^^-Labeled Proteins ... ............ 86 19. Apparatus for the Removal of Copper Following Oxidation of Protein Sulfur to Sulfate ...••• 92 20. Apparatus for Collection of Benzidine Sulfate in Planchet Fom ............ 9k 21. Self-Absorption for S^ in Benzidine Sulfate .... 98 22. Dog I. The Specific Activity of Sulfur in Whole Serum and in Total Serum Protein as a Function of Time , ............ 106 23* Dog I. The "Average Curve" of Figure 22 Plotted on a Semi-Logarithmic Scale .••••••••••• 108 2I 4. The Specific Activity of Sulfur in Whole Serum and in Total Serum Protein as a Function of Time 110 2S . The Specific Activity of Albumin Sulfur as a * . Function of Time ...................... 113 vii FIGURE PAGE 26. Dog II. The Specific Activity of Albumin Sulfur as a Function of Time • • • • .......... . . • 115 27 * Dog III. The Specific Activity of Albumin Sulfur as a Function of T i m e ........................ 117 28. Dog IV* The Specific Activity of Sulfur in the Beta Proteins as a Function of Time •••••• 119 29• Dog IV* The Specific Activity of Sulfur in the Gamma Fraction as a Function of Time • • • • • 121 30* Dog IV. The Specific Activity of Sulfur in the Alpha Proteins as a Function of T i m e ........ 121; 31. Dog IV. The Specific Activity of Albumin Sulfur as a Function of Time .................. 126 32* Dog V. The Specific Activity of Albumin Sulfur as a Function of T i m e ........................ 129 33• Dog VI. The Specific Activity of Sulfur in Whole Plasma and in the Albumin and Globulin Fractions Thereof as Functions of T i m e .................. 131 3lu Dog VII. The Specific Activity of Sulfur in Whole Plasma and in the Albumin and Globulin Fractions Thereof as Functions of Time • • • ........ 133 35* Dog VIII. The Specific Activity of Sulfur in Whole Plasma and in the Albumin and Globulin Fractions Thereof as Functions of T i m e ............... 135 36. Dog IX. The Specific Activities of Albumin Sulfur and Globulin Sulfur as Functions of Time . . . 139 viii FIGURE PAGE 37. The Turnover of Cystine Sulfur in Albumin, Globulin, and Body Protein in a Seven Kilogram D o g ......................• »••••• 150 38. The Apparatus of Cremer and Tiselius (1950) for Paper Electrophoresis ........................ 168 39* An Early Apparatus for Paper Electrophoresis Devised by Durrum (1950) ........................ 170 I 4 .O. An Apparatus for Paper Electrophoresis With Controlled Pressure and With Provision for the Removal of H e a t ..................................172 IpL • The Fractionation of Dog Serum and of the Crude Albumin Obtained The ref r can Using Cold Ethanol and Heavy Metals..............................* 211* H2 • Fractionation of Dog Serum With Heavy Metals in Aqueous Solution............................... 219 k3^ The Distribution of Protein After Fractionation of Dog Serum With Heavy Metals in Aqueous Solution 221 Idi* Apparatus for the Chromatography of Small Amounts (5 Mg*) of Protein With Gradient Elution • . • • • 223 1(5 • Chromatography of Crude Dog Albumin on DEAE-0- Cel lulose * 227 I 46. The Distribution of Protein After Chromatography of Crude Dog Albumin Using Acid and Alkaline Eluting Agents............* .................... 233 ix FIGURE PAGE 1*7. Electrophoresis of 0.20 Gram of Crude Albumin (G-£i*) in Starch ..................... 236 1*8. The Distribution of Protein in the Fractions Obtained by Electrophoresis in Starch of 1*#0 Grams of Crude Dog Albumin (G-51*) 2l*2 X LIST OF PLATES PLATE page I. Electrophoresis of two Samples of Serum From Dog I V ............................... • • • i;5 II. The Distribution of Serum Proteins in all Six Sheets of a Multiple-Sheet Run ................... 2*7 HI. The Separation of Albumin and Globulin at pH 5 in Multiple-Sheet Electrophoresis................. h9 IV. Analytical Electrophoresis at pH U.9 of the Albumin and Globulin Fractions Obtained From Dog Plasma by Multiple-Sheet Fractionation at pH 5 .......................................... 52 V. Analytical Electrophoresis at pH 8.6 of Globulin Fractions Obtained From Dog Plasma by Multiple- Sheet Fractionation at pH 5 ...................... 51; VI. Analytical Electrophoresis at pH 8.6 of Albumin Fractions Obtained From Dog Plasma by Multiple- Sheet Fractionation at pH 5 56 VII. Analytical Electrophoresis at pH 8.6 of Six Fractions of Dog Plasma Obtained by Multiple- Sheet Fractionation at the Same p H ............... 58 VIII. The Composition of Fractions Obtained From S^- Labeled Plasma, uNormalu Plasma and l f Mixed Plasma” ......................................... 72 xi PIATE PAGE IX, The Compos it ion of Fractions Obtained From S^-Labeled Plasma and "Mixed Plasma" .......... 7h X. The Composition of Fractions Obtained by Starch Electrophoresis of Crude S^-Labeled Dog Albumin . • 80 XI, Electrophoresis of Serum on Paper Between Large Metal Plates (12 inches x lii inches) ........... 176 XU, Human Serum From a Normal Subject and From a Patient with Multiple Myeloma Macheboeuf Apparatus , Incite Roof ............. 181 X U I* Human Sera, the Same Samples as in Plate XII. Spinco Apparatus ••••••••• .............. 183 XIV, Human Sera, the Same Samples as in Plates XII and XIII, Macheboeuf Apparatus, Asbestos Rood • • 185 XV, Human Serum, 200 Microliters, Applied Along an Oblique line. Asbestos R o o f .................... 187 XVI. Human Serum, 200 Microliters, Applied Along an Oblique line. Incite Roof ••••••••••. 189 XVII. Electrophoresis of Dog Plasma and Fractions Thereof at pH 1;.Q0 (Column I), pH I 4.. 8O (Column II), and pH 5.99 (Column;III)............................ 192 XVIII. Electrophoresis of Dog Plasma and Fractions Thereof at pH 7.U0 (Column I) and pH 8.6 (Column II) . . . 19U XIX. Electrophoresis of Serum at Ionic Strength 0.025* pH 8 . 6 .......................................... 196 xii PIATE PAGE XX* The Composition of Dog 1 1 Albuminu (Fraction V) Prepared by the Cold Alcohol Fractionation Scheme Designed fear Human Plasma ••••»••• 211 XXI. The Composition of Fractions Obtained From Dog Plasma by Various Methods ............ . • 216 XXII. The Composition of the Crude Dog Albumin (G-5U) Subjected to Chromatography. Human Serum is Shown for Comparative Purposes •••••••• 231 XXIII. The Composition of the Fractions Obtained by Electrophoresis in Starch of it.O Grams of Dog Albumin (G-f?lj.)............................. 239 XXIV. Electrophoresis on Paper of Fractions Obtained From Crude Dog Albumin by Electrophoresis in Starch....................................... 2hh CHAPTER I INTRODUCTION Preliminary Remarks. The metabolism of small molecules has been extensively investigated, and with notable success* This holds for the biosynthesis of these molecules as well as for their degradation* If, as an example, one glances at the table of con tents of "A Symposium on Amino Acid Metabolism” (l) one will find considerable reference to fruitful investigation of biosynthetic problems• Considerably less is known about the biosynthesis of macro molecules , at least proteins* Thus, in the "Proceedings of the Third International Congress of Biochemistry" (2) held in 1955 one will find (p. 92) a discussion by H* Borsook of "The biosynthesis of peptides and proteins •" This is in the nature of a hypothesis , as Borsook states* Comparatively little could be said on this subject to be established as fact* Some degree of success has been achieved in the measurement of the turnover of proteins, at least plasma proteins* This will be discussed at a later point in some detail* It may be pointed out here that even in this area there has been considerable un certainty in the interpretation of results* The major obstacle encountered in the investigation of the metabolism of these macromolecules is probably the lack of sufficient ly powerful and sufficiently simple methods of investigation, especial 2 ly when it is necessary to process large numbers of samples* In the work herein described the problem of methodology became an obstacle of such size that by far the major part of the time spent was devoted to the development of adequate methods of investigation* In the present study, time has permitted only a limited application of these techniques* It is hoped that the possibilities of the methods will be demonstrated by a more thorough investigation. Statement of the Problem* The work to be described is con cerned with two biological questions* For both of these the normal adult dog was used* The first question concerns the half-life of plasma albumin in this animal* This has been the subject of con siderable work, as will become evident in the historical section. However, there are some gaps and possible loopholes* For one thing ” albumin” does not seem to be a single homogeneous protein* The apparent half-life loses much of its presumed significance when there is more than one species of protein present, unless all species happen to have the same half-life* Thirdly, the apparent half-life obtained even for a homogenous protein is dependent on the method used for tracing the metabolism of the protein* It is therefore of value to compare the results obtained by different methods* The methods which were utilized in this investigation will be described later* A second question which posed itself, and which seemed amenable to the techniques already in use was this: What is the 3 extent, if any, of conversion of serum albumin into serum globulin? Or, otherwise stated, is the molecule of serum albumin or any peptide fragment thereof, used in the formation of a molecule of serum globulin? Terminology. One may ask what is meant by “conversion” and when will we say that a molecule of albumin is “used” to form globulin* This is closely related to the question of “synthesis” in the absence of net synthesis* These words deserve careful scrunity* We may begin by talking about extreme cases* Suppose that a molecule A (e.g. albumin) of molecular weight 48,000 combines some how with another molecule of the same species, and suppose that the resulting compound is a molecule G (e.g. globulin) of molecular weight 96,000* Then there is no question that there has been “synthesis” A of G from A, that there has been “conversion” of A into G, and that A has been “used” in the formation of G* Whether there has been “net synthesis” depends only on whether a previously existing molecule of G of the same species was converted during the same period to some other species. But whether this has occurred or not does not affect the statements regarding synthesis, conversion and utilization. Suppose that; instead of the above, the two molecules of A are hydrolyzed inside some cell to the component amino acids and that these combine in a completely different arrangement to form a molecule of G -which is secreted into the plasma, the amino acid re sidues of A being completely conserved in the process* 2A AA Then again we have synthesis of G, conversion of A, and utilization of A, Just as above, although the resulting G molecule is completely different from the previous one. On the other hand, suppose the cell hydrolyzes the albumin and that the resulting amino acids are released into the bloodstream, where they mix with the amino acids already present therein* The amino acids may then enter seme other cell and be used in the forma tion of a species of globulin, but albumin has in this case served only as a non-specific reservoir of amino acids which, in its absence, would be obtained from some other source* In this case we cannot say that G has been synthesized from A, or that A has been converted to G* If instead of 2A— G only one molecule of A is involved, A G, either directly or via amino acids, then we can still say that there has been utilization of A and conversion to G; and we may still include this in the category of synthesis of G from A. These are the extremes* Now suppose that A splits into fragments A^ and A2 and that one fragment, A^, combines with a protein or peptide, B, to give protein G. ( ) *2 I A A1 * *2 j or A -------- B G Here we cannot say that A was converted into G; but we would still say that A was utilized in the formation of G* And since the over all reaction is A + B >— G ♦ A^ G has been synthesized from A and B, Ag having been discarded in the process, as far as our present interests are concerned* If A^ can be obtained from some source other than A, for example by the breakdown of another protein , Ai LV 6 then we would have to distinguish the G molecules synthesized from A + B from those synthesized from oC + B. If A^ can be obtained by synthesis from free amino acids , then we must further distinguish those molecules of G which used A^ obtained by breakdown of A from those molecules of G which used A^ synthesized from free amino acids. If all, three processes occur simultaneously we may diagram the synthesis of G as follows: Graphic Representation of Protein Synthesis and Degradation. The above considerations can be generalized as follows, assuming the structural formula of protein G is completely known. Beginning at time t * o with free amino acids , suppose we know the time t at which each peptide bond is formed. This gives us a function of time which may be represented in graphical form. To do this, number the amino acid residues in sequence, beginning with any amino acid having its alpha amino group free. This is illustrated in Figure 1 for insulin (3,2;) • bet these numbers be the ordinate, and let time be the abscissa. Represent a chemical OC A Free Amino Acids B 7 FIGURE 1 DIAGRAMMATIC REPRESENTATION OF THE STRUCTURE OF INSULIN 8 U t l o 9 bond by a line joining the two residues involved, a peptide bond by a straight (vertical) line, and any non-peptide bond by a curved line. In this way we can obtain a clear picture of the synthesis of a protein* If we had such a graph for any protein, we would know what peptide subunits, if any, are involved in its synthesis and what is the sequence in which these units are joined to one another* The accompanying graph shows a hypothetical case of this sort involving three peptide units (I-IH) and one disulfide bond. With this representation the usual "types* of protein synthesis become quantitative variation of a single theme: bond formation as a function of time. Residue Number HT It must be emphasized that this does not depict a mechanism of protein synthesis. If we had such a graph it would not tell us what activated amino acid ^3 to join It would only tell us when this happened in relation to the rest of the molecular growth process• Catabolism of a protein molecule can be represented in the same way— in reverse* And if a unit obtained by the breakdown of one protein (A) were used in the synthesis of another (G) this could be indicated on a suitable pair of graphs* one for the de gradation of A* the other for the synthesis of G. These considerations are purely hypothetical and we have no hopes of being able to construct a graph such as the above for aqy protein in the immediate future* Our purpose was merely to clarify terminology and to depict in graphic form the kind of process which one may have in mind in connection with the problem involved in protein turnover* CHAPTER II HISTORY General♦ Whipple and his colleagues at Rochester have for many years (5) been engaged in the problem of the turnover of plasma proteins* They demonstrated, without benefit of isotopes, that the body (of the dog) is capable of manufacturing plasma proteins at a rapid rate (6,7). More recently they have shown that the liver is the site of formation of the albumins, and of a major part of the globulins --except for the gamma globulins* (8,10,11)* This later work is in agreement with previous results of Tarver and Reinhardt (9). The appearance on the market of isotopes of elements present in proteins made it possible to make measurements in normal, intact animals in connection with the rate of turnover of various proteins* There are two main approaches to the problem (12, p* 127li)« In the first a labeled amino acid is administered and its concentra tion in the protein under study is measured as a function of time* In the second a labeled protein is given, and its activity is like wise measured as a function of time* In the latter the protein may be labeled by supplying a "donor” animal with a radioactive amino acid and obtaining the desired protein from that animal after suf ficient time has elapsed for the amino acid to have been incorporated into protein* The first approach suffers from the defect that all of the 12 body proteins become labeled, not just the one being investigated (13). Labeled amino acid from the other proteins is then continous- 2y incorporated into the protein under study (ll*). The magnitude of this effect is difficult to appraise* An objection to the second method has been that the protein (e.g. albumin) introduced into the recipient animal may not be identical with its own, and that the animal may treat the injected protein as a substance foreign to its body (lit). In view of a lack of clear evidence to support this idea, it will be assumed that the animal does not distinguish between the two proteins (e.g. between its own albumin and that from another dog). A problem common to the two methods is that of being able to study a pure molecular species* In method 1 (injection of labeled amino add) this would necessitate the isolation of a pure chemical species from every sample (of blood) taken, for the contaminating species are radioactive* In method 2 it requires isolating a pure labeled protein once. Thereafter it is sufficient to guarantee that the composition of the samples taken is constant, and that the other species of protein in the sample have not become appreciably radioactive•. In most or all of the studies carried out on the turnover of plasma proteins the requirement for purity has not been met. In those investigations using method 2 it has been the practice to in ject labeled plasma, rather than an isolated protein (lf>, 16, 17, 18, 19)* It was therefore considered worthwhile to attempt a study by 13 method 2 of the biological decay of labeled albumin using a prepara tion which prior to injection was free, or nearly free, of globulins* It is recognized that 1 1 albumin1 1 may be a mixture of several species of proteins (20,21)* However, a large number of proteins which are known to be different from albumin and from each other have been eliminated, and this has yielded a mixture which is much less heterogeneous and which might even be treated biologically as homogeneous• In addition to a measurement of albumin the use of a pre paration of this protein free of globulins permits another important point to be investigated* That is the question whether the amino acids of albumin can be used for the synthesis of other proteins, e.g* globulins, without being broken down to the level of free amino acids (17)• It may simplify matters to state the problem in a negative way* A reasonable hypothesis in our present state of knowledge is that all of the plasma proteins are synthesized from free amino acids which are in equilibrium with those in the bloodstream* This hypothesis requires that an amino acid residue which is part of a globulin molecule could not formerly have been part of a molecule of albumin unless at some time in between these two states it existed free in the blood. It is intended to prove this hypothesis unlikely, thus making it possible that an amino acid residue can take a con servative route from albumin to globulin without running the risk of a free existence in the bloodstream* lh Conservation of Amino Acids in the Form of Protein* Whipple’s group found that ’ ’ plasma proteins given by vein can supply all pro tein needs of the body during a long protein fast*” (7)* Hemoglobin given intraperitoneally was also shown to be capable of sustaining nitrogen balance (22). Using 7 g* of plasma protein labeled with Cp^-lysine this group found that during a seven day period following intravenous administration only 2.$% of the C3"^ was excreted in the urine (17)* Considering that during the same period nitrogen excre tion was approximately 1 g* H/day, the small excretion of in dicates an efficient conservation of the amino acids administered in the form of plasma proteins. On this basis, and because of the considerable uptake of by tissue proteins, the authors came to the conclusion that plasma proteins do not break down to the amino acid level before their component amino acids are used in the forma tion of other proteins. When plasma was given orally instead of parenterally the conservation was very much lass, much more c1^ being lost as COg and in the urine (23). In dogs subjected to an inflammatory response, induced by the subcutaneous injection of turpentine, Yuile et al. found the half-life of albumin to be much less than in control dogs— about two days as compared to nine to eleven days, for the period between sixteen hours and three days after administration of the label (18). The albumin/globulin ratio was about one-third the normal value* The depression of the ratio of albumin to globulin in a variety of diseased states is known to occur (2lt)* In investigations on hemoglobin metabolism in dogs Tishkoff 15 et al. found that much of the globin released by breakdown of red cells Is "saved by the body and stored as reserves in the reticulo endothelial system, organs, and tissues.* (25)* Tarver and Morse fed rats with methionine labeled with and obtained results similar to those of Whipple’s group above, i.e. the loss of S3S was small (26). They state that "the formation of plasma protein may be considered to act as a buffer to prevent the loss of amino acid ... Plasma proteins probably mediate the equilibration or interchange of amino acids between the proteins of tissues." Abdou and Tarver administered labeled plasma protein to rats (15,27) by three routes: intravenously, intraperitoneally, and oral- In complete agreement with Whipple’s group these authors state that " an unequivocal demonstration is provided that plasma protein is more efficiently utilized when injected than after oral administration." However, Abdou and Tarver consider that the only "tissue" which shows a marked difference between intravenous and oral administration of plasma protein is the plasma. These authors take issue with Whipple’s group on the latter*s "conclusion" that plasma proteins are not broken down to the amino acid level during the incorporation of their amino acids into tissue protein. They consider that "the evidence is • • . open to other interpretations." (27). In their investigations Abdou and Tarver used both labeled plasma protein and labeled methionine. In their data there is a 1 6 strong suggestion of an apparent difference in half-life of plasma protein, depending on which of the two methods is used for tracing. With labeled methionine given (orally) to rata the "half-life1 1 for plasma protein was IwU days. Using labeled plasma protein the half-life was 2.6to 3*1 days. Steinbock and Tarver investigated the effect of diet on the turnover of plasma protein (16). They demonstrated a marked dependence of "half-life" on the protein content of the diet. For rats whose diet contained 6$% protein the half-life of plasma protein was 2.9 days; for animals on a 2$% protein diet the corresponding figure 7 was 5*1 days. Plasma Proteins Outside the Bloodstream. Wasserznan and Mayerson examined the relation between the plasma and lymph of dogs with regard to the specific activities of plasma proteins (28). They used r*^-albumin and I^^*-gldbulin. They found that for either protein the specific activity in the lymph became equal to that in the plasma within thirteen hours after intravenous injection of the labeled protein. Thereafter the specific activities remained equal, both declining at the same rate. In general agreement with Tarver and Whipple and other in vestigators Wasseman and Mayerson found two main phases to the decay curves obtained when specific activity was plotted on a logarithmic scale against time. The first phase was interpreted as being due to mixing of the protein in plasma with that in lymph. The second 1 7 phase was Interpreted as being due to the metabolism of the protein* This interpretation is in agreement with that usually given* In this investigation it was found that the length of time required for the specific activity in lymph to reach that in plasma depends on the amount of material injected. When the amount was large (e.g* 100 ml, 25% protein) the equilibration time was shorter (ga* 1^ hours) than when the volume was small (7 to 13 hours for 1 ml*). Forker, £haikoff , and Reinhardt also investigated the trans port of plasma proteins into lymph (29)* In contrast to Wasserman and Mayerson these authors reported that the specific activity in lymph "remained significantly less than that in plasma throu^iout the experiment (19 hours)." However, a much smaller number of cases was studied than in the experiments of Wasserman and Mayerson. Heart Fibroblasts in vitro. Francis and Winnick suspended heart fibroblasts in a medium containing free amino acids and protein from embryonic extract (30). With this system they studied the in corporation of amino acids from protein in the medium into cellular protein. They came to the conclusion that there was extensive utili zation of the nutrient protein without hydrolysis to the level of free amino acids* However, these authors mention the possibility that these amino acids may be attached to enzymes or phosphorylated and may therefore never be free to equilibrate with free amino acids in the medium. This possibility would allow for complete hydro lysis of the nutrient protein in the sense that every amino acid residue IB is freed from chemical bondage to any other fragment of the nutrient protein; yet an amino acid residue derived from nutrient protein is still distinguishable from free amino acid in the nutrient medium by virtue of its attachment to some other molecule* Hemoglobin* Muir et>al. have investigated the synthesis of the heme and globin portions of hemoglobin in the rat and rabbit (31)* They used radioactive glycine as the label* since this amino acid is a precursor to both parts of the hemoglobin molecule • They measured the molar activity of porphyrin isolated from hemoglobin and the activity of glycine from the globin part of the molecule* and obtained the value eight for the limiting ratio of these t*ro. On this basis* and sinee eight glycines are required for the synthesis of each porphyrin* they concluded that both parts of the hemoglobin molecule are synthesized from the same pool of amino acids* This striking result makes it likely that any precursor of globin is broken down to the level of free amino acids prior to its incorpora-* tion into globin. Proteins of the Mammary Gland. Askonas et al* have extensive ly studied protein synthesis in the mammary gland* They isolated the peptides from partial hydrolysates of casein and beta lacto- globulin after the simultaneous injection of various labeled amino acids into lactating animals (32* 33* 3h). In almost all cases they demonstrated equal labeling of any given amino acid residue in all parts of the protein molecule* They concluded from their results 19 that these proteins are synthesized from free amino acids in the bloodstream, and not from peptide fragments derived from plasma proteins. This conclusion is strengthened by the observation that equal labeling is obtained even when peptide fragments of casein are injected together with the tracer amino acid (35)* Plasma Proteins. In the course of an investigation on the metabolism of serum proteins in the rabbit Maurer * Niklas, and Iehnert injected S^^-methionine into rabbits and observed the specific activity of free methionine in serum as a function of time (36). They found a half-life of approximately twenty minutes for the free amino acid. These authors used methionine of very high activity (10 curies/gram), and measured the distribution of S?5 three hours after injection. From their data they calculated that the half-life of albumin is three times that of (total) globulin. Niklas & Poliwoda investigated the half-lives of albumin and globulin in human subjects using s35-methionine as tracer (37)* They noted that the globulin curves are not linear on a semi-log- arithmic scale, but tend to become parallel to the decay curve of albumin as time progresses. They suggested that this may be due to the conversion of albumin into globulin (see discussion). CHAPTER IU MATERIALS AND METHODS ELECTROPHORESIS IN SUPPORTING MEDIA (PAPER AND STARCH) Apparatus and Reagents. The Electrophoresis ^Cell.” The apparatus used for paper electrophoresis consisted of three main sections: a lower* middle, and upper section. The lower section was divided by a partition lengthwise down the center into two buffer compartments, cathode and anode* The middle section provided a means of supporting the paper. The upper section was simply a pitch roof, either asbestos or Incite. The details of design are given in Figures 2, 3 and h* For analytical (single sheet) runs the paper was suspended from the Lucite rod shown in the drawing. This was maintained at the highest possible position so that, with the buffer vessels about three-quarters full, the apex of the paper was some eighteen centi meters above the level of the buffer (38). Two small, separators (see drawing), one on either side of the midline, served to keep the paper in an inverted V shape. This is illustrated in the ac companying diagram (Figure 5). 21 FIGURE 2. THE APPARATUS FOR ELECTROPHORESIS IN SUPPORTING MEDIA THE LOWER SECTION T - Top View S * Side View E « End View BOTTOM SECTION 1/1 6" GROOVE y n s / s * i > “ < L . I I ! 1. I I 1 /2" - 1 2 " “ PART , / 2 " — 4" 4 3 / 4 " 1 SIDES ENDS BOTTOM PARTITION RAILS ROD (not shown) PIECES DIMENSIONS 2 4 3/4" X 12" 2 4 " X 9 l/2 " I 9 l/2"X 12" 1 4"X II 5/8" 2 I 'X II 1/2" I EDGE TAPERED 45° 2 3/8" X 14" THREADED (SHALLOW) NOTE: ALL FLAT STOCK IS 1/4" PLEXIGLAS. ROD IS 3/8"PLEXIGLAS (NOT SHOWN). ! \ ) no 2 3 FIGURE 3- THE APPARATUS FOR EIECTROPHORESIS IN SUPPORTING MEDIA THE MIDDIE SECTION T “ Top View S * Side View E * * End View MIDDLE SECTION 1 0 1 / 2" PART (ALL PLEXIGLAS) PIECES DIMENSIONS SIDES 2 1/8"X f'X 10" ENDS 2 1/8" X l"X 9 1/2" SIDES 2 l/4"X3/4"XI2" ENDS 2 l/4"X3/4"X8" ROD SUPPORTS 2 1/4"X l"X 6 1/4" ROD 1 3/8" X 10 3/4" (FOLLOWING NOT SHOWN) SEPARATORS,LARGE 2 SEPARATORS, MEDIUM 2 SEPARATORS, SMALL 2 5/16" S L O T GROOVE 1/4" XI "X1/4" SLOT 5/l6"X5" THREADED 6-32, FLATTEN ED SIDES l/4"X3 3/4H X 10 7/16" 1/4"X I"X 10 7/16" 1/4" X 3/4"X 10 7/16" t r ' 1 0 vs" 5 -32 THREAD ro • P " 25 FIGURE i *. THE APPARATUS FOR ELECTROPHORESIS IN SUPPORTING MEDIA THE UPPER SECTION T ■ Top View S * * Side View E « End View TOP SECTION ---------------- ----- PART (ALL PLEXIGLAS) T ends SIDES ______________________ CENTERBEAM PIECES DIMENSIONS 2 1/8"X 91/2“ ISOSCOLES TRIANGLE ( 6 0 ° ) 2 l / 4 " X fX 1 2 " GROOVES AT ENDS l / 8 " X l / 8 " X l " TAPERED AS SHOWN I l / 2 " X l / 2 " X I I 3 / 4 " TAPERED TO 60° AT TOP II 3/4" i/e" 27 ~ T ~ Mid JI e Sect ion L oW€h S ectio i n t_. _____ FIGURE $ For multiple sheet runs the Lucite rod was not usually used* Instead the sheets were laid flat on a piece of lucite (extra-large separator) which was supported on the rim of the middle section same ten centimeters above the level of the buffer* The asbestos roof was always used for multiple-sheet work. Details of procedure for both single-sheet and multiple- sheet runs will be found in a later section* For electrophoresis in starch the same apparatus was used as for paper work except that the center section was replaced by the arrangement illustrated in Figure 6* The lucite support (bottom) was not attached to the remainder (walls) of the starch container* This permitted the starch block to be freed from its container at 18 on 28 the end of a run. To do this the container was placed on a block of wood of such dimensions (20 x 25 x 8 cm) that only the Lucite bot tom rested on the wood, the remainder of the container being free. A downward pressure was then applied to the walls of the container, thus removing it from the starch. For starch electrophoresis the asbestos roof was always used. It rested on the top rim of the starch container. Electrodes. In order to minimize electrode effects Ag-AgCl electrodes were used and these were isolated from the buf fer by a special electrode vessel, as will be described below. The silver electrodes were made in the following way (39)* Silver wire, 22 gauge, was wound tightly around a large test tube (ca. 3/h x 8 inches), until most of the tube was covered. The test tube was re moved, leaving a coil spring of silver about six inches long. This was stretched somewhat along the axis of the coils to separate the adjacent turns of wire slightly. The coil was then placed in a beaker of 0.1 N HCl, one end being left out of the aeid. Four to six such coils were placed in the same beaker and the ends connected directly. The wire was connected to the positive pole of a source of direct current (low voltage). The negative pole of the supply was connected to a carbon electrode which was placed in another beaker of acid. The two beakers were connected by a bridge of HC1, and the source was turned on. The voltage was raised until 50 to 100 mill! amperes was flowing. 29 FKHJRE 6 THE STARCH BLOCK AND SUPPORTING FRAME *! 01 30 ji 31 When the electrodes were to be used as the positive pole the current was stopped in ca. one -quarter hour. When the electrodes were to be used as the negative pole the charging was continued for five to six hours or overnight. FIGURE 7 A cross section of the electrode vessel is shown in Figure 7. The agar-KCl “bridge” was made as follows: 0.1 M KC1 was 32 heated to near boiling and Bacto agar as added, 3 grams/100 milliliters KC1. Heating was continued, with frequent stirring, until the suspension was clear or nearly so* It was then allowed to cool to about fifty degrees, and poured into the electrode vessel until the "outsideM section was full* After a gel had formed and the temperature had dropped to ca. thirty degrees the electrode vessel was placed in a tray of the buffer to be used. 0.1 M KC1 was added to the "inside" (electrode side) of the vessel until its level was the same as that of the buffer on the outside. The agar was preserved in this way until use. A piece of neoprene rubber, about one-sixteenth inch thick, was cut to fit on top of the agar in the electrode vessel. Holes about one-quarter inch in diameter were cut in the rubber with a cork borer at very frequent intervals leaving about one- quarter inch of rubber between holes. This mat of perforated rubber was placed inside the electrode vessek and a coil of Ag-AgCl wire laid on top of the mat. Two such electrode ves sels were then placed in the lower section of the electrophoresis apparatus, one in each buffer compartment. The ends of the wire electrodes were brought to the outside and connected to the power supply. Buffer was added to each compartment of the lower section to the desired level, and 0.1 V KC1 was added inside the 33 electrode vessels to the same level ■When it was necessary to use high current for long periods of time, as in starch electrophoresis, the silver electrodes were replaced by a copper system. Copper wire (16 gauge) was wound into the fora of a spring in the same way as described above for the silver wire. The KC1 was replaced by the following solutions i Pole Hegative Positive 6U£1 2 1 M Trace W8£kKh06 0.2 M 0.2 M mi 0.1 M 0.1 M The system is otherwise the same as described above. Buffers. Except where otherwise stated,. : one of the following buffers was used A. High pH (liO) 1. Veronal, pH 8.6 to 8.8, 0.05li* (38) 3k This was made by adding 1 N NaOH to diethyl- barbituric acid while stirring until the desired pH was attained. In this way no non buffering anion (C1-) was introduced. 2. Veronal, pH 8.6 to 8.8, 0.075 This is the molarity recommended by Durrum (Ijl, p. l j . 0 6 ) and was found superior to (l) for single-sheet runs. It was made as described above for the more dilute buffer. B. Low pH 1. Acetate, pH to 5.1> 0.05 M. 1 Staining Reagent. Trichloracetic acid was incorporated into this reagent to denature the proteins on the paper (see page 111) . Final Gone. Acetic Acid h % (v/v) Trichloroacetic Acid 5 % (w/v) X This reagent was designed and tested by Dr. R. J. Winzler, Mr. William Murakami, Dr. Hal Frankl, and the author. 35 Final Cone . Bronrphenol Blue Mercuric Chloride k % (w/v) 0.05 % (w/v) (water soluble sodium salt) Procedure Preparation of the Paper. The procedure for multiple sheet electrophoresis will now be described in detail. Whatman 3 MM paper 18J x 22J inches is cut (by machine) into sheets 23 x U5 centimeters. A uniform pile of these is made and the long dimen sion (side) clamped tightly, with carpenter*s clamps, between two 21 * x hn blocks of wood. Silicone grease is then liberally rubbed into the sides of the filter papers. This is repeated on the other side. Greasing was found to prevent migration of the pro teins toward the sides of the the direction of the electric sheets,i.e. perpendicular to FIGURE 8 36 field. This is obviously a result of the prevention of evaporation of water from the edges of the sheets. A line is then drawn on each sheet perpendicular to its long dimension and dividing it in two. Lines parallel to the first are then drawn at one centimeter intervals. Fifteen such lines on either side of the midline are usually adequate. Each of these is marked with the distance from the midline. One side of the sheet is marked minus and the other plus. When vacant spaces are pre dictable, i.e. parts of the paper where no protein will be found, the lines are omitted from that section. The midline is distin guished by arrows about two centimeters from each side. JO 1ST + Jr 10 FIGUEE 9 Application of Sample for Preparative Purposes (l to 2 ml. Sample). A shallow tray (ca. 3/li” x 10” x lU") is half filled with buffer and the first sheet is drawn through the buffer. As the sheet is drawn through from left to right the part already wet is allowed to "hug” the right wall of the tray; most of the excess buffer is removed in this way. The wet sheet is laid flat on 37 three or four pieces of Whatman #500 filter paper* The other five sheets are wetted in the same manner and laid on top of the first* These are covered until needed with a sheet of Parafilm. Two pieces of plastic about 25 x 30 cm*each are arranged in a shallow V as shown* A space of approximately three centimeters is left between the two pieces* One of the wet sheets^" is then laid onto the two plastic pieces* with the midline between them. The sample is drawn into the micropipet and applied slowly to the midline, as evenly as possible* Control of the rate of flow is by mouth, by means of a thin rubber tube connected to the pipet* The pipet is moved up and down along the midline several times during the application. The position of the line of application with respect to the rest of the sheet, and avoidance of its contact with any other surface, are of great help in confining the sample to a small section of the paper (ca. 8 mm* x 15 cm. for a 200 microliter sample)* FIGURE 10 "The proper degree of wetness can be approximately judged as the point when the sheet suddenly stops to reflect light as a glossy surface, and becomes instead a diffuse reflector. The ratio of water to paper was unfortunately not measured. 38 The sheet bearing the sample is placed in the apparatus on its Lucite support* The sample is applied to the second sheet in the identical way and this is laid on top of the first, care being taken that the samples superimpose* The process is continued until the fifth sheet is in place* The sixth sheet, containing no sample, is put in place in the same way* u O FIGURE 11 When desired a tiny drop (ca. 1 microliter) of concentrated1 bromphenol blue (water soluble Na salt) in water is placed on the fifth sheet at one or two points of the midline or slightly toward the negative pole* The concentration was not measured* The solution was made by adding HgO dropwise to about 10 mg. of the dye, with shaking, until a (dark blue) solution was obtained* 39 Application of Electrical Field* The asbestos roof is put in place and sealed with tape to the bottom unit. The voltage is applied, usually ca. 5 volts/centimeter. As the run progresses the current rises and the voltage usually drops. The current rise is due to a sizable decrease in the electrical resistance of the buffered papers. This results from two effects* (l) an increase in the amount of buffer in and between the filter papers; (2) an increase in the ionic strength of the buffer as water evaporates* This is not compensated for by the flow of liquid from the buffer vessels, since this liquid is buffer (not simply water). The usual voltage drop is of course a response of the ordinary power supply to the increased current, representing the internal ir drop. It could be circumvented by incorporating a constant voltage control into the system* However, as will be shown below, the more nearly ideal condition is a constancy of the current (produced by pro gressively lowering the voltage). The ir factor of the uncoupen- sated power supply used acted as partial current control and was found sufficient for most of the large scale (6-sheet) runs. The duration of the run is ordinarily twelve to fourteen hours (overnight). A run can be carried through in six to eight hours at higher voltage (ca. 8 volts/cm.), but the separation between the different bands of protein is less and the resolution not as good. Conpletion of a run is indicated by the position of the spot of bromphenol blue. hO Location of Components. At the end of the run the voltage is turned off, the roof removed, and the plastic support with the six papers is placed on a couple of 2" x 1*" wood blocks as shown. The ends of the filter-paper sheets overlapping the Lucite are cut off. The top sheet is then removed, placed on a sheet of blotting filter paper until excess moisture is withdrawn, and then trans ferred to another sheet of blotting paper. Cut here Cut here FIGURE 12 The second blotter, holding the top sheet, is transferred to an oven preheated to 130 to 1U0 degrees. The oven switch is turned off so that the coils are not heating and no fan is blowing. After thirty minutes the paper is removed; during this time the temperature of the oven used dropped about twenty degrees. The paper is taken out, held against a window pane, and the dye used as tracer is outlined. The paper sheet is then stained with the bromphenol blue dye solution. h i It was sometimes desired to locate the positions of the protein bands immediately after the sheets were removed from the electrophoresis box* To do this the heating step was omitted and denaturation of the protein was accomplished by incorporating trichloroacetic acid into the stain at a concentration of $%• Staining time could be limited to five minutes* Excess stain was removed in the usual way by washing repeatedly with h% acetic acid* When the background was white the sheet was placed on a towel of filter paper (Whatman #500) to remove excess liquid* Finally the sheet was placed on a dry sheet of filter and set into an oven preheated to about 130 degrees for one-half hour. The stain containing trichloroacetic acid was used through out and found highly satisfactory. However, if protein is to be measured quantitatively by the amount of dye bound it should be considered that this acid may have a differential effect on binding by various protein fractions* The color of the pattern obtained is dependent on the procedure used for staining and washing, being primarily a function of acidity. Using the above procedure the dye appears green. This color is very suitable for photographic purposes, but for visual in spection the blue variation seems to be better. The dye is blue in an alkaline environment* To produce this condition the procedure recommended by Durrum was adopted ( 1 * 1 , p. 393)* This consists of a final rinse of the electrophoretic sheet for about two minutes in a solution containing NaQA.c at a concentration of 2% and H Q A . C at a ii2 concentration of 10%. In the subsequent step the acid is removed by the heat treatment, leaving the paper alkaline and the dye blue* Isolation of Components. While the top sheet is being treated to locate the components the remaining five sheets are wrapped tightly in Parafilm to prevent evaporation of water. When the guide sheet has been stained, the optimum positions for separa tion of the bands of protein are selected* A discarded microtome blade is used to cut through all five sheets at once. It is laid in position and struck with a hammer* During the cutting the sheets are still wrapped in Parafilm. The resulting strips are assembled for elution on a large sheet of Parafilm and then placed in the elution chamber. Elution is allowed to proceed until the the eluting agent is exhausted. In cutting the strips for elution, a region between albumin and alpha-1, J to 1 centimeter wide, was usually discarded, to prevent cross- 5 t >-i f S 1 <LYi d 2- corresponding to each fraction contamination* The five strips were arranged for elution as shown in Figure 13 (U2). The strips in the figure are shown as separated from each other FIGURE 13 U 3 for purposes of clarity* In practice there is no air space between strips one and two and none between strips three and five. Several drops of water applied to each "joint” served to keep the strips "glued” together. The lower ends of the three lower strips (3,1** and 5) were cut to a V shape, so that the eluate was received into the lower beaker as uniform drops. A volume of approximately five milliliters/centimeter width (five centimeters for all five strips) was found adequate to elute the major part of the protein, as was shown repeatedly by staining of eluted strips— very little stain "took", compared to strips which were stained without prior elu tion* Water was used for elution of albumin and saline for elu tion of globulin. Procedure for Analytical Purposes (0 to 1*00 microliters). When paper electrophoresis is used for analytical purposes, the procedure is generally the same, but there are certain important differences. In this case, of course, only one sheet of filter paper is used. It is marked in the same way as above at one centi meter intervals* The midline is divided to accomodate four to six samples with ca. two centimeters between every sample and the ad jacent one. The first and last samples are kept about two centi meters from the edge of the paper. The volume of sample is such as to contain about three-quarters of a milligram of protein,e*g* ten microliters of plasma. Bather large volumes can be used,e.g. 150 microliters; in this case the sample is applied to several hh passes of approximately ten to twenty microliters each, time being allowed between passes for the sample to soak into the paper* 1*5 to 2*0 centimeters of the midline is allotted to each sample* The shallow V trough described above is used for the application, and is especially valuable when large volumes must be handled* Reproducibility. The reproducibility of the electrophoretic separation from sample to sample in a single-sheet (analytical) run is demonstrated in the accomparying pattern (Plate I)* This pat tern Mas made with two samples of serum from the same dog* In multiple sheet work it is important that all of the sheets have the same distribution of protein*i*e* that any given protein in one sheet be directly superimposed on the same protein in the next sheet* That this is so is demonstrated in Plate II, which shows the distribution of protein in all six sheets of a multiple run* No protein was applied to the top sheet (f)* Composition of Isolated Fractions * Electrophoresis was carried out either at pH 8.7 or at pH $.0. In the latter case a good separation into albumin and globulin was achieved. In the former case there were always the four major components easily distinguishable* In addition alpha-1 was almost always distin guishable about l| centimeters behind the albumin* The beta frac tion was frequently separable into beta-1 and beta-2. The separation of albumin from globulin at low pH, in a multiple-sheet arrangement, is shown in Plate III* Each of the hS PLATE I ELECTROPHORESIS OF TWO SAMPLES OF SERUM FROM DOG IV L6 o 1 7 7 17 7 177 2-81 D o ^ p s r i f <b 1 2. .? ¥ ,r" £ 7 ?7/°; h i PLATE II THE DISTRIBUTION OF SERUM PROTEINS IN ALL SIX SHEETS OF A MULTIPLE-SHEET RUN.1 a * BOTTOM SHEET, f * TOP SHEET. pH 8.6 The albumin component is at the six-centimeter level, the gamma component at the zero line (starting line). ^•1 J I " ■ J J t* 1 k 10 PLATE III THE SEPARATION OF ALBUMIN AND GLOBULIN AT pH $ IN MULTIPLE- SHEET ELECTROPHORESIS. EACH OF THE FIVE LOWER SHEETS RECEIVED UOO MICROLITERS OF DOG PLASMA. THE PLATE SHOWS THE TOP (GUIDE) SHEET.1 1 MR refers to methyl red, BB to bromphenol blue. A drop of a solution of each of these dyes was placed at each of two points of the zero line at the start of the run. 5o 10\- % 1 C 51 five lower sheets in this run received UOO microliters of dog plasma, making a total of two milliliters. Experiments in which all six sheets were stained proved the nearly identical distribu tion of protein, as at pH 8.6. In addition, when the proteins were separated and eluted, and samples from the eluates were re run, the patterns shown were obtained (Plate IV). This shows the separation of the albumin fraction into two components and the absence of the faster (at pH 8.6) albumin from the globulin fraction* The near absence of albumin from the globulin fraction was also shown by electrophoresis at pH 8*6 (of the globulins obtained at pH 5) (Plate V). The composition of the albumin fraction is shown in Plate VI. The presence of an alpha com ponent and a small amount of beta protein is clear. Plate VII shows the analysis at pH 8.6 of fraction obtained at the same pH. It was necessary to use large volumes of all eluates, because of the high dilution. The gradation of mobility is nevertheless very clear. The correlation of the mobility of each isolated fraction with the corresponding frac tion of whole serum is also reasonably good. 52 PLATE IV ANALYTICAL ELECTROPHORESIS AT pH k*9 OF THE ALBUMIN AND GLOBULIN FRACTIONS OBTAINED FROM DOG PLASMA BI MUL TIPLE-SHEET FRACTIONATION AT pH 51 'The numbers 1 and 3 refer to two of four replicate sasqples of plasma which were subjected to the same fractionation procedure* J *f.13 f 5 AH { ( • I JET pH r-o{i)M % PLATE V ANALYTICAL ELECTROPHORESIS AT pH 8.6 OF GLOBULIN FRAC TIONS OBTAINED FROM DOG PLASMA BY MULTIPLE-SHEET FRACTICWATION AT pH 5.1 The numbers 1 to k refer to four replicate samples of plasma which were subjected to the same fractionation procedure. 6* sLu TXy 3zr T T ‘ f * '& _3 ? H Daj y d h bui,'rtf 1J 0 1^3 V S~ L 56 PLATE VI ANALYTICAL ELECTROPHORESIS AT pH 8.6 OF ALBUMEN FRAC TIONS OBTAINED FROM DOG PLASMA BY MULTIPLE-SHEET FRACTIONATION AT pH 5.1 The numbers 1 to U refer to four replicate samples of plasma which were subjected to the same fractionation procedure 57 7- 3 , i 9 All 3. 1 z H 58 PLATE VII ANALYTICAL ELECTROPHORESIS AT pH 8.6 OF SIX FRACTIONS OF DOG PLASMA OBTAINED BY MULTIPLE-SHEET FRACTION ATION AT THE SAME pH. ( U j i l ) wjM»y v 1 ^ (% i{ 7lt $ { s j z ) 00% & ^L2l£) ^7° ffa 00 h fa? II) *1° o 0/7 I A 3> o Si J2t < ? X i £ * S4 L S V 65 60 FRACTIONATION OF CRUDE ALBUMIN I Reagents (1) Acid Citrate Dextrose (ACD) Trisodium citrate (friHgO) 26.7 g. Citric Acid 8.0 g. Dextrose 22.0 g. HgO to 1000 ml. One milliliter of the above was used for every six milliliters of blood. Final (2) Reagent a Cone. Sodium Acetate, l i M 200 ml. 0.8 M Acetic Acid 10 M I 4 OO ml. U*0 M HgO to 1000 ml. pH ■ 1*.0 (3) Ethanol-Acetate Ethanol, 9$% 2$0 ml. 23.75# (0.0852 mf) Sodium Acetate )Reagenf c 0.002 M Acetic Acid )(abtre)2*5al‘ 0-01 M HgO to 1000 ml. 2 used for protein fractionations described in the Appendix are included in this list. 2 mol fraction 61 (li) Reagent A1 (Lever et al. ii3) Final Gone • Ethanol, 95# 200 ml. 19.0# Sodium Acetate, 1 M 1 + 0ml. Q.OU M Acetic Acid, 1 M 3.5 ml. 0.0035 M Ho0 to 1000 ml. pH 5.8 (5) Ethanolic Zinc Reagent (Human) Ethanol, 95# 200 ml. 19% (0.0665 mf) Zinc Acetate Dihydrate 5k *8 g. 0.25 H HO to 1000 ml. 2 (6) Ethanolic Zinc Reagent (Rat) Ethanol, 95% 270 ml. 25.7# Zinc Acetate Dihydrate 5k .8 g. 0.25 & HgO to 1000 ml. (7) Ammonia Buffer Ethanol, 95% 200 ml. 19.0# Ammonia, conc. 57 • ml. Ammonium Chloride 3*1 g« H20 to 1000 ml pH - 10.5 "Hnol fraction. Final Cone. 62 (8) Aqueous Zinc Reagent (Suspension),pH 6.9 Zinc Hydroxide 0.01 mole 0.1 M Acetic Acid, 1 M (8.75 ml.) 0.088 H to pH 6.9 H2° to 100 ml. (9) Aqueous Mercury Reagent (Suspension), PH 7 Mercuric hydroxide 0.01 mol 0.1 M Hg0 to 100 ml. Preparation of S^-Labeled Albumin Chemical Fractionation. The preparation of S^-labeled albumin (dog) is based on the results described elsewhere (p. 210) using unlabeled dog plasma and fractions thereof. The procedure used is basically a two-step process similar to that used for rat albumin by Ulrich et al.(IUi). The bulk of the globulins was first removed by chemical fractionation and the resulting crude albumin was then purified by electrophoresis in a starch medium. To avoid the necessity of handling large volumes of material during the chemical fractionation the procedure of Hans Nitschmann (k5)"** was used as the preliminary step. Reagents containing ethanol or a 1 The author wishes to thank Dr. Karl Schmid for calling his attention to this valuable paper. 63 heavy metal were added in the frozen state, as described else where (Appendix G). A small dog (1;.0 kg.) was chosen and injected with 68 milli- 35 grams of L-cystine, containing two millicuries of S . One day after the injection the animal was exsanguinated from the carotid artery. The blood was collected into 35 milliliters AGD buffer. The cells, 120 milliliters, were removed as usual and a major part of the plasma (total: 210 ml.) was subjected to the cold alcohol fractionation as modified by Nitschmann (ii5)| zinc was used to precipitate the albumin. The procedure is outlined in the ac companying flow sheet (Figure 1U) • To eliminate as much S-^-labeled globulin as possible, and to dilute the activity of the S^^-labeled globulin not eliminated as far as possible, the radioactive albumin obtained was mixed with unlabeled. globulin and this "mixed plasma** was subjected to a second fractionation. The unlabeled globulin was obtained by fractionating plasma from a normal dog. This fractionation was carried out simultaneous ly with that described above and is given in Figure 15 • The procedure used for fractionation of the * > mixed plasma*' is depicted in the accompanying flow sheet (Figure 16). Half of the "normal globulin" obtained above was used for this fractiona tion. Ethanol was removed from the fractions, along with other reagents, by dialysis. Immediate dialysis against water was 200 ml. S35 _ AGD Plasma 5U ml. 9$% EtOH 1.5 ®1• Acetate buffer 10 ml. HgO to 19% EtOH to pH 5*8 -5 > degrees 1 S^-Globulin 1 S35-Albumin (I + II + III) (IF + V + VI + * 19% EtOH -5 degrees 1 S33-Globulin \ S^-Albxamin (I + II + III) Extract FIGHRE Ui FRACTIONATION OF S3*-1ABEI£D PLASMA (DOG) IK CO ID ETHANOL 65 S-^~Album±n (IV + V + VI + .••) EtOH to i | 0£ 1 M AcOH to pH 1*.8 [ S-^-Alburaln (IV + V) 20 ml* H20 30 drops 1 M NaOH to pH 5.8 t Suspension S^-Albumin t S3^-VI r X Host of the protein appeared to have gone into solution* FIGURE l l j . (Continued) FRACTIONATION OF S33-LABEIED PIASMA (DOG) IN COID ETHANOL + ••• P.l volume zinc reagent Ammonia buffer to pH 7-5 Overnight ” J S^-VII ♦••• 6 6 300 ml, "Normal” ACD-Plasma 91* ml. 80$ EtOH 2.25 ml* Acetate buffer to 19$ to pH 5.8 1 1 Normal” Glob«~Hn (I + II + in) 19$ EtOH •5 degrees "Normal” Globulin (I + II ♦ III) H 0 2 I "Normal” Globulin (a suspension) "Normal” Albumin (IV + V + VI + •••) Albumin extract "Normal” Albumin (IV ♦ V ♦ VI) FIGURE 15 FRACTIONATION GB UNIABEIED PLASMA (DOG) IN COID ETHANOL 67 ’ •Normal" Albumin (IV ♦ V ♦ VI ♦ •*•) 0.1 volume Overnight Zinc reagent Stirred slowly -5 degrees • ’ Normal1 * 17 + V * » Normal” VI + VII + ••• 0.1 volume Zinc reagent Ammonia buffer to pH 7.5 Overnight * f f VI VII + ••• FIGURE 1& (Continued) FRACTIONATION OF TJNLABELED PLASMA (DOG) IN COLD ETHANOL 68 1 1 Normal” Globulin (a suspension) Albumin Extract J of total EtOH 1 1 Mixed Plasma” Stirred slowly Centrifuged ” Mixed Globulin” 100 ml* Stirred slowly Centrifuged 1 hour Reagent A1 ca« 2 hours degrees Suspension of S^^-Albumin to 13% -5 degrees (Milky suspension) ca. 19 hours 2 hours -j> degrees 303 ml* S3£-Albumin (clear) 1 ”Mixed Globulin” Albumin extract 0*15 Volume Zinc reagent Overnight t M2.3=1. M- } FIGURE 16 FRACTION OF "MIXED PLASMA" (DOG) IN COID ETHANOL 69 303 ml* s3?-Alburain (clear) Zinc reagent 30 ml Stirred slowly li- hoars Centrifuged S^-Albumin V - 8 degrees Zinc reagent Ammonia buffer Overnight to pH 7.5 -5 degrees 1r M- 2-3 (Frozen Suspension) t t vi vn ♦ ••• FIGURE 16 (Continued) FRACTIONATION OF , ! MIXE3) PLASMA1 * (DOG) IN GOLD ETHANOL 70 avoided since this would have to be carried out at zero degrees or higher, -where the proteins would likely be denatured. Therefore NaCl was added to the protein solutions to a concentration of two to three M. The resulting solutions were then dialyzed for two to three days at -7 degrees against twenty-five liters of 1 ; M Na£l; then at 2 degrees centigrade for about two days against twenty to twenty-five liters of 0.02 M versene (disodium salt of ethylene- diaminetetraacetic acid) (1*6) preadjusted with NaOH to pH 6*2 (li3)5 finally at 2 to I ; degrees against a similar volume of H^O for about three days. The dialysing liquid was stirred thoroughly by con tinuous pumping* and was changed several times, the new "wash” being precooled before each change. After dialysing against distilled water the contents of each bag was emptied into a flask and the water removed by lyophilization. The product was white. The yield of protein in each of the major fractions was as follows: S3* - Plasma Held (g.) Globulin; "Water-Soluble" 1.3 Fraction VI 0.2 "Normal” Plasma Globulin; "Water-Soluble1 1 1.5 Albumin Fraction VI 1.1 71 "Mixed Plasma" Globulin, ‘ Water-Soluble" M- 2=1 H- 3-h s3^-AIburain (crude) Yield (g.) 2*2 0*2 Negligible 3-7 The composition of some of the above fractions is shown in Plates VIII and IX* pared by the above procedure was next purified by electrophoresis in starch (1*7). This procedure will be described in detail* To 21l;0 grams of Aroodtocrat potato starch (Morningstar- Nicol Inc ♦) was added, with stirring, two liters of veronal buffer, 0*05 M, pH 8. 6* This yielded a paste occupying some 31*20 milli liters* Roughly two-thirds of this was used* The sponges were put in place in the starch container* The starch was then poured into the container (Figure 6), yielding a rectangular block of starch approximately 20 x 29 x 5 centimeters* Excess water, which rose to the top of the starch block, was removed by blotting with filter paper* The crude albumin was dissolved in approximately fifty milliliters of veronal buffer* 53*5 grams of starch was added with stirring to provide a paste* An amount of starch corresponding to 1 The author wishes to thank Dr* J* W. Mehl for suggesting electrophoresis in starch for the purification procedure* Electrophoresis in Starch*^ The s3f>-labeled albumin pre- 72 PLATE VXXI THE COMPOSITION OF FRACTIONS OBTAINED FROM S3^- LABEIED PLASMA,1 "NORMAL" PLASMA,1 AND "MIXED PLASMA"1 .ese preparations are described on pages 62-71 73 PLATE IX THE COMPOSITION OF FRACTIONS LABELED PIASMA AND «MIXE3) OBTAINED FROM S3£ PLASMA”1 iese preparations are described on pages 62-71# J e t P»*!H 75 /3- / . u / ( v ^ W»o-s.|- M- 3-? 5 " A I k u x i l i , Ba*f* #- l i'r~ Ate s'- PL*-*-. i 76 that of the albumin-starch paste was removed from the midline of the block with a spatula* The sample was then poured into the ‘ ’ ditch” so formed* The starch block was evened out with a little fresh paste and covered with two pieces of filter paper* Contact between each end of the starch block and the cor responding buffer was provided by the porous sponge, as recommended by Kunkel and Slater (1*7) * The asbestos roof was put in place and the power supply turned on. The electric field was applied for 2|r days* The voltage was varied between seventy-five and ninety volts (five to six volts/cm.); the current varied between 90 and 180 milliamperes. Twice during the run the polarity of the electrodes was reversed, the starch container being at the same time turned around* The electrodes and buffer were completely replaced with fresh material about half-way through the run. After electrophoresis the starch was removed from the con tainer (page 21) and cut into one centimeter wide sections, which were placed in Erlenmeyer flasks* An approximately equal volume of water was added to each section and the suspension mixed thorough ly* A few drops of toluene were added and the flasks were placed in the cold room (ca. four degrees) overnight. The sponge at the positive pole was squeezed out thoroughly and washed, since it was suspected that some albumin might have migrated into it* The solution so obtained was treated as the starch extracts were treated* The starch in each flask, which had settled, was resuspended 77 by shaking, and poured into one of several cylindrical chromomato- graphic columns, the columns were between twenty-eight and thirty- four millimeters in diameter, with a sintered glass disc at the lower end. Prior to pouring the starch a “pad1 1 of dicalcite (Speedex) was made to overlay the sintered glass, Water was added to wash the column of starch until the total volume of eluate was in the neighborhood of 200 milliliters. The eluate was filtered through Whatman #1 filter paper, nine to eleven centimeters in diameter, on a Buchner funnel, then through Whatman #f>0 filter paper of the same diameter, and finally through Munktell OK, 2.2 centimeters in diameter. The Munktell paper had been pretreated with a tiny amount of fine celite. This was done by filtering a few milliliters of a celite suspension through the filter. The elution procedure proceeded dropwise. Filtration through the Whatman papers was moderately fast, requiring two to five minutes for a few hundred cubic centimeters. Filtration through the Munktell paper was rapid. The filtrates were considerably cleared by the filtration. Many were water-clear. The following were turbid: the four fil trates for the sections between -1 and + 3 cm. and the “filtrate** from the sponge. To 1.0 milliliter of eluate from starch was added six drops of 1 M NaOAc, two to three drops 10M HOAc, and one drop concentrated (12 M) H310^. To all tubes from-9 to + f > cm., which showed little 78 or no "response1 1 to HC10^a was added concentrated phosphotungstic acid (PTA), 0.2 ml3.11 liter. The results are shown below. Tube No. Precipitate witii HCIO^ PTA -9(-9 to -8 cm.) 0 -8 0 ++ •7 0 ♦ -6 0 0 -5 0 0 -U 0 ♦ -3 0 + -2 0 ♦ -1 (-1 to 0 cm.) 0 ++ 0 (0 to 1 cm.) 0 + + * ♦ • x 0 ++++ 2 0 ++++ 3 0 ++♦ I * 0 *+ $ 0 ++ 6 Slightly turbid 7 ++ 8 +++ p ++++ + Sponge + 10 79 One milliliter aliquots of selected sections were treated, at -f> degrees, with Zn , to a final concentration of approximately 0*02* M, and EtOH, to a final concentration of forty per cent* After standing at -5 degrees for about two days the precipitate in each was centrifuged down, dissolved in buffer (pH 8*6 veronal) or water, and subjected to electrophoresis on paper at pH 8*6* The results are shown in Plate A* The filtrates were dialyzed against three changes of circulating distilled water for approximately three days* They were then lyophilized. A small sample from each section was dis solved in buffer and subjected to electrophoresis on filter paper* The results show the success of the separation on starch* Most of the albumin was obtained free from globulin. The contaminated albumin was not used* The results of the electrophoretic fractionation are sum marized in the accompanying graph (Figure 17) • Of the 3*7 grams of protein applied only 2*0 grams were recovered* Of these 1*1 grams were globulin-free albumin* It is clear, though highly un fortunate, that the remaining protein, 1*7 grams, was lost into the positive buffer vessel* This portion was undoubtedly albumin of high purity. The possibility of nearly complete recovery of protein from starch had previously been demonstrated (see Appendix C). The protein (nearly 100/5 albumin) from section ten (9 to 10 cm*) was dissolved with U*0 milliliters saline plus 2*0 milli- 80 PLATE X THE COMPOSITION OF FRACTIONS OBTAINED BY STARCH ELECTROPHORESIS OF CRUDE S^-LABELED DOG ALBUMIN 8l 82 FIGURE 17 DISTRIBUTION OF PROTEIN AFTER EIECTROPHORESIS OF CRUDE S3*- LABELED ALBUMIN IN STARCH MEDIUM JOOrMj FVot e i o 100 - 600- roo - ¥o* 300 • 200- 100- |TotaJ AIL oCl Alt + oCZ Alt • f <*2 T “ 8 Alt hck)4 -sol. Z Z L 12. cm 00 Ui 8 U liters HgO. This was centrifuged at 5 degrees* A dark brown precipitate and an overlying gray precipitate were discarded* The supernatant was clear, slightly yellow. The -albumin from the sponge was dissolved with li* mil liliters saline and centrifuged for thre^-quarters of an hour at 5 degrees ceftfcigrade. The residue consisted (again) of some brown material and an overlying layer of gray material. The supernatent was clear, green. To remove any residual zinc from the albumin prior to in jection, the protein solution was passed through a mixed-bed ion exchange resin (1*3* U5). A column of MB-1 about 2 cm. (diameter) x 20 cm. was used. This was done in the cold room (+ 1 * degrees) and all materials were precooled before use. SPECIFIC ACTIVITY OF SULFUR General. The concentration of the radioactive element (S35>) in a given protein fraction could be determined on a sample of the protein, or on amino acids (cystine) isolated from such a sauple, or on S isolated from the protein in a manageable fora. An attempt was first made to count the protein itself, to avoid the necessity of any further treatment. However, no success was achieved in the preparation of a smooth planchet of protein which was suitable for counting, and which at the same time contained sufficient radioactivity to be easily measurable. It should be remembered in this connection that small amounts of S35 and total 85 S were used throughout* It was decided to oxidize the protein sulfur to sulfate, in which form it could be isolated as either the barium or benzidine salt (U8,U9,5>0,5l). Several conventional methods of sulfur analysis (50,52,53) were considered but most were unsuitable for routine analysis with the instruments available* Of a few methods tried the only one which showed promise was the wet oxidation in HG10i l -HN03 (5U. Apparatus* (l) The oxidation tube is pictured in the accompanying diagram (Figure 18) attached to a condensing tube. The tubes were made from a pair of 12 x 150 mm Pyrex tubes with matched male and female ground glass Joints* The Joints were chosen for perfection of fit. The female half was made into a container by sealing off the end. The male half was attached to a 12 millimeter piece of Pyrex approximately 60 centimeters long. The condensing tube in a West condenser was removed and replaced with that described above. Glass hooks were provided as shown so that the Joint could be sealed tightly with a pair of springs* The springs provided with the ground glass Joint were found too weak; springs of much higher tension were purchased in a hardware store. (2) A small heater was made by wrapping resistance wire (nichrome) around a ceramic tube of 13 millimeter bore. Sufficient wire was used so that the total resistance was 10 ohms. 120 volts from the house supply, when placed across 86 FIGURE 18 THE OXIDATION TUBE AND CONDENSER FOR WET DIGESTION OF S-^-IABELH) PROTEINS1 ^Sixteen of these units were operated simultaneously, the water line and heaters being in a series-parallel arrangement* Wctej'- •oki Xoi- Springs 88 four such heaters in series, provided a suitable rate of heating# Reagents (1) Oxidation Reagent (5W (Modified Pirie*s Reagent) Perchloric acid, 60% (w/v) 1 volume Nitric acid, 70% (w/v) 3 volumes (2) Copper Wire, 28-32 gauge (3) hydrochloric Acid, 6 N (10 Dowex 50 Resin, 100 mesh, sodium cycle. (5) Sodium hydroxide, 1 N (6) Acetic Acid, glacial (7) Benzidine, 1%, in Acetone (8) Borate Reagent Sodium Tetraborate (10 HgO) 1% (Final conc.) Sodium hydroxide 0.1 N (Final conc.) (9) Color Reagent Sodium-beta-napththoquinone-lj.-sulfonate 0.15 g* H20 100 ml. Benzidine dihydrochloride was purified as described by Hawk et al. (55* p* 588). The clean product is pure white. Ben zidine was prepared from the dihydrochloride just before use by neutralizing an aqueous solution of the latter and filtering the benzidine which precipitated out onto Whatman #56 filter paper. A seven to nine centimeter piece of paper and a Buchner funnel 89 were used. The paper and precipitate were then dropped into a flask containing sufficient acetone to give a 1 % (w/v) solution of benzidine. The resulting solution should be colorless, or nearly so* Sodium beta-naphthoquinone-U-sulfonate was purified ac cording to the detailed directions given by Folin (56, p. 389). The purified solid is bright orange* The solid was dissolved in water just before use to a concentration of 0.15 grams/100 milli liters (55, p. 587). Isolation of Protein from Solution. The amount of sulfur available for an analysis was small, about ten micromoles, and the following procedure was found suitable* The sample of pro tein, eluted from paper with twenty to thirty milliliters of solvent, was placed in a fifty milliliter conical centrifuge tube and to it was added perchloric acid to a fined, concentration of 0.6 Jt. The suspension was then placed in a boiling water bath for ten to fifteen minutes. (Omission of the heat treatment resulted in poor recovery of protein, at least for the albumin fraction). The suspension was centrifuged for ten to fifteen minutes, re suspended in 95# ethanol, and centrifuged again. The precipitate was finally suspended in ether and centrifuged once more* The ether was poured off, the tube drained onto a pad of absorbent paper, and then allowed to stand at room temperature for a few minutes. By this treatment the protein was obtained in the form 90 of a small ball which did not adhere to the glass tube and which could be transferred in one step to the oxidation tube* Oxidation. After the protein sample was transferred to the oxidation tube a two centimeter length of copper wire, twenty- eight to thirty-two gauge, was added (5h)* A small dab of silicone grease resistant to high temperature was applied to the male member of the joint* The tube was attached to the condenser and turned several times around to spread the grease evenly* The springs were then attached to the hooks and the water to cool the condenser was turned on* The oxidation tube, containing the sample of protein and attached to the condenser, was half-submerged in an ice bath* One milliliter of modified Pirie1 s reagent (5h) was drawn into a two milliliter syringe and added to the tube through the condensing tube* After a moment the ice bath was removed* A glass tube of thirteen to fourteen millimeter bore was cut so that when raised into position around the oxidation tube the lower ij to 2 cm. of the latter was left exposed. The heater was then raised as far as the glass jacket would permit and the voltage applied. Refluxing was allowed to continue for eighteen hours (5U). The heaters were shut off by an automatic timer. It was found convenient and efficient to oxidize sixteen samples simultaneously, in four groups of four, a series-parallel arrangement being used. 91 Removal of Perchloric Acid* When the heater(s) had cooled the tube was removed and its contents transferred to a ten milliliter beaker* The transfer was made quantitative by rinsing the oxida tion tube with one milliliter of water two to three times* the rinsings being added to the beaker* The latter was then placed on a hot plate covered with a thin (ca. one-sixteenth inch) layer of asbestos* Heating was regulated by a Powers tat transformer at a level just sufficient to provide continuous removal of acid vapors from the sample* The evaporation was carried out in a hood* After the sample was completely dry the transformer was removed from the line and the hot plate set on "high” for one-half to one hour* The sample became black. It was cooled briefly, one milli liter of 6 N HC1 was added, and the heating process repeated, again to blackness* Finally, one milliliter 6 N HC1 was added once more, but this time heating was discontinued when the sample was dry and copper-colored• Removal of copper * The copper introduced for the purpose of oxidation was found to interfere in a subsequent step, namely the isolation of benzidine sulfate* It was therefore necessary to remove the copper; this was done with a small column of Dowex fifty resin (57, P* 11|2). A column of resin in the sodium form was used for this purpose. The arrangement is illustrated in Figure 19* 92 To the dry sample was added | to 1 milliliter of water. The re sulting solution was transferred to the resin column, followed by J to 1 milliliter rinsings from the beaker. The tube was then stoppered and pinch- clamp #1 was opened until a fine stream of mercury flowed from the capillary into the pressure tube. The level of mercury in the reservoir was about 50 centimeters above the resin column. After a few milliliters of mercury had entered the pressure tube its rate of flow slowed to a dropwise flow. The pressure applied in this way to the solution was sufficient to force it through the resin at a fairly rapid drop-rate. Without this pressure the flow rate was many times slower. Cl asm R eceiVer FIGURE 19 93 The eluate was collected into a thirty milliliter beaker* The ten milliliter beaker used for oxidation was rinsed with water and the rinsings passed through the column into the vessel con taining the previous eluate • The washing process was repeated once more. Isolation of Sulfur. 1 N NaOH was added to the eluate dropwise until it was alkaline. One to two drops were usually sufficient* The beaker (eluate + rinsings) was placed on a hot plate and evaporated to near-dryness * Complete drying was care fully avoided because some sample is lost by sputtering if this is done* i *.0 ml. of HgO was added, and this was followed by 1*0 milliliter of glacial acetic acid and 9*0 milliliters of benzidine reagent (55 * P• 587 )• The beaker was covered tightly with Para- film and placed in the cold room ( I j . ° €) overnight * Some precipita tion was usually visible immediately after adding the benzidine reagent* The precipitate was collected on a disc of filter paper* Munktell 0& paper was used and the discs were cut out with a large cork borer* The size (of the borer) was chosen so that the discs fitted into the "metal Buchner funnel” used for filtration, Tracerlab 8B. The disc of paper was first weighed and then placed in the funnel and washed with blank solution, i*e* with H^O - secetic acid - acetone in the ratio s 1: 9. One milliliter of a sus- 9k pension of fine celite in the above solution was then drama through the filter and, without delay, the sample was poured in. The Hoke needle valve used for control of the vacuum was opened very slightly, about an eighth of a revolution. The filtrate was collected in the manner shown* It was almost always crystal- clear and was then discarded. However, if any turbidity was observed the filtrate was pas sed through the filter disc again* The precipitate was washed with blank solution and then with acetone* The "Buchner” funnel was covered with a piece of Kleenex and a £0 milliliter beaker (in verted), and air was drawn through for five minutes to dry the sample. FIGUBE 20 The sample, in the form of benzidine sulfate, was white and perfectly even except at the rim, where a slight elevation was present. This sample was used both for "counting” and for determination of sulfur. 95 Determination of Radioactivity ("Counting"). Since most of the samples were of low activity a gas (Q-gas) flow counter was used for counting. A triple-chamber counter made by Tracer lab was available. The disc of paper bearing the sample of benzidine sulfate was lifted with a fine pair of curved forceps and placed in an aluminum planchet. The latter was then lowered into the counting chamber, care being taken to center the sample. The counting procedure was the least accurate measurement in the determination of specific activity. In early work (Dogs I and II) the variation between replicate counts was especially 210 large. After this a "standard” bismuth disc (Tracerlab) was used to check the performance of the instrument before use. A plateau curve was run before every set of measurements and the instrument set at the center of the plateau voltage. This was regularly 1225-1325 volts. A series of replicate counts was then made with the bismuth standard to check for reproducibility. A similar check for reproducibility was often made also with a high counting sample of or (soft beta radiation) (58). With samples of low activity 512 counts were usually taken. With samples of high activity six or eight times as many counts were taken. It was necessary to apply two corrections to the counting rate measured! (l) a correction for self-absorption (and back- scatter) (1*8,1*9,50,51*59) and (2) a correction for the decay of 96 S35 (1 * 9). The latter would not be necessary if all samples could have been counted at the same time. Since this was not possible all counts were corrected to the figure which would have been ob tained at a given time. This correction is singly made on the basis of a semilogarithmic plot of the decay of (half-life; 87.1 days, references li9 and 60). The correction for self-absorption was made on the basis of a series of samples (in duplicate) containing a constant amount of S-3^ and varying amounts of benzidine sulfate (6l). These were prepared exactly as were the unknowns, starting with sulfate salts (see table)s lOmM. ( N V 2S0U 11 1, 000 cpm Na2S35 0h h2o Glacial Acetic Benzidine Reagent 0.20 ml. 1.00 ml. 2.8 mU 1.0 ml. 9.0 ml. .kO 1.00 2.6 1.0 9.0 .60 1.00 2.2i 1.0 9.0 o co • 1.00 2.2 1.0 9.0 1.00 1.00 2.0 1.0 9.0 2.00 1.00 1.0 1.0 9.0 After these samples had been collected and counted the sulfur determination was run in the usual manner. The ratio of counting rate (over and above background) to the amount (micro moles) of benzidine sulfate found colorimetrically is called SAp (specific activity found). Let SAq (specific activity calculated— no absorption, 100$ recovery) be the reciprocal of the number of micromoles of sulfate originally added as salt. SA^ is a steep function of the amount of sulfate in the sample (for small samples) and, if there were no absorption, SAp would be the same function, except for a constant factor. To focus our attention on the self absorption effect take the ratio SAp/SA^. This would be constant if there were no absorption. Otherwise stated the ratio SAp/SAQ Number of micromoles of S added Activity x dumber of micromoles of S found is the activity observed corrected for ary loss of which may have occurred during isolation. The results are plotted in the accompanying graph (Figure 21). There is some spread in the results. A "maximum curve” and a "minimum curve" were drawn so as to include the majority of the points between them. A curve was then drawn in such a way that, at any point (abscissa x), the ordinate be the average of the corresponding ordinates for the "maximum" and "minimum” curves. This is the "average curve". Using the latter, let C be the ratio of SAp/SA^ at ary point (abscissa x) to the value of SAp/SA^ for a sample of ten micromoles. C — ^Ap/SA^Jx FIGURE 21 SEIF- ABSORTION FOR S& BENZIDIHS SUIFATE 99 ShrlSAc (C o u n ts pel* M i n u t e ) 10,000 8,000 6,000 *f,000- 1,000 0 - 0 1 0 10 Micromoles S e n x iJ in e Sulxu-te on P l» n c ll# t 100 Then C is the correction factor for self-absorption (and back- scatter) for a sample containing x micromoles of benzidine sulfate. The (uncorrected) specific activity is divided by C. Betermination of Sulfur* Sulfur was determined by the method of Letonoff and Reinhold (55* page 587 )• This is in reality a benzidine determination based on its reaction with sodium beta- naphthoquinone-U-sulfonate to yield a highly colored orange-brown product. The paper disc containing the precipitate of benzidine sulfate was first weighed. A Sartorius balance was very convenient. Since the disc had been weighed before collection of the precipitate* the weight of the latter could be calculated. This weight was in error by the amount of celite present on the paper disc. An approx imate correction was made on the basis of a blank. The disc was then placed in a tube or flask and borate reagent was added so that the final concentration of benzidine was ca. 0.6 mM. The suspension was placed in a water bath at fifty to seventy degrees for | to 1 hour* with occasional shaking. The benzidine sulfate went into solution slowly* but some material remained suspended. This was the celite. The suspension was clarified by filtration through another disc of Munktell OK paper on the metal "Buchner" funnel. For efficiency of operation in collecting and washing the benzidine sulfate precipitate and in clarifying the benzidine solu tion* three or four metal Buchners were operated simultaneously 1 0 1 under very mild vacuum, the vacuum controlled by Hoke needle valves. For the benzidine determination one milliliter of the benzidine borate solution was used* For the standard curve 0 to 10 milliliters of borate reagent was added and the volume made up to eleven milliliters with water* The remainder of the procedure is as described in Hawk et. al. ($S>). Occasionally a precipitate formed in the reaction tube. This was dissolved by addition of two milliliters (more) of acetone to all tubes, in cluding the standard. The color measurement was made in a Coleman #11 spectrophotometer at a wavelength of L l90 millimicra. The adequacy of the procedure for sulfur determination is demonstrated by the following considerations s (l) The standard curve for benzidine dihydrochloride is linear up to about 0*7 micromole, after which it levels off* The slope of the standard curve was generally 1.3 optical density units per micromole of benzidine* (2) Known amounts of ammonium sulfate carried through from the isolation step forward gave 95% recovery. (3) Samples of ammonium sulfate introduced into the procedure at the oxidation step gave seventy to ninety per cent recovery. (h) Samples of cystine and methionine gave satisfactory recovery of sulfur. (5) Samples of bovine serum albumin gave values for sulfur close to those in the literature (62). (6) Several samples of orosomu- coid (mucoprotein) prepared in different ways were analyzed suc cessfully for sulfur content. University of Southern California Library 10 2 It may be called to mind that the determination of specific activity is not dependent on the recovery of sulfur. CARE AND TREATMENT OF ANIMALS It was not possible to select dogs for uniformity of genetic constitution. All of the animals had been inoculated for distemper and for rabies and had been isolated for at least ten days prior to use. Those used were presumably in good health. Each animal received six ounces of Purina Kibbled Meal, three ounces of Miller’s Puppy Meal, and three ounces of fresh ground horse meat per day. This diet is twenty-two per cent protein. Water was always available. Blood samples were taken from the superficial veins of the hind legs and forelegs. These veins were also used for intra venous injection. Blood samples were usually taken in the morning, before the animals were fed. For total exsanguination a dog was first anesthetized with nembutal given intraperitoneally. When anesthesia was sufficiently deep An incision was made on the ventral aspect of the neck, ex posing some three inches of trachea. Both carotid arteries were then located and isolated with loops of thread. One was tied off. The second was clamped shut with a hemostat and a cut was made in it about one inch distal to the clamp and extending approximately one-third of the way around the vessel (circumference). Through this cut was inserted a plastic (Polyethylene) tube cia. one-eighth inch in diameter. The end of this tube had been cut at an angle of about forty-five degress to allow for easy insertion. The tube was tied in place and its free end was put into a receiver containing some fifty milliliters of ACD solution. The hemostat was then removed from the artery. It was sometimes necessary to hold the plastic tube at its entrance into the vessel to prevent the opening of the tube from being closed by the wall of the 1 artery. 1 The author wishes to express his thanks to Dr. T. M. Lin for teaching him this technique and for other valuable aid generously given. CHAPTER IV RESULTS Information relative to the material injected into each animal is summarized below: Injection Animal Dog Weight (kg) Material and Amount Route I - S^-L-cyst ine, 150 microcuries IP II - S^^-L-cyst ine, 200 microcuries IP III 13. s3^-L-cystine, lliO microcuries IP IV 13. S^-L-cystine, 200 microcuries IP V 1U. Crude S-^-Labeled Dog Albumin (Gr-5U)>2.5 x 106 counts per minute#6.0 grams Protein (in 100 ml.) IV Donor #2 l*.o S^-L-cystine, 2 millicuries (68 mg. in U*7 ml.) IP VI 6.5 S-^-Labeled Dog Albumin, U x 10° counts per minute IV VII 8.5 S^^-Labeled Dog Albumin, 3*6 x 10° counts per minute IV VIII 7.0 S35-Labeled Dog Albumin, 3.6 x 10® counts per minute IV IX 6.U S^-Labeled Dog Albumin, 3.0 x 10® counts per minute IV IP = intraperitoneal IV = intravenous 1 0 $ In the determination of turnover it is highly desirable to measure several variables at the same time. However, such an in vestigation requires the labors of several trained people* Since in the work herein reported only one individual was available, it was necessary to limit the scope of the analyses as much as pos sible. As a result the biological data obtained are confined largely to measurements of the specific activities of the albumins and the globulins as a function of time. In the first series of investigations each of four dogs was injected intraperitoneally with S^-L-cystine. The radioactiv ity of the albumins and of the globulins was then measured over a period of about thirty days. In the second series each of five dogs was injected with S-^-albumin from a donor dog and similar analyses were carried out. The accompanying figures diagram the results obtained after injection of S^-L-cystine into dogs. In the first (Fig. 22) the plot is made with a linear scale for both abscissa (time) and ordinate (specific activity). In the next (Fig. 23) the specific activity of total serum protein (Dog I) is plotted on a logarithmic scale. Figure 2h gives the corresponding curve for Dog II. The different values plotted for a single time represented by a given symbol (e.g. 0) are various counts taken on the same sample and illustrate the difficulty which was encountered in obtaining re producible measurements of radioactivity. It was subsequently learned from the Nuclear Corporation that much counting difficulty 106 FIGURE 22 DOG I* THE SPECIFIC ACTIVITY OF SULFUR IN WHOLE SERUM AND IN TOTAL SERUM PROTEIN AS A FUNCTION OF TIME1 OWhole serum (first aliquot). □ The same serum (second aliquot). O' Total serum protein precipitated from the same serum (third aliquot) with percloric acid (0.6 M). Ef A duplicate sample of total serum protein obtained from the same serum (fourth aliquot). Where two or three values fall close together this is indicated by including the corresponding points within one symbol. Where two or more values are equal; the symbol is filled in to appear solid. Specific Activity (Ceuntj per Minute per Micromole S u/fiir r 00 ■ 1 0 0 - 00 - 10 10 0 ijro M icrocuHe.s S - L “C y*ti*e XvttKaperitovieaJ^r H o 1 0 8 FIGURE 23 DOG I. THE "AVERAGE CURVE" OF FIGURE 22 PLOTTED ON A SEMI-LOGARITHMIC SCALE 109 ic A&tjVity ( Counts f>e»- Minute pt \ r SuljMf) 800 400 200 too o zo 10 1^0 Mic.racw~#es S C y s tin e I » t r * - p e r it ^ ^ e c U / 11 0 FIGURE 2k DOG II. THE SPECIFIC ACTIVITY OF SULFUR IN WHOLE SERUM A3!® IN TOTAL SERUM PROTEIN AS A FUNCTION OF TIME1 O Whole serum (first aliquot)# (3 The same serum (second aliquot)• & Total serum protein precipitated from the same serum (third aliquot) with perchloric acid (0*6 M)# GT A duplicate sample of total serum protein obtained from the same serum (fourth aliquot)# Where two or three values fall close together this is indicated by including the corresponding points within one symbol. Where two or more values are equal the symbol is filled in to appear solid# Ill S p e c ific A c t iv ity ( Count* fir M jmrfrc f AH M icromofe S u l f u r ) 600 f fOO ■■ ZOO • 100 t 3JT *LQQ I v l icirQcui~ies S - L~ C y . s tin€ I h tr^peH totje^Hy 11 2 was caused by the poor quality of Q-gas available. In addition, it is felt that the values obtained early in these experiments are less reliable than the later figures, due to the experience of preparing a good planchet containing a sufficient amount of benzidine sulfate. Figures 25 and 26 illustrate the results obtained with albumin from dogs I and II by electrophoresis on paper at pH 8*6. Figure 27 illustrates the corresponding graph for dog III. This is a later experiment and the improvement in results may be noted. From this result one can state that the decay of albumin in this dog was linear (on a semi-logarithmic scale) with a half-life probably not less than thirteen and one -half days and not greater than fifteen days. Each serum sample from dog IV, after electrophoresis at pH 8.6, was separated into its electrophoretic components, and the specific activity of each of these was determined (counts/ minute per micromole sulfur). Figure 28 illustrates the results obtained with the beta proteins. Two points are worthy of note* First, there is a sharp contrast between the decay rate of each fraction in the first few days after injection and during the second week thereafter. Second, the decay rate of beta-1 was much faster than that of beta-2 during the first few (four) days, but the difference was much less during the second week. Figure 29 illustrates the results with the gamma fraction. 113 FIGURE 25> DOG I. THE SPECIFIC ACTIVITY OF ALBUMIN SULFUR AS A FUNCTION OF TIME1 1 O Albumin obtained from serum# 0 Albumin obtained from a duplicate aliquot of the same sample of serum. Where two or three values fall close together the corresponding points are enclosed in one symbol. Where two or more values are equal the symbol is filled in to appear solid# SPECIFIC ACTIVITY (counts per minute per micromole sulfur) IOOO 800 600 400 ALBUMIN HALF-LIFE: 1 4 DAYS 200- 100- 0 DAYS 20 10 150 MICROCURIES S^-L-CYSTINE INTRAPERITONEALLY us FIGURE 26 DOG II. THE SPECIFIC ACTIVITY OF ALBUMIN SULFUR AS A FUNCTION OF TIME O Albumin* 13 Albumin from a duplicate aliquot of the same sanple of serum* Where values fall close together the corresponding points are included in one symbol* Where two or more values are equal the symbol is filled in to appear solid. 1x6 SPECIFIC ACTIVITY (counts per minute per micromole sulfur) IOOO 8 0 0 - 6 0 0 4 0 0 - ALBUMIN HALF-LIFE! 15 DAYS 200- 100- 0 10 20 DAYS 200 MICROCURIES S 3 5 -L-CYSTINE INTRAPERITONEALLY FIGURE 27 DOG III. THE SPECIFIC ACTIVITY OF ALBUMIN SULFUR AS A FUNCTION OF TIME1 1 Where values fall close together the corresponding points are included in one symbol# Where two or more values are equal the symbol is filled in to appear solid. 1X8 SPECIFIC ACTIVITY (counts per minute per micromole sulfur) lOOn 80 LBUMIN HALF-LIFE: 13 1/2-15 DAYS 6 0 - 4 0 - 20 20 30 40 DAYS 1 140 MICROCURIES S35-L-CYSTINE INTRAPERITONEALLY 119 FIGURE 28 DOG IV. THE SPECIFIC ACTIVITX OF SULFUR IN THE BETA PROTEINS AS A FUNCTION OF TIME1 1 O The beta-1 fraction. A The beta-2 fraction. 120 SPECIFIC ACTIVITY (count perminuteper micromole sulfur) 1000 800- 600- 400 200 24 doys 132 19 days /31 3 days f31 100 7 days f3 2 20 0 10 DAYS T 200 MICROCURIES S3 5 -CYSTINE INTRAPERITONEALLY 1 2 1 FIGURE 29 DOG IV, THE SPECIFIC ACTIVITY OF SULFUR IN THE GAMMA FRACTION AS A FUNCTION OF TIME SPECIFIC ACTIVITY (counts per minute per micromole sulfur) lOOO-i 800- 600- 400- discord 200- l2doys days 100- 20 10 DAYS 0 200 MICROCURIES S35-L-CYSTINE INTRAPERITONEALLY 122 1 2 3 It is immediately elear that, unlike the beta fractions, the decay of gamma is linear from the second to the fourteenth day after injection* The results with the alpha fractions (Figure 30) are un certain but it appears that alpha-1 showed an initially rapid decay rate followed by a much slower rate. The alpha-2 fraction, on the other hand, probably did not show this apparent change in rate of decay. The following graph (Figure 31) shows the results with the albumin fraction from this dog. Again, although the initial decay rate is slower than for the globulins, the f , slowing up” of the rate of decay with time is clear* Perhaps the most striking observation is that, although the rates of decay during the first few days differ widely for the various fractions, all except the gamma have a half-life of eighteen to twenty-four days during the second week following in- • a £ jection of the S-' -labeled amino acid. Dog V was injected with a partially purified preparation of s3£-labeled dog serum albumin* This was contaminated with alpha- 2 and beta proteins, both actives Specific Activity Protein (%) (cpm/micromole S) Albumin 67 550 Alpha-2 26 1010 Beta 7 902 12k FIGURE 30 DOG IV. THE SPECIFIC ACTIVITY CF SULPHUR IN THE ALPHA. PROTEINS AS A FUNCTION OF TIME1 O The alpha-1 fraction. A The alpha-2 fraction. SPECIFIC ACTIVITY (counts per minute per micromole sulfur) IOOO 800H 600 4 0 0 4 200 I00H 80 H 1 8 days oC 2 60H 20 days ct 40H 3 days 10 20 0 DAYS t 126 FIGURE 31 DOG 17. THE SPECIFIC ACTIVITY OF ALBUMIN SULFUR AS A FUNCTION OF TIME 127 SPECIFIC ACTIVITY (counts per minute per micromole sulfur) IOOO n 800- 600- 400- 200- 26 days 10 days 100- 10 20 DAYS O t 2 00 MICROCURIES S35-CYSTINE 1NTRAPER1T0NEALLY 128 The graph of specific activity vs time for this dog (Figure 32) shows the same sort of change in rate of decay as was observed previously when S -cystine was injected. It is noteworthy that the half-life for the period three to six days after injection of labeled protein is very large in this case* Dogs VI to IX (inclusive) were injected with purified S - labeled dog albumin, prepared as described in the section on methods. Serum samples from these dogs were separated into an albumin fraction and a globulin fraction as described. All four dogs were injected with the same preparation, but dog IX received the protein a few weeks after the others* This dog gave evidence of shock shortly after the injection and was found dead the following morning. This was the only dog from which samples were taken shortly after administration of S3 ^-labeled albumin. The small ratio of the specific activity of globulin to that of albumin shortly after injection is confirmation of the electrophoretic purity of the preparation injected and of the success of the electrophoretic separation. The data for the specific activities of albumin and globulin as a function of time are given for dogs VI, VII, and VIII in the accompanying graphs, using the usual semilogarithmic plot (Figures 33 to 35)• These data cover the period from the third to the twenty-fourth day after injection of S-^-labeled albumin. The points indicated by squares represent the values obtained using the hot 129 FIGURE 32 DOG V. THE SPECIFIC ACTIVITT OF AIBUMIN SUIFUR AS A FUNCTION OF TIME 1 3 0 SPECIFIC ACTIVITY (counts per minute per micromole suHUr IOO-k 801 60H l/^i 32 days 401 20 2 4 6 DAYS CRUDE S35-LABELED ALBUMIN, C fl. 2.5 MICROCURIES INTRAVENOUSLY 131 FIGURE 33 DOG VI. THE SPECIFIC ACTIVITY OF SULFUR IN WHOLE PLASMA AND IN THE ALBUMIN AND GLOBULIN FRACTIONS THEREOF AS FUNCTIONS OF TIME O Albumin precipitated with (unheated) perchloric acid. G3 Albumin precipitated with hot perchloric acid. 7^ Total plasma protein precipitated with perchloric acid. O' Globulin precipitated with (unheated) perchloric acid. GfGlobulin precipitated with hot perchloric acid. SPECIFIC ACTIVITY (counts per m inute per m icrom ole sulfur) IOOt 132 80 60- 40H 20- 10- 8- 6- 4- 2- O ALBUMIN HALF-LIFE; 10.4 days o V PLASMA T 1 /2 10.8 GLOBULIN r l/2 ^ 22 t s 35 To 2^ DAYS S3° LABELED ALBUMIN, Sfi. 4.0 MICROCURIES, INTRAVENOUSLY 133 FIGURE 3h DOG VII. THE SPECIFIC ACTIVITY OF SULFUR IN WHOLE PLASMA AND IN THE ALBUMIN AND GLOBULIN FRACTIONS THEREOF AS FUNCTION OF TIME O Albumin precipitated with (unheated) perchloric add. [ 7}Albumin precipitated with hot perchloric acid. XTotal plasma protein precipitated with perchloric acid. C^Globulin precipitated with (unheated) perchloric acid. G^Globulin precipitated with hot perchloric acid. SPECIFIC ACTIVITY (co u n ts per m inute per m icrom ole sulfur) IOO-i 80- 60- ALBUMIN HALF-LIFE: 1 1 .7 DAYS 4 0 - 20- X PLASMA 10- 8- 6- 4 - GLOBULIN Ti/a 120 2- 0 |0 20 DAYS IS 35 LABELED ALBUMIN.fifl. 3.6 MICROCURIES, INTRAVENOUSLY 135 FIGURE 35 DOG VIII. THE SPECIFIC ACTIVITY OF SULFUR IN WHOLE PLASMA. AND IN THE ALBUMIN AND GLOBULIN FRACTIONS THEREOF AS FUNCTIONS OF TIME © Albumin precipitated with (unheated) perchloric acid. G Albumin precipitated with hot perchloric acid. * Total plasma protein precipitated with perchloric acid, o'Globulin precipitated with (unheated) perchloric acid. GfGlobulin precipitated with hot perchloric acid. SPECIFIC ACTIVITY (counts per minute per m icrom ole sulfur) 100 80 60- ALBUMIN HALF-LIFE! 9.0 DAYS 4 0 - 20- PLASMA T i/2 9.9 days 10- 8- 6- 4 - GLOBULIN 23 2- 0 1 0 20 DAYS t S35 LABELED ALBUMIN,ca. 3.6 MICROCURIES, INTRAVENOUSLY 137 perchloric acid (0.6 M) precipitation of protein. The other points were obtained with protein samples precipitated by HC10|^ (0.6 M) without heat. The samples obtained by the latter method contained little material and the results are therefore not as reliable as those obtained by the former procedure# Alb 0 to 1 day VI VII VIII IX - 1*3 day Tj. Alb 3 to 2h days 10#U 11.7 9«0 - days Plasma 10.8 - 9#9 - days T § Glob 22. 18. 21*. - days The data for the four dogs receiving purified S*^-labeled albumin are summarized in the accompanying table. Some points may be noted now about the data presented* Al though there are a few values which are obviously incorrect, a general picture is nevertheless discernible. The half-life of albumin is between nine and twelve days for each of the three dogs# The decay of albumin seems to be linear,or nearly so, on a semi-log plot# The specific activities of total plasma protein decayed at approxi mately the same rate as the specific activity of albumin. The specific activity of the globulin fraction varied between five and fifteen per cent of the albumin activity for the period between three and twenty-four days after injection of S^-labeled 138 albumin* The globulin fraction appears to have decayed with a half-life of approximately twenty days, about twice that of albumin. For dogs VII and VUI, and possibly for dog VI, the specific activity of this fraction appears to have reached a maximum between the seventh and ninth day after infection of S^-labeled albumin. In the case of dog IX, where measurements were made during the first twelve hours after injection, the picture is entirely different (Figure 36). Here albumin decayed with a half-life of about lj- days. The globulin activity was three to five per cent of albumin and was higher twelve hours after the injection than it was at the fourth hour. 139 FIGURE 36 DOG IX, THE SPECIFIC ACTIVITIES OF AIBUMIN SUIFUR AND GIOBUUN SUIFUR AS FUNCTIONS OF TIME IJ+O SPECIFIC ACTIVITY (counts per minute per micromole sulfur) 400n ALBUMIN “HALF-LIFE": 3 1 hours (1.3 days) 200- 8i 4 - 2- OIED (Shock) HOURS 10 5 LABELED ALBUMIN, £ 2 . . 3.0 MICROCURIES, INTRAVENOUSLY O CHAPTER V DISCUSSION Design of the Experiments. In tracing the metabolism of proteins one has a wide choice of amino acids and some choice in the element to be used as tracer. Sulfur has the advantage that it occurs in only two of the amino acids , methionine and cystine (and cysteine)* Thus, oxidation of a protein and isolation of the sul fate produced yields information about these two amino acids which is uncomplicated by the status of the other components of the protein, neglecting any form of bound sulfur other than amino acid sulfur. Methionine sulfur is converted rapidly into cystine sulfur, but the reverse does not hold (63,61*,6f>). Therefore if one uses s35>-cystine ^ tracer one can assume that the results obtained reflect the metabolism of this amino acid (cystine +cysteine) alone. S -cystine was therefore chosen as the tracer amino acid. If one uses S^^-methionine both the cystine and methionine of the proteins become labeled. This offers an attractive possibility for an investigation of the relative rates of uptake of two amino acids into different proteins and into different parts of the same protein (32,33 »3k)* It was originally intended to include the former as part of the present work,i.e. to compare the uptake of cystine and methionine into plasma albumin and globulin. A means of separating the cystine sulfur from the methionine sulfur was developed for this purpose.^ However, time did not permit this part of the work to be carried out. Such an investigation would yield valuable information as to whether or not various proteins (and various parts of the same protein) are built from the same ^pool” of amino acids. It may be emphasized that using sulfur it is necessary to separate only two amino acids from each other, the presence of any of the others being of no consequence. The labor involved is thus much less than when it is necessary to isolate an amino acid free from all others. In measuring the specific activity of protein sulfur methio nine sulfur was not removed. Assuming a constant composition for any given protein fraction, this merely means a dilution of the cystine sulfur. Since the amounts of sulfur measured were quite small the addition of a constant proportion of inactive sulfur was considered advantageous. One group of dogs was injected with S^-labeled cystine and the other with S-^-labeled albumin. Since it was desired to obtain information about reincorporation of s35 from other proteins into albumin it would have been highly desirable to do both experiments on every animal. Thus,one could first measure the metabolism of / albumin when it was the only protein initially labeled and one could then, after a suitable intermediate period, inject the same animal 1 A nearly identical method has been published (66). 1h3 with the labeled araino acid and measure the metabolism of the albumin again. It would then be possible to compare the apparent rates of metabolism of the same experimental material using the two techniques. While this approach is much to be preferred it was not used simply because -labeled albumin was not available until near the end of this work* Interpretation of Results * For dog I?, which was injected with S^-L-eystine 3 the time course of the specific activity of albumin sulfur (Figure 31) has an initial rate of decay corresponding to a half-life of ten days* However, the decay rate is not linear (on a semi-log plot) but decreases as shown* This is what one would expect on the basis of continued incorporation of S from other sources into albumin* Dog V, which was injected with a crude preparation of S^ - labeled albumin containing other S^-labeled proteins, shows a similar change in decay rate with time* The specific activity of the contaminating proteins injected was nearly double that of the albumin and they constituted one-third of the injected protein* The initially very rapid, and the subsequent very slow, decay of the albumin cannot be simply interpreted as due to reincorporation of 35 Sr"' from "body protein", since the latter was not originally labeled. The observed result can be partially explained as being due to In- 35 corporation of S*^ from the globulin fractions of the injected material into the albumin fraction. However, this makes it difficult lWt to explain the very slow decay, since this is not in keeping with the data of others following the injection of whole plasma (15,1?, 19*27). The possibility of the presence in the donor animal of an albumin component which is metabolized slowly by the recipient animal comes to mind. The initially rapid decay in this animal is also somewhat puzzling* Considering the large amount of material injected one would not expect the effects of mixing of intravascular with extra- vascular albumin to be manifest beyond the first day (28)* On comparing the two groups of animals, those injected with S^-cystine (dogs I-IV) with those injected with S^-labeled albumin (VI-VIII), the data suggest that the half-life of albumin in the former is about fourteen days while that of the latter is about ten days. The simplest interpretation of these results is that with the former method of tracing there is continuous in corporation of S3* from other sources into albumin, so that the time course of specific activity is not a true reflection of the rate of metabolism of the protein. This would also explain the changing slope of the decay curve for the albumin of dog IV (Figure 31). Why decay should be linear in one case (dog HI) and not in the other (dog IV) is not clear. The importance of re incorporation for the globulin components is clear from Figures 28 to 30. Only the gamma fraction, and possibly the alpha-2 fraction, appear not to be affected. One may here recall the "synthesis of all plasma protein fractions except gamma globulins 1 h S by the liver1 1 (10,11). It may also be noted again that the apparent rates of decay of the various protein fractions are much closer for the later phases of the curves than for the early phases. This is what one would expect if the controlling factor in the apparent rate of decay shifted from the rate of metabolism of the various fractions to the rate at which S35 is released from some 1 1 external” source. The implication of reincorporation as responsible for the difference between the two groups of animals with respect to the apparent half-life of albumin is not unequivocal. For the experiments with S*' -labeled albumin, dogs of small size were chosen so that the amount of radioactive albumin available would be sufficient. It is known however (12, p. 127?) that, in comparing different species, the half-life of the plasma proteins is related to size, being larger for the larger animals. Whether this applies to different individuals within a species is not known to the author. A com parison of dogs VI, VII, and VHI makes it unlikely that the dif ference in size could alone account for the observed difference in half-life. Another factor which cannot be ruled out is the effect of diet. The amount of protein in the diet has been shown to be an important factor controlling the rate of metabolism of the plasma proteins (16). Since the dogs were fed a standard diet the larger animals received less protein,per unit weight, than the smaller animals. This would tend to give rise to a slower decay rate for 11* 6 the plasma proteins in the larger animal,i.e. to a longer half-life* It is difficult to evaluate the contribution of this factor, but from the evidence cited (16) it would appear that with the low protein diet used the amount of protein fed per unit weight of animal could account for only a small difference in half-life. The effects of diet become greater when comparing a high-protein diet (e*g* 6$%) to one of much lower protein content (e.g* 2$%)• rt zi Following the injection of S^ -labeled albumin the specific activity of the globulin fraction was measured as a function of time* The object was to estimate the probability of a conversion of amino acids from albumin into globulin without their release into the bloodstream* long after this work was completed the author became aware of work along nearly identical lines carried out by Maurer and Muller with rats (67)* The analysis given by these investigators seems very satisfactory. This analysis will therefore be usedjwith minor modification, for the interpretation of our data* The general picture presented by the data obtained (Figures 33 to 36) is that albumin has a half-life of about ten days, that the globulin fraction attains a maximum specific activity around the seventh day, and that this activity is four to eight per cent as large as the initial (extrapolated) specific activity of the albumin. Part of this could be due to contaminating albumin, but from Figure 36 this would account for at most» figure of three per cent. More over, the shapes of all of the globulin curves speak for incorpora tion of radioactivity from some non-globulin source and a net loss Hi7 of activity which is much slower than that characteristic of globulin decay. The activity observed to be present in the globulin fraction of dog IX could arise in one of two ways. Either the preparation injected contained a small amount of radioactive globulin or the separation of the two protein fractions on paper was incomplete, leaving some albumin (S^) in the globulin fraction. This might be expected to occur at pH U.O (20). Whether it occurs at pH 5*0 seems doubtful. The "tailing” phenomenon (68) is probably not a contributing factor here since the globulin moved away from the albumin to a part of the paper which had not previously seen albumin. This leaves us with the likelihood of the presence of a small amount of S-’ -'-labeled globulin in the material injected. If the activity due to this globulin is 0.03 A^* (see below) at the beginning of the experiment, it will be less than 0.01 on the seventh day, when the globulin activity is at a maximum. Therefore, at worst, our data indicate for globulin a specific activity one to five per cent as high as the initial specific activity of albumin, and at best a value of four to eight per cent. For a seven kilogram dog the plasma volume may be taken as 280 milliliters and the plasma protein content as 20 grams. (69). Taking the rate of metabolism of globulin as a whole as being 2.5 times as large as that of albumin (12,28) one can calculate the following as reasonable approximationss liiS Albumin Globulin Reference' Intravascular 8.6 g. ll.ii g. 17 Interstitial it.8 g. U.O g. 28 Total 13*1* g. l5.lt g. 28 Half-life 10 days k days 12,28 k (Decay const.) 0.0693 day*"^- 0.173 day~^ From the extensive data of Partridge, Vessel, and Grossley on the composition of dog plasma proteins (70,71) and from the data of others (72,73) on the free amino acids in the circulation, the following can be calculated: Albumin Globulin Free^ Cystine 6.52 3.3# 6.7 mg. Methionine 1.2# 2.0# 3.8 mg. Cystine Sulfur 1.7# 0.87# 1.8 mg. Methionine Sulfur 0.26# O.itf# 0.81 mg Amino Acid Sulfur 2.0# 1.3# From these two sets of figures one can arrive at the following distribution of cystine sulfur: references cited give the location of the information on which basis the given figures were calculated. 2 Total amount free in the circulatory system. All values are calculated for a seven kilogram dog. Ui9 Albumin 230 mg* 131* mg. Globulin Free 1*8 mg (in the circulatory system) Assuming that the residues of all proteins in question are in equilibrium with the free amino acids in the circulatory system one arrives at the representation of the metabolism of cystine sulfur shown in the accompanying diagram* The turnover rates of albumin and globulin were calculated using the decay constants given above* The turnover of cystine sulfur of body protein was calculated assuming that this is 200 times as large as the corresponding rate for albumin (67)* i£o 230 mg. Albumin- Cystine- Sulfur Cystine- Sulfur 13U mg. Globulin- Cystine- Sulfur 1*8 mg Free- 3180 mg./day Body Protein - Cystine- Sulfur FIGURE 37 Letting A represent the total amount of albumin (cystine sulfur) in the animal at any time t, A* the amount which is radio active, and dA^ the amount which is catabolized in time dt, and assuming the steady state, - <1A* _ 4* where A is A at time t — 0. o “ = where Ta « half-life of albumin U L* = A*e^at dA* . ~ kaA*e " V dt “ * — k A*e dt • dA - -a-0 If a fraction f of the cystine arising from the albumin dA*, which has decayed between time t and t ♦ dt, is in corporated into globulin 152 assuming the incorporation time is negligible relative to the time dt. At any later time T some of this globulin will have decayed, leaving an amount dG* _ (dG*). e-kg (T-t) ” t — fk_A e • e m O - i v > ' V . dt . The total activity in the globulin fraction at time T can be obtained by integrating: 5 T i*_ fkA* e^8T | • dt G*= fk&Aoe ”^g e k -k g a _ -k T G* = * V o e 8 kg " ka E (k -k )T _ , e g a A * fk G - 4 — A k_ - k o g a [■ e -kaT -k„T ] To find when G* is a maximum set = 0. dT 153 § = - [ - v * v - v ] - k - k. g a , -kT _ . -kT k e a — k e & a g h - e (ka-kg)T kg (ka - kg)T - In (ka / kg) T _ la(ka / kg) _ \n (kg / ka) k — k k - k a *g g a ■ i f This is the time at which G has its mi value* To firkt this value we substitute the value of T back into the equation for G * . * -k T a* fkA e 8 v j — a o______ k - g * (kg - ka)T - 1 G* - max — fk A* e -kgT a o k - k g a t2 - 1 a * “ "k^T a s f A e 8 XSh This is the same activity that would be present in the globulin if the fraction f of the original albumin activity were converted into globulin at t = O, and if none of the remainder of the albumin activity were ever incorporated into globulin. In our case 0 = 13U milligrams of globulin- cystine - sulfur and A s 230 milligrams of albumin- cystine - sulfur* Hence 0/A s 0*58 or 0 = 0.58A Therefore specific activity of albumin at t = 0* Knowing these we can calculate ft a 0.58 A Here G /G is the specific activity of globulin and k0 / k the 155 • ' T ' Taking 'a s 10 days = h days , G*max / G A and — zr± J- r. = 0.05 A / A o f » 0*58 (0.05) (fc.62) « 0.13 Thus the observed maximal activity of globulin, relative to the initial activity of albumin, indicates that about thirteen per cent of the cystine gradually released from the latter is con tinually incorporated into the former. The turnover of body protein cystine is about 200 times as large as the turnover of the cystine of plasma albumin (67), and therefore 200 x 15^2 or about lUO times as large as the turn- 23.2 over of cystine of plasma globulin. On this basis we would expect that about 0.1% (l/liiO) of the activity released from albumin would be continually incorporated into globulin. The value found for f is therefore fifteen to twenty (ca. Id) times as large as one would expect if the amino acids arising from the decay of albumin were released into the bloodstream prior to incorporation into globulin. To review the above analysis: we have compared the in corporation into globulin of cystine from two sources, (l) free cystine,(2) plasma albumin cystine. This comparison indicates the likelihood of a much larger incorporation from albumin than would 156 be expected on the basis of the incorporation of free cystine* Such a result indicates that the cystine from albumin, or a part of that cystine, is never liberated to mix with the pool of free cystine* It it The time T at which G = G can be calculated as follows s T “ ln fe<Aa^ - I'TV'Tg ) k - k 1 ® 2 (iter- — 3- g a vT g T S - ' T g ln (^a/ Tg) ln 2 ( ^"a - ' T g) — 10(i») ln 2*5 _ 1*0 log 2*5 ln 2 (6) 6 log 2 hO (0*398) _ 6 (0.301) " 8*8 dayS This agrees reasonably well with the value of seven to eight days found experimentally, considering that we have assumed the globulin to be metabolized as a homogeneous substance* It must be remarked, however, that this agreement cannot be cited as evidence in favor of the conservation of the amino acids of albumin, for it will be noted that T for 0 =0 maic is independent of f. CHAPTER Fl SUMMARY AMD CONCLUSION The turnover of plasma albumin was studied in two groups of normal adult dogs* The results are expressed in terms of half- 35 life* The four dogs in the first group received S -L-cystine by vein* Samples of plasma obtained at various intervals of time from three to over thirty days after the injection were subjected to electrophoresis on paper at pH 8*6 by a technique especially designed for this purpose* This is an adaptation of the method of Macheboeuf et al. The albumin fractions so obtained, and when possible some of the other fractions, were analyzed for specific activity of sulfur isolated in the form of benzidine sulfate* For the albumin fraction the values obtained for half- life were approximately fourteen, fifteen and fourteen days respec tively for the first three dogs* The curve for dog IF was not linear on a semilog plot* The early phase ( two to four days) showed a half-life of ten days and the late phase (nine to fourteen days) corresponded to an apparent half-life of twenty-four days* Dog V received an injection of plasma proteins containing albumin and alpha and beta globulin fractions, all radioactive. The albumin fractions isolated from plasma samples obtained during the first week following the injection exhibited a non-linear decay curve* The early phase (one to two days) was rapid, with a half 158 life of about two days. The later phase (three to six days) was surprisingly slow, with an apparent half-life of thirty-two days. The globulin fractions of dog IV were isolated and analyzed in the same way as previously described. The gamma fraction seemed to have a constant rate of “decay" with a half-life of eleven to twelve days, and the (X^ fraction a half-life of about eighteen days. The <X ^ f3 ^ and j3 g fractions exhibited non-linear two- phase curves. The second phase had an apparent half-life of nineteen to twenty-four days in all cases. For the first phase the values were three, three, and seven days for the (3 ^ and |3 2 fractions, respectively. The second group of four dogs received S-^-labeled dog albumin by vein. This was purified by cold alcohol fractionation followed by electrophoresis in starch. A modification of the method of Kunkel and Slater (1*7) was used, permitting large amounts (four grams) of protein to be purified. Tfc© milliliter samples of plasma obtained at various time intervals up to twenty-fbur days following the injection were separated into albumin and globulin fractions by electrophoresis on paper at pH 5. Except for the pH used the technique was as described above. The half-life of the albumin fraction was nine to twelve days. It thus appears likely that following the injection of labeled amino acid there occurs a continuous incorporation of labeled ma terial into albumin, from non-albumin sources, of sufficient mag nitude to elevate the half-life to an apparent value about twenty 2S9 to fifty per cent higher than the true value# An attempt was made to measure the incorporation into globulin of labeled material derived from albumin. While it is not possible to come to a definite conclusion, it appears likely from the results that there is a conservative route from albumin to globulin, a route which does not involve complete breakdown of the former to the level of amino acids free in the bloodstream. This idea is supported by the fact that the apparent half-life ob tained for the globulin fraction in these dogs is the same (eighteen to twenty-four days) as that obtained in the second phase of the globulin decay curves for dogs injected with labeled amino acid. BIBLIOGRAPHY 1. McElroy, W. D., and Glass, B., A Symposium on Amino Acid Metabolism, The Johns Hopkins Press, Baltimore (1955)• 2. Iiebecq, €., Proceedings of the Third International Congress of Biochemistry, Brussels, 1955, Academic Press Inc., New York (1956). 3. Thompson, E. 0. P., in The Physics and Chemistry of Life, Simons and Schuster Inc., New York (1955)* U. Sanger, F., Smith, L. F., and Kitai, R., Biochem. J., 58, vi (1951*5. 5* Whipple, G. 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Biochem. Biophys., lit, h65 (19U7). 72. Denton, A* E., and Elvehjem, C. A., J. Biol. Chem., 206, Wi9, U55 (195«. --- 73* Braun, P., Foldi, M., Kisfaludy, S., and Szabo, G., Nature, 177, 1133 (1956). APPENDICES 7ii. Cremer, H. D., and Tiselius, A., Biochem. Z., 320, 273 (1950). 75* Durrum, E. L., J. Am. Chem. Soc., 72, 29h3 (1950). 76. Macheboeuf, M., Rebeyrotte, P., Dubert, J. M., and Brunerie, M ., Bull. soc. chim. biol., 35» 33k (1953). 77. Macheboeuf, M., Dubert, J. M., and Rebeyrotte, P., Bull. soc. chim. biol., 35, 3hf> (1953). 78. Kunkel, H. G., and Tiselius, A., J. Gen. Physiol. 35, 89 (1951). 79. Cohn, E. J«, Gurd, F. R. N., Surgenor, D. M., Barnes, B. A., Brown, R. K., Derouaux, G., Gillespie, J. M«, Kahnt, F. W., Lever, W. F., Liu, C. H., Mittelman, D., Mouton, R. F., Schmid, K., and Uroma, E., J. Am. Chem. Soc., 72, U65 (1950). 80. Michaelis, L., Biochem. Z ., 23h, 139 (1931). 81. Sherwood, G. E. F., and Taylor, A. E., Calculus, page 139, Prentice-Hall Inc., New Xork (19U5). 165 82. Kelts* A** and Me hi, J. W., J. Am. Chem Soc., 76, 1*0C>U (195U). 83* Cohn, E. J., Surgenor, D. M., Schmid, K., Batchelor, W. H., Isliker, H. C., and Alameri, E. H., Discussions of the Faraday Soc*, 1^, 176 (1953)* 81*. Tull is, J* L. (ed«), Blood Cells and Plasma Proteins, Their State in Nature* Memoirs of the University Laboratory of Physical Chemistry related to Medicine and Public Health, Harvard University, #2, New York (1953)* 85* Lowry, 0* H*, Rosebrough, N* J*, Farr, A* L«, and Randall, R. J., J. Biol. Chem., 192, 265 (I95l). 86. Sober, H. A., and Peterson, E. A., J. Am. Chem. Soc., 76, 1711 (195W • 87. Peterson, E. A., and Sober, H. A., J. Am. Chem. Soc., 78. 751 (1956). ^ 88. Sober, H. A., Gutter, F. J., Tffyckoff, M. M., and Peterson, E. A., J. Am. Chem. Soc., 78, 756 (1956). 89* Wineler, R. J., Devor, A. W., Mehl, J. V., and Smyth, I. M., 27, 609 (19^8). APPENDICES 16? APPENDIX A DEVELOPMENT OF THE METHOD OF PAPER ELECTROPHORESIS To follow the time course of specific activity it was necessary to separate the plasma proteins from each other« The method of paper electrophoresis seemed an obvious choice for this purpose. The chlorobenzene method of Cremer and Tiselius (7U) was tried but found to be much too cumbersome for routine use* The method of Durrum (75>) was tried and gave good separation of albumin from globulin, but little or no resolution among the globulins. A diagram of each type of apparatus used is presented (Figures 38,39 and 1 | 0). It seemed at the time that the appartus of Durrum was bas ically unsuitable because of the continuous evaporation of water which took place from the surface of the filter paper. This re sulted from the heat generated by the passage of the electric cur rent • If thus appeared essential to seal the paper surface from the atmosphere, and to provide some means other than evaporation for re moval of heat. The method of Cremer and Tiselius satisfied these requirements, but was otherwise unsuitable, as mentioned* The arrangement illustrated was evolved'*' as a substitute (Figure I 4 .O). This apparatus is a device for applying a selected 2 pressure evenly to a strip of filter paper. The bars were half- 1 By Mir. Burt Kallman and the author. 2 The idea of using springs to produce the pressure originated from Dr. J. W. Mehl of this department. 16 8 FIGURE 38 THE APPARATUS OF CREMER AND TISELIUS (1950) FOR PAPER EUSCIROPHORESIS1 1 B = * Buffer • The paper was clamped between glass plates and submerged in chlorobenzene • C i ^ e w u r 4* Tiv*eliui (nfo) CMoh®t enx.O\« 1 7 0 FIGURE 39 AN EARIY APPARATUS FOR PAPER ELECTROPHORESIS DEVISED BY DURRUM (1950)1 1 Suspension of the paper as shown Is referred to as the "hanging curtain" technique • 172 FIGURE 1*0 1 AN APPARATUS FOR PAPER ELECTROPTHORESIS WITH CONTROLLED PRESSURE AND WITH PROVISION FOR THE REMOVAL OF HEAT ST 1 7 ii inch aluminum* 2 x 10 inches * the inner surfaces of which were ma chined and polished by hand to match each other within a few thousandths of an inch* The face of the lower bar was insulated by 0.00f > inch Teflon* The upper bar was separated from the paper by one-eighth inch Incite. A hole three-eighths of an inch in diameter was drilled through the upper bar and through the Incite six centimeters from one end to allow addition of the sample after the paper was tightly clamped in place Experiment with varying amounts of pressure applied to the paper showed that a pressure of three to four pounds per square inch was optimum* If the pressure was less than this the paper became too wet with bufferj if much more the mobili ty of the proteins was reduced* Using this apparatus it was found possible, with considerable care, to separate three samples of human plasma into seven components* albumin, alpha-1, alpha-2, beta, fibrinogen, gamma, and a small component with a mobility slightly greater than that of albumin. The potential power of the method for analytical purposes was thus shown. However, the technique was still limited to a volume of fifty micro liters or less • For practicality one milliliter of plasma was a minimum* An attempt was made to increase the capacity by using a multiple-sheet arrangement 5 this was so time consuming that it was considered impractical. 3 This means for adding the sample is due to Dr* R. J. Winzler, formerly of this department. 175 The results of another attempt to increase the capacity of the method are shown in Plate XI. This is the same device as described above except that one-half inch steel plates 12 x 11* inches were used instead of the aluminum bars. This permitted the use of correspondingly large sheets of filter paper* The lack of resolution among the components is apparent. The turning point in the search for a suitable method of paper electrophoresis was the appearance of a pair of articles by Michel Macheboeuf and coworkers, in which they demonstrated the equilibrium nature of electrophoresis on paper (hanging curtain) and the importance of evaporation to the process (76,77)* Mache boeuf *s experiments on the fractionation of plasma were repeated with good results* For this purpose an enlarged Burrura apparatus was used, which had been made previously when Durrum1 s technique was being tried. The only part of the apparatus which was changed was the "roof*. The flat Lucite roof^ was replaced by a pitch roof of asbestos as described by Macheboeuf • This gave the apparatus illustrated in Figures 2,3, and 1 *. There are several attractive features to Macheboeuf1s ap paratus. Firstly, it is much simpler to use than the system pre viously investigated, in which the paper was clamped between two bars. Secondly, the temperature of the protein solution remains ^The flat roof was not due to Durrum. It was used in the enlarged Dumanapparatus because of the greater ease of construction. 17 6 PLATE XI EIECTROPHDRESIS OF SERUM ON PAPER BETWEEN LARGE METAL PIATES (12 INCHES X 11* INCHES) |)%*&■$: ■ ■ 178 near that of the environment, and no special apparatus is required to maintain this temperature* Thirdly, every spot of protein is compressed in the direction of the electric field, thus greatly improving the resolution between proteins* Fourthly, the position of a given protein at the end of a run is much less dependent on its position at the beginning than in any system of electrophoresis in which evaporation is prevented* Thus Macheboeuf1 s scheme is not nearly as utouchy” as other systems* Because of the above features it seemed possible that the Macheboeuf system with asbestos roof might make possible multiple- sheet electrophoresis, thus increasing the capacity of the apparatus* Accordingly 200 microliters of serum was applied to each of five papers (Whatman 3 MM) in the same position on each and these were put into the apparatus one on top of the other* Bromphenol blue was added to the serum (78) and the usual voltage applied. When the dye had migrated about half-way down one side, the power supply was turned off and the sheets developed as usual. It was thus shown that the separation was reasonably good on every sheet and that every band was in the same position on every sheet* Repetition of the experiment gave the same results* It was later shown that the volume of serum or plasma used could be doubled. The resolution among the globulin was not as good as before but the albumin was still well separated, and four components were always clearly distinguishable (albumin, alpha, beta, and gamma). The capacity of the method was thus raised to 179 2.0 milliliters. With five sheets some difficulty was encountered in getting them to lie close to one another. It was found that equally good results were obtained when the sheets were laid flat on a piece of Lucite. Only two of these (the first and fifth) were allowed to dip into the buffer $ the others were cut to just the size of the Lucite support. The latter was elevated some ten centimeters above the level of the buffer. It was also found convenient to lay a sixth sheet of filter paper (also Whatman 3 MM)* containing no sample, over the other five. All six sheets were marked with pencil lines at every one centimeter level while still dry. As the run (and evaporation) progressed some of the protein moved upward from the lower sheets into the top sheet originally devoid of protein. At the end of the run the top sheet was removed and stained to indicate the positions of the various bands of protein. The lower sheets were then cut for elution according to the pattern of the top sheet. Control of Evaporation. Approximately one year after starting to use Macheboeuf fs scheme, the Spinco apparatus for paper electrophoresis (Ijl, p. 371) became available. On comparing the two methods it was apparent that the latter possessed the dis tinct advantage that the distance of migration of every protein component was two or three times the corresponding value in Mache boeuf1 s system. The only essential difference in construction 1 8 0 between the two was in the ”roo£,t l the Spinco apparatus having a solid Incite roof* It thus appeared that the two methods were basically the same and differed only quantitatively in the rate of evaporation of water from the surface of the paper. To prove that this was the case the asbestos roof was replaced with a solid lucite roof* Two samples of serum were run in this system and in the Spinco apparatus* The results are shown in Plates XII and XIII. Plate XIV shows the same sera run with the asbestos roof. It is very clear that the roof is indeed an important controlling factor in determining the distance of migration and the separation of components* To further analyze the effect of rate of evaporation, the M ob lique -lineM experiments carried out by Macheboeuf et al* were applied to the two systems,i.e. solid roof and porous roof. The accompanying patterns, Plates XV and XVI, show that the positions of equilibrium for any two components are considerably further removed from each other in the system with the solid roof than in that with the porous roof. Because of its obvious advantage the solid lucite roof was adopted thereafter for all analytical (single-sheet) runs. However, for multiple-sheet fractionations, where the separation is greater than with single sheets in any case, the asbestos roof was retained* m PLATE XII HUMAN SERUM FROM A NORMAL SUBJECT AND FROM A PATIENT WITH MULTIPLE MYELOMA MACHEBOEUF APPARATUS9 LUCITE ROOF. 182 10 jJ. N»hm « • / © 10/1 m»k . Nyi> \/dl Mo// - Myl- ro 183 PLATE XIII HUMAN SERA, THE SAME SAMPIES AS IN PLATE XII. SPINCO APPARATUS N o hWte 1 8 5 PLATE XIV HUMAN SERA; THE SAME SAMPLES AS IN PLATES XII AND XIII. MACHEBQEUF APPARATUS, ASBESTOS ROOF. 186 10 } d k/3H>, s * - f. sv yt4 N f t i n m * . 1 0 y»X UuH.M y- HilfrJA* 5 g t ( f > n : 18 7 PIATE XV HUMAN SERUM, 200 MICR01ITBBS, APPLIED ALONG AN OBLIQUE LINE. ASBESTOS ROQE 188 189 PLATE XVI HOMAN SERUM, 200 MICROUTERS, APPLIED ALONG AN OBLIQUE UNE. INCITE ROOF 1 9 0 L V > v - t ^ V M— S • IPSHgH-- - rrrr~ITT 191 £H. The effect of pH on the separation of proteins by electrophoresis on paper is demonstrated in Plates XVH and XVIII* These patterns were obtained with dog plasma and with fractions obtained from this plasma by Method 10 of €ohn et. al. (79)* The buffers were acetate-veronal buffers (80), approximately 0.050 M in every case. They were made up as follows: Volume (Milliliters) Reagent Reagent per Liter of Buffer________ Sodium Acetate, 2.0 M 12.5 12.5 12. 6.6 12. Acetic Acid, 10 M 25. $*9 2.2 2.1 * 0.30 Sodium Diethylbarbiturate, 0,60 M h2* h2. h3. 61.5 1 * 1 * . pH li .00 U.80 5.99 7.1*0 8.73 It is clear from these results that the resolution of components is improved as the pH is raised from i*.0 to 8.7. This is what one would expect from classical electrophoresis. It may be noted that at pH 6.0 the two alpha components are not resolved. This resolution is achieved at pH 7 *h and 8.7 Ionic Strength. The effect of low ionic strength on the electrophoretic behavior of serum proteins on paper is shown in the next pattern (Plate XIX). The sample of serum was applied four centimeters to the positive side of the mid line, but this has been demonstrated to have little effect on the general appearance of the pattern. It is clear that cutting the (initial) ionic strength 192 PLATE XVII ELECTROPHORESIS OF DOG PLASMA AND FRACTIONS THEREOF AT pH 14.00 (COLUMN I) y pH I 4.8O (COLUMN II), AND pH $.99 (COLUMN HI). 193 H-,0 0 > r > S -JP-t H k > 0 0 © L . 6 1 2 . J ^ 191* PLATE XVIII EEEGTROPHDRES IS OF DOG PLASMA. AMD FRACTIONS THEREOF AT pH 7.1*0 (COLUMN I) AND pH 8.6 (COLUMN II). 195 f X 7 - 3 b i - T - 3 0 ACS) ACJ) 53^ P U H 7 4 0 JST-tTT - 1 +;£->; 1-1 - i f i f . 1S>6 FIATS XIX EI£ETROPHORESIS OF SERIN AT IONIC STRENGTH 0.02$, pH 8.6 197 w m 198 of the buffer in half, from 0.05 to 0.025, has a pronounced effect on the mobility of the albumin component, but little effect on the beta and gamma components. The highly irregular appearance ob tained at the lower ionic strength is probably coincidental, and not a necessary disadvantage of this condition. The use of low ionic strength has been found to be very useful in connection with the electrophoretic separation of other materials. 199 APPENDIX B AN INQUIRY INTO THE NATURE OF ELECTROPHORETIC MIGRATION IN PAPER Macheboeuf et al. give a mathematical analysis of the mi gration in paper of a substance of mobility yu- in an electric field of strength E (77) • With this as a basis an attempt will be made to demonstrate how one may choose the electrical parameters to ef fect optimum separation of components, other conditions being given* In general keeping with the designations of Macheboeuf et al*, we define the followings Symbol Units Meaning M Apex (Midline) of paper ir Position of equilibrium for uncharged sub stances P Position of a given substance at any time t x cm Distance 'TTP* The positive sense is chosen as that from the negative pole to the positive pole. a cm Distance TT M (T g/cm^ Amount of water in the paper under unit surface area - A P g/cm Rate of evaporation of water through unit sec surface area -1 k sec* (3 / <T , the fraction of water in the paper which is evaporated in unit time i amp/cm Electrical current flowing in paper per unit width* It is given a positive (absolute) value. 200 Symbol P E v Units ohm eq volt/ cm 1 cn^sec cm^ volt sec cm Meaning Electrical resistance offered by the volume under a one-centimeter square of surface, one edge of the square being perpendicular to E cm width (ohm x ohm) cm length Electrical field strength (volt/cm length)* It is given a positive (absolute) value* The fraction of heat generated electrical ly which is removed by evaporation Velocity of substance under consideration Electrical mobility in paper, Vg/E. j j l is assigned a positive value for a negatively charged substance* The value of x for which v * 0 - T T M O CL -f From the analysis of Macheboeuf et al* (77) we may obtain 1 the following relations: fiE 2IU+0 ( 1) (2) The factor f does not appear in the paper cited (77)• It was there assumed to be equal to 1* Equation (3) shows that the position of equilibrium of a substance with respect to that of a substance of zero charge is proportional to its electrical mobility J*- and inversely pro portional to the current flowing. It is important to note that it is (otherwise) independent of the field strength and resistance# From equation (3) i - 2hhQ CT ( \ f I 3* ; and (i) =2U«Gr ( £ \ (W v ■ 0 * \ * / Hence (i)T . Q _ xeq 0r ' O I (1 I 1 (5) Other conditions being given, it is desirable to know what current to choose (by adjustment of the voltage of the power supply) to give a maximum rate of movement* For this purpose we set 9 i = 0 and solve for i, x being held constant* For equation (2) 202 From equation (3) eq _ - 214*0 CT>a = ” xeq fi2 i From equation (l) u _ P_fiE _ f ? i2 v V 2 W < r " 2lfeV 9 ^ _ „ f P i = ? is ■ g j - 2 2i | U o c r 2 i _|v - kv ( ^ e q j ♦ 2 _ x) Setting •(xeq - 2x) * = 0 - eq * 2x for v dv -51 xeq ' 2x for v “ vmax ................(6) But according to equation (5) i - f — ) (i) V / v - 0 Therefore the optimum current (i) opt “ ( & ) (i) T - 0 ■ £(i) v - 0 W T - 0 “ 2(i)opt ....................... (7> Thus, the maximum rate of movement at a given position (x) is achieved with half that current which would immobilize the 203 substance at that position* This means that if a substance at any position x is moving with maximum velocity under conditions where the current flowing is i, merely doubling this current will complete ly immobilize the substance. From equations (i;) and (?) Hence, the optimum current is inversely proportional to the distance of the sample from the TT line. This means that to achieve maximum migration during a run it is necessary to control the current with a variable regulator so that it decreases con tinuously in such a way that equation (8) is always satisfied* There are two important applications of these considerations. The first can be considered as a refinement. If, following separa tion by electrophoresis on paper, it is desired to improve the re solution among components, this can be done by raising the current so that equation (U) is satisfied or nearly satisfied. In practice the current was usually raised to double its previous value and maintained at the new level for about three hours. This procedure serves to concentrate the components into better defined bands. of regulation imposed on the power supply,i.e. constant current regulation versus constant voltage regulation. It has been found ( 8) The second application concerns the choice of the type In practice that, if a constant voltage is supplied, the current rises considerably during the course of a run* Since we have found that for maximum rate of migration the current must decrease con tinuously, it appears that constancy of current is preferable (to constancy of voltage)* Current regulation is provided with the Spinco unit and was adopted for the analytical (single-sheet) runs herein described* It has been assumed above (second application) that CT and p (and JX) are constant* If they vary our conclusion is open to question* Actually both <T and p (and probably JXS) do vary during the course of a run* Their variation is in fact responsible for the rise in current under conditions of constant voltage. The largest change in CT occurs where the paper dips into the buffer, while the largest increase in p occurs at x ■ 0 (77)* An analysis of the situation taking into account the variations in CT and p with x (which is itself a function of time t) will not be under taken here* We may now inquire in some detail as to what value of i is the optimum under the condition of constant (as opposed to optimal) current* It will again be assumed that CT and p (and ) are independent of time* These assumptions leave much to be desired* How serious deviation from constancy is must be determined by experiment* In the meantime it is felt that the analysis which follows may give a reasonable approximation to reality, at least under certain circumstances. When CT and P are independent of 205 both x and t (which is a somewhat greater restriction than we have imposed) constant current implies constant voltage. By equation (3) constancy of i implies constancy of so that these conditions are interchangeable. Now , fiE ^ “ 2Uko <r and by equation (3) f± m 2hh0 CT x eq k E v x: eq By equation (3) 21^0 Oybt E . ip . . . k v t (* rF) Substituting into equation (2) / \ 2hh0<rp \jJ^ / I - x x — T - ^ r ^ ^- 2-) Writing equation (2) in differential form ^5 * * k_ (x - x) dt ^ eq ' Holding x0(^ constant (constant current) d (xeq“ *) = - dx = - (*eq - x) dt 206 i<X1 ' *> - . V „ <*.„ - *> If the starting line is x r* G <xeq - x) ] Xl - - V ] t - T t = 0 ln/Xeq - *1 \ V / - V 2ldiQ <rpjj£- r f x 2 eq “ £ 2Wt0 or ^ J\2 xeq ^ I -23 • ( 10) and set To find the optimum (constant) value of i hold constant 3 t eq 3t ■ f ■JaT^ 214*0 f /<■ \xeq " *1 Setting. X0/, - Xn eq 1 2 In 207 Let fig___ xeq - X1 Then *i ^q “ X1 I - 1 I - 1 * 2 In I or In I * J (I - 1) One solution of this equation is 1*1. This holds if = 0 or if x s OO former is a trivial special case, and it can be eq * 9 shown (by series expansion of the logarithmic term for T) that if x = OO (i = 0) T * * O O , The second solution was found ap- eq proximately by graphing y^ ® In I and J2 m i (I • 1) and finding the point of intersection y^ = From this approximate value (3*55) a more precise solution was obtained by the equation yi + 3 X A I “ y2 + $r ^ 1 which is essentially Newton's method of approximation (81). This gave the value I = 3 013 j which satisfies the equation I - 1 * 2 In I precisely. It can be shorn, by taking the second derivative, that this solution corresponds to a minimum value for T. eq eq - 3.513 2.513 x - 3 .5 1 3 x-L 208 x ■ 1*398 x, eq 1 2 kk°.JZ/±- - 1.398 X j _ i “ 1750 ....... Equation (H) gives the optimum constant current which ought to be employed to move a substance of mobility /*■ from x * 0 to x ■ * x-^, s * and A being assumed constant* The time required to do this is \ - ----- ~4 9 (1.398 x.)2 In ( i ) ^ min^i oons't 210*0 CT f JX* ^ X 1 ' 1.95 fx.2 ■ ----— 1=-- In 3.513 21*1*0 <T f U 2 2 " 0*802 x 10“3 ^X1 ( i ^9^60 crp ul2 ~ 2 (T . )• -tJ. “ 1*01 x 10"3 lxl l W l WnsH o-f ^ 2 ......... (12) To find out how much efficiency is lost by operating at optimal constant current rather than at optimal variable current (equation 8) we will compare the last result with the minimum possible time required* Under optimal conditions x « 2x * ..........(6) eq 209 By equations (2) and (9) dx „ 6io <rp>c dt fx Tr oJ dt min T rain 6io crpyu.1 xif o J x dx I . -I 0 610 crp>c 0.82 x 10-3 > [ fx^2 1&) fxi‘ 1220 crpyx1 (13) (' Imln)l cons't _ 1.01 x 10"3 fX]_2/ q-p 0.82 x 10-3 fx-^2/ crpjuJ- “ min (**rain)i consft 1.23 (li+) ^min The above result assumes that (T , p , and are constant. The comparison made shows, under these conditions, that the ad vantage of maintaining the current at its optimal value at every instant during a run is not large* The same result can be achieved, with the expenditure of a little more time, if the current is held constant, provided that the value at which it is held is properly chosen (equation 11). APPENDIX C 210 DEVELOPMENT OF METHODS FOR THE PURIFICATION OF DOG ALBUMIN Chemical Fractionations. Several methods of fractionation were considered for obtaining purified S^-labeled dog albumin. These included the use of (a) salt, (b) cold ethanol, and (c) heavy metals, both in the presence and in the absence of ethanol. Because of the purity of bovine albumin achieved by the Pentex Corporation using cold alcohol it was thought worthwhile to try the same pro cedure on dog plasma. This procedure was kindly carried out by the Pentex Corporation. The results are shown in Plate XX. Consider able contamination with beta protein is evident. This is not sur prising, in view of the variability of plasma among different species. The presence of a highly soluble beta protein in rat plasma may be called to mind (82). An attempt was made to remove contaminating protein from the Pentex preparation by refractionation under varying conditions. None of these was successful. The best preparation was obtained by precipitation of the majority of the contaminating protein with divalent zinc ion. However, some beta was still left with albumin in the supernatant, and about half of the albumin was lost to the precipitate. With S^”labeled albumin this would mean a loss of half of the radioactivity which had been made available as albumin Reagents, see pp. 60-62. 211 PLATE XX THE COMPOSITION OF DOG "ALBUMIN" (FRACTION V) PREPARED BY THE COLD ALCOHOL FRACTIONATION SCHEME DESIGNED FOR HUMAN PLASMA i h ' 4 py Do^ Allu/n RtfurtLii'tj yf^ 4) J O yi ° i V r\,6 7 & < \ Io f - I J ro yl & ro/JL y ~ S ~ o J J % - X 5«>-u )o Attuw>i Rcftirifi* i (t % ) t ox ^ 3 h )r c 7 % ° i io by the donor animal* A word about fractionation technique may be in order* To avoid high local concentrations of ethanol and heavy metals, re agents containing these are usually added dropwise while the protein solution, cooled in a bath to near-freezing, is stirred (Gohn et al 79) • This procedure is difficult with small volumes of material unless special apparatus is used. The following technique was used as a substitute and appeared quite satisfactory. The reagent (con taining ethanol or a heavy metal) was introduced into a centrifuge tube (heavy wall, conical tip). It was then cooled in a dry ice- acetone mixture until it just froze. The protein solution was then poured into the centrifuge tube and the contents of tube stirred gently until the frozen reagent was nearly melted. The tube was then placed in a bath maintained at the proper temperature (e.g. - 5° C), or in a room maintained at this temperature, until ready for centrifugation. In this way the protein was exposed to the re agent in a more or less uniform gradient fashion and at the lowest temperature possible. Ethanol Systems. Since the Pentex preparation was not pure, a five milliliter sample of dog serum was fractionated to the stage of crude albumin using the procedure of Keltz and Mehl designed for rat plasma (82 )j this was then divided into five parts and each part sub-fractionated in a different way. The procedure used is outlined in the accompanying scheme (Figure Itl) • 5.0 ml .Serum (Dog) 20 ml.Ethanol-Acetate 2U f - 5° c a: i (i ♦ li + h i ) U ml.HgO f c 5 t B .3 (IV + V) 0° G \ A 2 (IV + V + VI ...) pH 5.7 2.0 ml Zinc reagent (rat) _ 5° c G 6 (volume: 6 ml) t B i t (VI + ....) r D 11 1 ml. € 6 (Various) 4 D 12 0.10 ml Zn (QAc)«>0.10 M -5° G 0.10 ml EtOH, 95% 1 E 2} E 2lt FIGURE la THE FRACTIONATION OF DOG SERUM AND' OF THE CRUDE ALBUMIN OBTAINED THEREFROM USING COLD ETHANOL AND HEAVY METALS1 The fraction on the right is the supernatant in each case • 215 For the subfractionations, 1.0 milliliter of the crude albumin (C 6) was used and treated with one of the combinations of reagents, listed below, at -5 degrees, to the final concentrations indicated. The reagents were put in the tube first, frozen, and then the pro tein was added, as described on page 21iu Reagents for the Fractionation of Dog Albumin Reagent Cone. Volume2 (Micro- liters) Final Concentration a b c in Tube d e EtGH 95# 1603 15.2 15.2 15.2 15.2 11.W Ba(0Ac)2 1.0 M 10 0 0 0.010 0.010 0.010 M Zn (OAc)^ 0.10 M 100 0 0 0 0.010 0.010 M NaOAc 2.0 M 10 0 0.020 0.020 0.020 0.020 M HOAc 1.0 M ca. 7.3 0. 0.0073 0.0073 0.0073 0.0073 M The use of divalent barium in an attempt to keep albumin in solution while precipitating the globulins is based on its use with human plasma (79; 83» p. 182; 8U* P* 2i3)» The concentrations of all reagents used are likewise based on this procedure. Plate XXI shows the composition of precipitates D H (column I) and also of various fractions obtained from precipitates A 1 (column II) (by procedures not described). No pure albumin fraction was obtained at stage D 11. Electrophoresis of precipitates E 23 1 Unless the final concentration is zero. 3 Except for tube e. 216 PLATE XXI THE COMPOSITION OF FRACTIONS OBTAINED FROM DOG PIASMA BY VARIOUS METHODS & CM i -Sjf f * S k F ~ L X 6 I umrr£OQ / i a ^ a Of ^fMay i tZ3 u - ' * w- +.-H - H - Ki < - tc< t - £ - ' - * 4 - 0 r*M +■ n D a I p * 1 h i a ( iid: (on paper) showed a similar lack of purification* It may also be noted that divalent barium was not effective in the removal of globulin from albumin. Heavy Metals in Aqueous Solution. Following the appearance of references (83, p,. 180% 81*, pp. 36,37,1*0) to the fractionation of plasma proteins by the use of divalent zinc and mercury at neutral pH in aqueous solutions, a few one milliliter samples of dog serum were subjected in duplicate to the procedures given in Figure 1 *2 and in the following table. Reagents for the Fractionation of Dog Serum Series h20 Aqueous Zinch Dog Serum Total Volume A 0 2.0 ml. 1.0 ml. 3*0 ml. B 3.0 ml. 2.0 ml. 1.0 ml. 6.0 ml. € 10.0 ml. 2.0 ml. 1.0 ml. 13.0 ml. Series Aqueous, Mercury^ 3.1 M HCIO^ A 100 JUL. 0.6 ml. B 100 JJL1. 1*2 ml. € 100 jXl. 2.6 ml. The major fractions obtained were analyzed by electropho resis on paper and by determination of total protein by the method 219 1.0 ml. Serum (Dog) Zn f 1 - 1 (Zn-Glob) 1 - 2 (Alb) Zn-wash 1) f Mg 2-1 1 f I r Saline Versene 2-2 (Alb) 2=2 2-1 * 2) 3-1 3-2 Fibrinlike r 3-5 4-41 Ba^ to 10 M i f 3-6 3-7 HdOi 1 3-8 Saline Versene^) k-9 h-10 f Saline Versene ^ NaOH 1 to near AcOH J neutrality kJ2 None 1 k'±k FIGURE 1 *2 FRACTIONATION OF DOG SERUM WITH HEAVY METAIS IN AQUEOUS SOLUTION --------- T)--------- This is the same as the zinc reagent used in the first step except that it was diluted to correspond to the con centration of zinc in the serum*zinc suspension. 2 ) Ethylenediaminetetraacetic acid, disodiura salt (1 * 3,1 * 6), 0.1 M. Several drops were used. 220 The beta and gamma proteins were largely precipitated by the zinc reagent, whereas the albumin remained soluble. However, considerable amounts of all of the globulins remained in the more soluble fraction. The recovery of protein and its distribution among the various fractions is shown in Figure 1*3* The small amount of protein in the fractions intermediate between the three major fractions may be noted. Ion Exchange €hromatography. Since the chemical methods attempted did not show promise of satisfactory success in obtaining a pure preparation of albumin, fractionation by ion exchange chromatography and electrophoresis were considered. Dr. Sober (86) was kind enough to furnish a sample of DEAE-0-Cellulose (the diethyl- aminoethyl ether of cellulose) and directions for its use prior to detailed publication of his method (87 #88). The procedure was adapted to small amounts of material with the apparatus shown in Figure 1 * 1 * . The following points may be noted in connection with Figure 1 * 1 * : (1) The protein sample was introduced into the column directly, before the column was closed with the rubber stopper (holding the glass tubing). (2) The valves containing glass beads serve to adjust the pressure at the start of the run to atmospheric pressure in both containers which are closed to the atmosphere. Before such a valve was provided for the column, merely closing it / 221 FIGURE h3 THE DISTRIBUTION OF PROTEIN AFTER FRACTIONATION OF DOG SERUM WITH HEAVY METAIS IN AQUEOUS SOLUTION^ The fractionation procedure is outlined in Figure U2 and in the table on page 218. Duplicate samples (1 and 2) were run for each series (A,B, and C). 222 1 «n b » B E • O e a > r- C D — C M V J o - 4 — * J H--- J O s l - o • a to VJ *£• 5 o- % £ N I < M m o •4 . cn po cvj ^ CD — CM C fc CD o o -7 CM f 1 a — CM < < i . v v — o 4 v F occ *+* o SeFWM • * I * 223 FIGURE Ut APPARATUS FOR THE CHROMATOGRAPH! OF SMALL AMOUNTS ($ MG.) OF PROTEIN WITH GRADIENT ELUTION Hypodermic NceJle 2 2 ^ X X <r* D u o a, •M S u i r ~ A * with the rubber stopper developed sufficient pressure inside to force the protein solution and then air into the resin* Omission of the valve from the mixing vessel led to a similar complication: when the vessel was closed sufficient pressure was developed to force liquid out of it, without allowing fluid from the reservoir to enter. The liquid level in the mixing vessel then dropped below the level of the glass tube* (3) The glass beads have a diameter about two millimeters larger than the unstretched diameter of the rubber tubing* They are forced into the rubber tubing, and the tension of the rubber holds them in place* To open the valve the rubber tubing is held tightly between two fingers and pulled slightly to stretch it, so that an opening is created between the bead and the inner wall of the tubing. (h) The reservoir is a glass tube, about lj inches in diameter, open at both ends* (5) After atmospheric pressure is established throughout, flow is induced by raising the reservoir until the desired flow rate is obtained* (6) The complications mentioned are not important with large columns and large samples. Prior to use the resin was washed successively with 0.1 N NaOH, HgO, and 0.005 M phosphate buffer of pH 7*0. Fraction V (crude albumin) prepared by the Pentex Corporation from dog plasma was used as starting material* This was dissolved to give a solution ten per cent in protein, 7 mM in phosphate, and of pH 7*0. A fifty microliter sample (5*0 mg.) of the fresh solution was applied to the column and this was forced into the resin material by air pressure, 226 being careful not to introduce ary air* Two samples of the dilute phosphate buffer of similar volume were added, each being forced in to the resin in the same fashion* Gradient elution was then begun, the eluate being collected in four-inch test tubes in a fraction collector at the rate of approximately one-half drop per minute* Two drops were collected in each tube* Every even-numbered tube was analyzed for protein by Lowry *s procedure (85), and the protein of every odd-numbered tube was subjected to electrophoresis on paper at pH 8.6. To increase the sensitivity of the protein determination the sample (2 drops) was not diluted, and only one milliliter of reagent C and 0.10 milliliter phenol reagent were used. The results of this fractionation are shown in Figure U5* Hinety-six per cent of the protein applied (5*0 mg.) was recovered. The presence of at least four components in the chromatographic pattern is clear. The results of the corresponding electrophoretic analyses show that the second broad double peak, comprising close to three-fourths of the total protein, is composed largely of albumin. The beta and alpha-2 proteins were removed in the earlier eluates, together with some albumin. The resolution of the peak containing alpha-2 and beta proteins from that containing most of the albumin is clearly good. A second run with the same material some two weeks later gave an elution pattern similar to the first, except that only sixty-eight per cent of the protein put on the column was recovered in the eluate. It may be noted that the material applied in the 227 FIGURE b$ CHROMATOGRAPH! OF CRUDE DOG ALBUMIN ON DEAE-O-CELLULOSE M1CR06RAMS PROTEIN PER TUBE 400 TOTAL RECOVERY! 95.8 % OF INPUT (5.0 mg) 300 200 ALBUMIN +cCI eg.5% 100 50 216% 2.4% 50 40 30 20 10 0 TUBE-# (100 d ro p s ) 228 229 second case, when subjected to electrophoresis, exhibited only albumin and a beta conponent, whereas the fresh solution used in the first case was resolved by electrophoresis into albumin, alpha-1, alpha-2, and beta components. In the above fractionations the solvent in the reservoir was alkaline (pH 8.7). The primary mechanism of elution is, in this case, discharge of the quaternary nitrogen of the resin. An alter native procedure is to increase (algebraically) the charge on the protein molecule by lowering the pH (88). Both of these methods were applied to a preparation of crude dog albumin (G-5U). The eluting solvents used had the compositions given below. Solvents for Elution at Low pH D D^O nho E E60 E80 F NaCl, mM 20 30 ho 5o 60 80 100 NaHgPO^, mM 50 50 5o 5o 50 50 50 /2 (xlO3) 70 80 90 100 110 130 i5o pH (measured) k. 51 h.hl h.hk h»k2 luU2 lw38 230 Protein Solvent A1 A2 Ah Na Cl, mM 0 5o.o 100. 150. 200. NaH2P0^, mM 2.86 2.57 2.29 2.01 1.73 Na^HPO^, mM 2.11* 10.0 20.0 30.0 1*0.7 /2(x 103) 9.28 82.6 162.3 21*2. 321*. pH (measured) 7.0 7.1*0 7.61* 7.80 7.93 Fifty microliters of the crude albumin (G-5U) was applied in each case and 1# 5 milliliters of each of the above solvents was used for elution. The composition of the crude albumin is shown in the accompanying electrophoretic pattern (Plate XXII), where it was run alongside a sample of human serum for comparison# The column used was 0.1* centimeter in diameter x 1 * centimeters in length* The eluates weate analyzed for protein by LowryT s procedure (85) and for composition by electrophoresis at pH 8.6. The results are shown in Figure 1*6. The recovery of protein was eighty-seven per cent with the alkaline elution and eighty-nine per cent with the acid elution. In general the order of elution was the same with both solvent systems# Although the separation of albumin was by no means complete it is clear that there is a potential for complete separation using a gradient elution process. Again the order of elution was beta, alpha, and albumin* 231 PLATE XXII THE COMPOSITION OF THE CRUDE DOG ALBUMIN (G-5U) SUBJECTED TO CHROMATOGRAPHY. HUMAN SERUM IS SHOWN FOR COMPARATIVE PURPOSES1 nHe-Atm” refers to the use of a helium atmosphere inside the electrophoresis apparatus. This was tried following a report claiming increased distance of migration in the presence of helium. Such a result was not observed, but this may be due to improper use of the gas. 232 Cl/ 7 1? • Seh We- /Uj* 2 33 FIGURE U6 THE DISTRIBUTION OF PROTEIN AFTER CHROMATOGRAPH! OF CRUD^ DOG ALBUMIN USING ACID1 AND ALKALINE 2 ELUTING AGENTS^ 1 Broken line* 2 Solid line* 3 The abscissa and the ordinate are the same for both experiments* 231* MICROLITERS 6-5 4/T U B E OF ELUATE ( 1.5 ml.) 20 16 12 8 ALB. + ALB. - c C2 -hC2 +&? ALB. + <£ 2 ALB. + a lbT] 2 1 /9~ i -------- : NO ALB. ZL t Al D 3 t t A2 A3 D30 D40 6 * I t T A4 E E60 ESO t F 12 ml.ELUATE It may be noted that there is a clear separation of two beta components in the electrophoretic patterns of G-5U* The minor beta component occupies about the same position as the beta of human serum. The major beta component occupies a position between the beta and gamma components of human serum. Electrophoresis in Starch. The second physical method attempted for isolation of albumin was electrophoresis in a starch medium. The basic system of Kunkel and Slater was used (1*7)* How ever, to increase the capacity of the system it was not closed to the atmosphere, but left open as in the method of Macheboeuf et al. (76). To accomplish this the same apparatus as for paper electro phoresis was used, including the asbestos roof, except that the center section was replaced by a trough of starch (Figure 6). In the first such fractionation 2*0 milliliters of crude albumin (G~51|, ten per cent protein) was applied to a block of starch \ cm. x 10 cm. x 16 cm. The buffer was 0.05 M veronal, pH 8.6. After electrophoresis the starch was cut into one centimeter sections and each section extracted with 5*0 milliliters of the buffer. Each extract was analyzed for total protein (85) and was tested with perchloric and phosphotugstic acids (89), and six of the eight extracts were analyzed by electrophoresis on paper. The results are shown in Figure U7* It can be seen that the majority of the albumin was freed Cf contaminating protein (*5 to +7 cm). These cuts contained no 236 FIGURE 1*7 ELECTROPHORESIS OF 0.20 GRAM OF CRUDE ALBUMIN (G-Sl*) IN STARCH1 HO correction was made for possible absorption by broraphenol blue, and the abscissa of the graph was not cor rected for endosmosis. 237 PROTEIN IN EXTRACT (5.0ml.) 30 -mg.PROTEIN MATERIAL SOLUBLE IN HC104 AL 20 ALB + POLE - POLE 10 - 8 cm. 6 2 4 0 - 2 - 4 material soluble in perchloric acid. The preceding three centi meters, which contained the alpha component, had a considerable amount of protein which was soluble in perchloric acid. The part of the starch containing only beta protein had no perchloric acid soluble material* The recovery of protein was approximately thirty- seven per cent* In a second run with the same preparation ij.,0 grams of protein was applied to a starch block about six centimeters deep. The system used was otherwise the same as before, A total of 300 milliliters of water was used for extraction of each centimeter section of starch. This was done in two stages; the starch was first extracted with ca. 15>0 milliliters of water for several hours, and the suspension was then filtered through sintered glass. The process was repeated. A one milliliter aliquot from each extract was precipitated in the cold (ice-bath) with acidic sodium per chlorate, and the precipitate was dissolved in water and subjected to electrophoresis on paper* The conposition of each section is shown in the accompanying pattern (Plate XXIII-a) • The supernatant of the perchloric acid precipitation was treated with phosphotungstic acid to a concentration of 5% (89)* As in the preceding run, the maximum amount of perchloric acid soluble material was found in the sections behind the albumin. The bulk of each extract was dialyzed against distilled water and lyophilized* A sample from each section was subjected to electrophoresis* The results are shown in Plate XXHI-b* 239 PIATE x x m THE COMPOSITION OF THE FRACTIONS OBTAINED BY ELECTROPHORESIS INSEARGH CF i*.0 GRAMS OF DOG ALBUMIN (G-$h) XXIII-a. PERCHLORATE PRECIPITATES OF STARCH EXTRACTS XXIII-b. DIAIYZED AND LYOPHILEZED EXTRACTS The results of this fractionation are summarized in Figure i±8. As in the preceding case the majority of the albumin was nearly free of contaminating protein. It is noteworthy that 3*8 of the U.O grams of protein were recovered (9$%)• Prior to removal of water by lyophilization 100 microliter samples of each fraction (300 ml.) were applied to Whatman 3 MM paper., using the trough described (page 37) to prevent excessive spreading of the sample. The results are shown in Plate XXIV. It msy be noted that, considering the volume of sanple applied, the protein spots are remarkably conpact; and, as a consequence, the resolution among components is fairly good. It is evident that the sanple underwent a “compression" in the direction of the electric field in the course of the elctrophoretic process. This is a distinct ad vantage of the Macheboeuf system not shared by those schemes in which evaporation is prevented. 2k 2 FIGURE ii8 THE DISTRIBUTION OF PROTEIN IN THE FRACTIONS OBTAINED BY ELECTROPHORESIS IN STARCH OF h.O GRAMS OF CRUDE DOG ALBUMIN (G-SU) I2 0 0 t mg. PROTEIN IOOO-- 8 0 0 -- - CIO4 -soluble 600 - 4 0 0 -- 200-- 2 1 4 . 3 ALB C104 insolu A LB a I albJ + oC A LB . -8 -6 -4 -2 0 2 4 6 8 cm . 2hh PIATE XXIV ELECTROPHORESIS CM PAPER OF FRACTIONS OBTAINED FROM CRUDE DOG ALBUMIN BY ELECTROPHORESIS IN STARCH Si^yok Ei limits 100 1° V C*n V A \O0 loo i d joyu^. ¥ it c&i .fe C 6 T £>n VVW^ 6 - r ¥ Sfayck £ T lutfts A .k r V A y * -t

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Creator Mozersky, Samuel M (author)

Core Title The half-life of plasma albumin and the possibility of its partial conversion into globulin as studied in the normal adult dog

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Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

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Tag Biology, Veterinary Science,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-1219

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Rights Mozersky, Samuel M.

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