University of Southern California Dissertations and Theses

The isolation and characterization of rat plasma albumins

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The isolation and characterization of rat plasma albumins

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Content THE ISOLATION AND CHARACTERIZATION OF RAT PLASMA ALBUMINS 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 by Alan Kelts August 195H This dissertation, w ritten by ................iO£n_K^ts.................. under the direction of. 1 ^.®.Guidance Committee, and approved by a ll its members, has been pre sented to and accepted by the Faculty of the Graduate School, in p artial fu lfillm e n t of re quirements fo r the degree of D O C T O R O F P H IL O S O P H Y Date.. Guidance Committee I Sal. t ACKNOWLEDGMENTS It is with sincere gratitude that I acknowledge the invaluable help, patience and understanding of Dr. John Mehl during the course of these investigations. I wish to thank my wife, Miriam, for her patience during my graduate study period* I am grateful to the United States Public Health Service, which agency furnished the funds without which these studies would not have been possible* TABLE OF CONTENTS PAGE I. INTRODUCTION...................................... 1 A* Purpose and Scope* ........... 1 B. Historical ••••• ............... • ••••• 2 C* The Interactions of Proteins and Zinc*......... 6 II. MATERIALS AND METHODS.............................. 11 1* Plasma ........•••• •••• 11 2* Reagent Solutions* •••*•••••••••• 11 3* Biuret Analysis* . • «•••* 11 iu Spectrophotametric Measurements* ••••••• 12 5* Measurements of pH • ••••••••••*•« 12 6* Cold Bath.................................. 12 7. Preparative Centrifugation ............. 12 8* Determination of Zinc •••*••••«*• • 13 9* Light Scattering Measurements * ............. 13 10. Ultracentrifugal Measurements. • ••••••• 15 11* Diffusion Measurements • ••••*••«••• 21 12* Electrophoretic Measurements «•««*•*«• 23 13* The Fractionation of Rat Plasma* «••«••• 23 ill. Equilibrium Dialysis Procedure •••*•••• 25 15« Partial Specific Volume •••*•••••*•• 26 16. Nitrogen Determination* •••* 27 17* Determination of Free Sylfhydryl Groups. • • • 27 V PAGE III. EXPERIMENTAL................................... 28 1* Removal of Zinc from Fraction V* . • ......... 28 a* Dialysis techniques. • ••••••••••• 28 b* Use of ion exchange resins • •••••••• 28 2. Refractionation of Fraction V. • ••••••* • 29 a. Solubility in alcohol solution......... • 30 3* The Addition of Zinc to the Refractionating System *•«•••••*•••••*•*•*•• 32 lu Equilibrium Dialysis of Zinc and Rat Plasma Albumin* •••••••««*•••••••#•• 35 5* The Nitrogen Content of Rat Plasma Albumin • • • 39 6. Electrophoretic Studies with Rat Plasma Albumin* ••••••••••••••••••*• UO 7* The Determination of Molecular Weight by Light Scattering ••••••••••«•««••••* U3 8* Ultracentrifugal Results *•••••••.*•• 2*9 9* Diffusion Results* •••••••*••••«•• 53 10* Calculation of the Molecular Weight by Sedimentation and Diffusion* •••«*••••• 53 11* The Shape and Hydration of Rat Plasma Albumin* * 57 12* The Determination of Free Sulfhydryl Groups* * * 59 IV. SUMMARY AND CONCLUSIONS.......................... 62 V. BIBLIOGRAPHY................................... 65 LIST OF TABLES TABLE PAGE I. Proteins Separated from Human Plasma by Reversible Complex Formation with Bivalent Cations, by Association with Specific Polyelectrolytes or Polysaccharides in Water at pH 7*2 and 0°C* ♦ • • • 8 II* The Binding of Zinc to Rat Plasma Albumin * * * * * 36 III* Comparison of the Electrophoretic Mobilities of Rat and Human Albumins • •*«•••«••••* 1*6 IV* Light Scattering Data for Rat Plasma Albumin* • * * 1*7 V* Ultracentrifugal Data for Rat Plasma Albumin* • • • 5>1 VI* Diffusion Data for Rat Plasma Albumin* • • • « • • $$ LIST OF FIGURES FIGURE PAGE 1* Solubility of Human Mereaptalbumin in Solutions Containing Zinc • •••••••••••••••.. 7 2. Standard Curve for the Colorimetric Determination of Zinc with Dithizone at 535 myu.. ....... ll* 3* The Ultracentrifuge Control Panel and Optical System Termination. ••••••••••••••.• 16 U. The Ultracentrifuge in Operating Position • • • • # 17 5A. The Ultracentrifuge Drive Unit and Vacuum Chamber with the Lock Ring in Place #.••#•••••.* 18 533. The Ultracentrifuge Drive Unit and Vacuum Chamber with the Top Raised to Show the Rotor in Position * 19 6* The Electrophoresis and Diffusion Apparatus Showing the Special Stirring Device in Position*........ 22 7* The Solubility Behavior of Fraction V from Rat Plasma in 30 Per Cent Ethanol, Y/2 0*01 • . • * • 31 8* The Binding of Zinc to Rat Plasma Albumin. R Equals Moles of Zinc Bound Per Mole of Protein. • • • • • 38 9* Electrophoretic Patterns of Fraction V at pH 8.6 V/2 0.1 Veronal Buffer • •••••••••••• 1*1 10. Electrophoretic Patterns of Fraction V, at pH 1 *. 5 V/2 0.1 Acetate Buffer 1*2 viii FIGURE PAGE 11. Electrophoretic Patterns of Rat Plasma Albumin Fraction Concentration Equals 1 gm. Per 100 ml. . . ........... UJU 12. The pH Mobility Curve for Rat Plasma Albumin. • • • • kS 13. Plot of Hc/V Versus c for the Determination of the Molecular Weight of Rat Albumin by Light Scattering^ M Equals 6l,000. •••••••••••• SO 3lu Plot of the Sedimentation Values for Rat Albumin at Varying Concentrations. Extrapolation to Zero Concentration Yields an S20 Value of U.35* . • • • • S2 1S* Diffusion Pattern of Rat Plasma Albumin at 96,960 Sec. Concentration Equals 0.5> gm. Per 100 ml........................................... Sh 16. Plot of the Diffusion Constant for Rat Plasma Albumin at Varying Concentration. •••••••••• 5>6 17. Optical Behavior Showing Complex Formation of Albumin and p-chloromercurib enz oate ..••••••• 60 I. INTRODUCTION Purpose and Scope In view of the tremendous amount of time and energy which has been devoted to the study of proteins in the past, it is surprising to find that little is known concerning the nature of the protein constituents of the blood of the smaller animals* It is obvious that, for purposes of biochemical research, small animals such as the rat are the most readily available and widely used* Thus it was felt that a method for the separation of the plasma proteins of the rat would prove to be a valuable aid in the study of problems involving the biochemistry of proteins* The purpose of the work which is to be described was threefold, namely, to add to the fundamental knowledge of the character of proteins as obtained from native sources, to provide a biochemical tool for the stucfor of the metabolism of proteins, and, finally, to provide the author with a working knowledge of the character of native proteins and the methods for their efficient separation* After investigating the methods available for the separation of complex protein mixtures such as plasma, the method developed by Cohn et al*, at Harvard (8) appeared to offer the most promise* Earlier work by the author on the application of these methods to the separation of rat plasma proteins with special reference to the albumin - containing fraction V has been reported previously, (2U, 25)* The work reported in this dissertation has been concerned 2 with extending the fractionation to yield a homogeneous albumin preparation, and with the study of some of the properties of this albumin* Since the behavior of rat albumin toward zinc has proved to be of considerable importance in the purification of the albumin, this was investigated by the technique of equilibrium dialysis and has provided interesting results, indicating an association constant for the first binding site of the order of 10? • B. Historical The employment of solvents of low dielectric constant such as alcohol and acetone in the precipitation of proteins is not new* Since early in the nineteenth centuxy such reagents have been used by chemists to precipitate proteins, wash them free of impurities and prepare them for analytical study* The accompanying denatura- tion of the protein was not usually a matter of concern, and those precautions essential for the preparation of native proteins were seldom taken* One of the first relatively successful uses of alcohol for the preparation of an undenatured protein was the concentration of diphtheria antitoxin in the cold by Mellanby in 1908 (32)* In 1910, two years later, Hardy and Gardener (17) published accounts of experiments involving the precipitation of plasma proteins with ethanol or with acetone at low temperatures, and their subsequent 3 washing with ether and ethanol to remove lipids* The resulting solid protein preparations were readily dissolved to give stable, elear solutions in water or physiological saline* These results marked a major advance in protein chemistry* However studies by McFarlane (30) and by Pederson (3k) with the ultracentrifuge have demonstrated marked changes in the sedimentation diagrams of plasma proteins after treatment with alcohol and ether to remove lipids* Later, Wu (LtO) successfully employed low temperature ethanol precipitation for the preparation of undenatured serum proteins from the sheep and the horse* ferry, Cohn and Newman (11, 12) employed aqueous ethanol systems at -£°C and at low ionic strengths i for the study of the solubility of egg albumin and horse hemoglobin* These workers were able to show that the solvent action of neutral salts under these conditions was much greater than in water, and that these proteins could be recrystallized after exposure to from | fifteen to twenty-five percent ethanol at -5>°C for long periods, j provided the ethanol was removed before raising the temperature* The first report on a truly systematic fractionation of plasma was made in 19U0 by Cohn et al* (7) on bovine plasma* A similar system, employing ethanol in the cold at low ionic strengths, was evolved in Cohn's laboratoiy for human plasma protein fractiona tion* Although some material was published in 19 l i2j. concerning the nature of some of the products obtained and their uses clinically (21, 22), a full detailed report was not published until 19U6 (£)• The report in 19U6 by Cohn and co-workers presents brief accounts of the first five methods attempted and a fully detailed description of so-called Method 6, which was adapted to the large scale frac tionation of plasma* The physico-chemical properties of some of the fractions obtained by the use of Method 6 were reported by Oncley et al. (33). Method 6 of the Harvard group has served as the basis of a fractionation procedure for other species* The method was modified by Gjessing, Ludewig and Chanutin (13) in order to study serum changes produced in rats following thermal injury and in dogs following injury by turpentine, heat, and bis (beta chloro-ethyl) sulfide* In 195>0 (lU) at Chanutin* s laboratory the method was further modified in order to follow the changes occurring in the rat plasma proteins following radiation damage* The method which was evolved did not result in very clearcut fractions and provided less than maximal yields* Method 6 served as the basis for a method of preparing rat plasma albumin by Roberts (37) and by Tarver and Lee (28)* Fractionation procedures continued to be evolved by the Harvard group* In 19*>0 Cohn et al*, published an account of Method 10 in which advantage was taken of the interaction of proteins with barium ion and zinc ion (8)* These modifications made possible the use of 5 lower concentrations of ethanol* Method 10 proved to be applicable to the preparation of protein constituents from relatively small amounts of plasma* A further modification of Method 10 involves the use of filtration of the precipitated proteins in the cold* With the development of Method 10, and a study of the reactions of plasma proteins, there has been a renewed interest in this field* Schmid (38) has been able to fractionate the components present in fraction VI prepared from Method 10 and has separated an acid glycoprotein as the lead salt* The availability of the mercaptal- bumin of Hughes (9), which reacts stoichiometrically with one mole of mercuric ion and can be crystallized as a mercury salt, has led to the investigation of the binding behavior of this protein toward several other metal ions* Gurd and Goodman have studied the binding of zinc ion to mercaptalbumin (l£)* Gurd and Mir ray (16) have investigated the combination of plumbous ions with mercapt albumin and have concluded that these ions are bound to the carboxyl groups of the protein* * The specificity of the interactions between plasma proteins and metallic ions such as zinc and mercury have led to the development of Method 12 for the separation of human plasma proteins (9)* The method is unique in that the procedure is carried out at neutrality in a completely aqueous system using zinc ion, mercuric ion and lead subacetate* 6 C. The Interaction of Proteins -with Zinc The combination of zinc ions with human serum albumin was taken advantage of in Method 10 (8)* It is now known that zinc combines with sixteen groups of the serum albumin molecule and that these are the imidazole groups of the protein. The average association constant of the zinc-imidaz ole reaction is of the order of 1000 (15>)* This complex is sufficiently stable for the zinc to precipitate the albumins from an ethanol-water mixture at neutrality. Apparently the amount of ethanol necessary for precipitation need be no greater than mole fraction O.OUO (about 12% by volume) near pH 7*0 (9)* At this pH zinc combination is maximal, and as the pH is decreased a higher ethanol concentration is necessary for precipitation. The solubility of human serum albumin in ethanol-water mixtures in the presence of zinc is compared, in Figure 1, with the solubility of free albumin. The zinc-albumin complex is many times less soluble, even at lower ethanol concentrations. Completely reversible complex formation may be carried out with other plasma proteins and zinc (see Table I). The zinc complexes of -lipoproteins, oC -glycoproteins, alkaline phosphatase, serum esterase and /^-metal-combining protein, like the zinc-albumin complex, are water soluble; the complexes of /£-lipoproteins, -globulins and fibrinogen are insoluble in water near pH 7 at 0°C even at the salt concentration of plasma. The zinc complexes of S 9 0 1 1 0- COMPO S IT /O N O F S Y S T E M TOTAL PROTEIN / Z G /L Z n + + M 'T E M P E R A T O R E - S ° C O E T P A N O L 0.047 MOLE FRACTION 0*01 V /2 . 0.0 2 . N a C I m o 50 6 o pH 70 ao F / G . n S O L U B IL IT Y O F H U M A N IsAFRCA PTA L B U M IN /N S O L U T IO N S CO N TAINING 'Z IN C . C9> TABLE I PROTEINS SEPARATED FROM HUMAN PLASMA BY REVERSIBLE COMPLEX FORMATION WITH BIVALENT CATIONS, BY ASSOCIATION WITH SPECIFIC POLYELECTRQLYTES OR POLYSACCHARIDES IN WATER AT pH 7*2 AND 0°C Trace Protein Major Protein Components Components of Concentrated Fraction Fraction Dissolved by Stabilized by in Fraction Adsorbed by Eluted by III III IV-1 Fibrinogen Antihemophilic Globulin Cold Insoluble Globulin /3, -Lipoprotein Precipitated as Zinc Complex Citrate NaCl -Non-Lipid Proteins - Globulins Glycine and C0o Glycine or sugars Prothrombin Serum Prothrombin Conversion Accelerator Desotxyribonuclease Caeruloplasmin Plasminogen Isoagglutinins BaSfy Citrate Stroma Lactose 00 OC,- Lipoproteins Glycine Glycoproteins Glycine Mucoproteins Glycine Precipitated* as Zinc- Mercuiy-Complex Albumin NaCl Plasma Inhibitor Serum Esterase Fraction 7 VI VII Trace Protein Major Protein Components Components of Concentrated Fraction Dissolved by Stabilized by in Fraction Adsorbed by Eluted by -Metal Combin- NaCl ing Protein Serum Albumin Non-polar Alkaline Glycine anions Phosphatase Precipitated as Lead Complex Acid Glycoprotein Polypeptide Plasmin Inhibitor Peptidase CO prothrombin and of the isoagglutinins are insoluble in water at ionic strengths of 0.03 and lower, but are rendered appreciably soluble at higher ionic strengths (9)* The serum albumins, as well as many other proteins, lose zinc to glycine when the amino acid is present in excess. The reaction of zinc with glycine and other oC-amino acids is dependent upon the for- J i mation of a zinc complex. The zinc diglycinate is readily crystal- | j lized and appears to have an octahedral configuration (9). ! t The complex formed between ary of the zinc-activated enzymes and j i their substrates may or may not be with the octahedral configuration j of the respective proteins. All but the terminal <X-amino groups of proteins have generally been thought to be bound in the peptide chain. It is possible that the spatial relationship between adjacent distal amino and carboxyl groups may permit complex formation of proteins with zinc. However, only one amino acid in proteins, namely j t I cystine, would appear to be capable of contributing a carboxyl group j and an amino group in this position. Realization of this suggested 1 the possible site of combination of zinc in certain enzymes or in hormones such as insulin. If this cystine were the site of a zinc complex, reduction to cysteine, or oxidation to cysteic acid, might split the metal-combining group from the protein. On this basis cystine appears not to be the site of zinc binding in insulin (10). Zinc does not combine equally strongly with /3 - and yiamino acids nor with dicarboxylic acids (9). The effect of non-polar side 10 chains on the dissociation of hydrogen ions suggests that such sub stitutions would weaken the association with zinc. Zinc combined with the imidazole groups of serum albumin is still able to combine with glycine, forming a mixed complex. Following treatment of serum albumin with urea a combination with zinc was noted (9) which was greater than expected on the basis of the imidazole groups of the molecule. Whether under these condi tions internally bound imidazole groups, or amino and carboxyl groups of cystine, or other amino acids, become available is being investigated (9) and may prove to be a valuable tool in determining the spatial relationships between the metal-binding reactive groups of proteins, and thus of their structure. Zinc is removed readily from many proteins not only by increasing the concentration of glycine or another metal binding agent, but by increasing the acidity, by the use of complexing agents such as citrate and ethylene-diamine tetracetate, or by cation exchange resins. With the possible exception of the occurrence of intra molecular binding, in which both valences of the zinc become attached to different groups in the same protein molecule, followed by distor tion of the natural configuration of the protein, the quantitative removal of the metal has appeared readily achievable. Irreversible changes in the protein due to the removal or binding of zinc have thus far not been detected. 11 II* MATERIALS AND METHODS 1* Plasma The plasma employed in these investigations -was obtained via cardiac puncture from normal adult male albino rats of the TJ*S*C* strain* Ten ml* of blood was drawn into a syringe containing 2 ml* of a solution consisting of 26*9 gm. of trisodium citrate (£*5> HgO), j i 8*0 gm* of citric acid and 22*0 gm* of glucose per liter of distilled ! : i ! water. The formed elements of the blood were separated by centrifu- j * j gation in a Serval angle head centrifuge* Fractionation of the | plasma (hereinafter designated A*C*D* plasma) was usually initiated ; within two hours after collection, since the apparent instability of j I rat plasma made storage undesirable* ! 2* Reagent Solutions Reagent solutions were freshly prepared at room temperature | (25>°C) for each fractionation from C.P* 9$ per cent ethanol, C.P* reagents and distilled water, as specified, and were precooled to j j -5>°C before using* ; 3* Biuret Analysis Biuret determinations were carried out by a method in which each series of readings was standardized against a standard preparation of rat serum albumin* Determinations were made on aliquots of the original sample by adding an equal volume of a reagent containing 37*£ weight per cent sodium hydroxide and 1*37 weight per cent cupric 12 chloride* Cupric chloride was substituted for cupric sulfate to prevent precipitation of the barium present in some samples. The absorbance of the biuret reaction mixture was determined at m u* lu Spe c trophotomet ri c Measurements Spectrophotometric determinations were carried out in Beckman model DU and model B spectrophotometers# 5>. Measurement of pH A Cambridge "research model1 1 pH meter with glass electrode and saturated calomel half cell was employed in the determination of pH. The instrument was standardized against phosphate reference buffer at pH 6.86 or 0.0$ M potassium acid phthalate solution at pH h at 25>°C., and all determinations were made at room temperature, which was close to 2f?°C. 6* Cold Bath All fractionation procedures were carried out at -f>°C in a large bath filled with 20 per cent ethylene glycol, placed in a -5>°C cold room and stirred constantly. 7* Preparative Centrifugation The separation of the solid fractions from the fractions remain ing in solution in the liquid phase was carried out in an Inter national model SB centrifuge with an angle head. Centrifugation was carried out in the -5° cold room. n 8* Determination of Zinc Total zinc concentrations were determined by extraction with dithizone (diphenylthiocarbazone)• The procedure was as follows: to 1 ml. of sample were added h ml. of 2 M acetate buffer, pH U.6. The resultant mixture was extracted with 10 ml. portions of a solution containing 10 If of dithizone per ml. of carbon tetra chloride until no color change occurred in the dithizone solution. The resulting red or pink colored solution was placed in the spectrophotometer and the absorbance determined at 3> 3 5 > r a ja. The values obtained were compared to a standard curve (see Figure 2). Protein present in the sample was first precipitated with 2 M trichloracetic acid, a procedure which has been found to remove zinc quantitatively from its combination with the protein (15>). The resulting suspension was then centrifuged, sufficient carbonate added to neutralize the trichloracetic acid, and determinations were carried out on the supernatant solution as described* 9# Light Scattering Measurements The method of Brice, Halwer and Speiser (U) for the determina tion of molecular weight by light scattering was followed. The in strument used was that manufactured by the Phoenix Instrument Company. The refractive increment was determined by differential refracto- metiy. The use of both instruments was graciously provided by the Department of Chemistry. All protein solutions were filtered twice through an ultra-fine sintered glass filter under vacuum (as \ 1U ^ 05 k N <o £ 0-4 ft / % Z.N /=-/G.2. STANDARD CURVE FO R THE COLOR [M E T R IC £>ET£?RM/NA T/O N OF TINC W /TH O/TN/'ZONE A T S 3 S 772 ^ f 15 provided by a water aspirator) before determinations were attempted* 10* Ultracentrifugal Measurements The determinations of sedimentation velocity were carried out in an air-driven centrifuge following the design of Beams and Pickels (36)* The instrument was originally constructed by the Statham Instrument Company of Los Angeles, California, but has been modified to receive the optical system manufactured by Specialized ! Instruments Company* The instrument is shown in figures 3* U, 5>A and 55* The angular velocity was determined with the aid of an electronic 1 | | tachometer manufactured by the Hewlett-Packard Company* The sensing i I | element consisted of a light source and photoelectric cell, inter- ; ruptions in the light source being provided by the top of turbine j drive, one-half of ihich is painted white and one-half black* j Tachometer readings were corrected by reference to a spinning disc j painted with alternating white and black segments and rotating at a | i known speed* | The temperature sensing device used was essentially that devised | 1 by Pickels (36)* The indicating pyrometer used was of the potentio meter type and was manufactured by the Leeds and Northrup Company. The pressure in the vacuum chamber was determined with the aid of a thermocouple vacuum gauge tube manufactured by the General Electric Company, utilized as directed by the manufacturer* The E^ig# 3* The ultracentnfuge control panel and optical system termination 17 Fig* i|* The ultracentrifuge in operating position 18 Fig. 5A. The ultracentriruge drive unit and vacuum chamber with the lock ring in place 19 Fig* 5>B* The ultracentrifuge drive unit and vacuum chamber with the top raised to show the rotor in position Key to Figures 3* hi. and $B 1. Mercury vapor light source. 2. Air turbine assembly. 3. Top of turbine. lw Top of vacuum chamber. 5. Drive oil collector cup. 6. Aluminum analytical rotor. 7. Main body of vacuum chamber. 8 . Hydraulic lift piston. 9. Optical bench. • o H Vacuum gauge tube housing. 11. Vacuum chamber lock ring. 12. Speed indicator light source. 13. Speed indicator photocell. 1U. Cold trap. 15. Oil diffusion pump. 16. Fore pump. 17. Electronic tachometer. . CO H Motor driven disc used for tachometer calibration 19. Vacuum gauge control unit. . o C M Pyrometer. • . H C M Centrifuge control panel. 22. Film holder. 21 indicating millivoltmeter was not calibrated in pressure -units, but the readings were checked against the calibration curve supplied by the manufacturer* The pressure obtained was in the vicinity of 10"^- microns and resulted in a temperature rise of approximately 1°C per hour at the rotational velocities employed* j I Kodak spectrographic plates were used throughout for all photo- j graphs* The optimum time for exposure proved to be 3 seconds with a I ; slit opening in the light source of 0*0081 1 . All patterns were en- j : | j larged before measurements were attempted* The total enlargement j i ; factor used was 10:1* i ’ | ; 1 11* Diffusion Measurements j ! ! The diffusion constant of the rat plasma albumin (subfraction 2) | was measured in a standard U c*c* Tiselius cell* Measurements were i made ateontinously decreasing concentrations and extrapolated to j ; I zero concentration* In order to minimize disturbances during the i i diffusion runs due to vibration in the bath, a special Stirling device was constructed (see figure 6)* j I Wienerfs method was used to calculate the diffusion constant* The method depends on the fact that the diffusion constant is related to the area and maximum height of the diffusion concentration gradient by the formula Do,h "(02 A2 UTltH* where **> « enlargement factor Fig. 6. The electrophoresis and diffusion apparatus showing the special stirring device in position 23 A ■ Area of curve H = Height of curve t * elapsed time when the quantity C&2 A^ /h 7 iis plotted as a function of t a straight line results* The diffusion constant Do,h is given by the slope of the line* 12* Electrophoretic Measurements All electrophoretic determinations were made in a Tiselius apparatus with a 2*5 ram* cell purchased from the Pyroeell Manufactur ing Company, using the method previously described by Mehl and Winzler (31)* Measurements of boundary displacement were made directly on the photographs of the boundaries* The enlargement factor proved to be 1*1* 13* Hie Fractionation of Rat Plasma As has previously been reported (23)? the fractionation of rat plasma albumin is a modification of Method 10 (8) of the Harvard group for human plasma* The solutions used for the fractionation are as follows: Solution I* 200 ml* of 95 per cent ethanol and 2*5 ml* of acetate buffer ( \/Z 0*8, pH U*0) per liter* Solution IX* 280 ml* of 95 per cent ethanol and 5U*8 gms* of zinc acetate dihydrate per liter* Solution IU* 270 ml* of 95 per cent ethanol, 20 ml* of 1*0 M sodium acetate, 2 gms* of zinc acetate dihydrate, 3*8 gms* of barium 2k acetate and enough glacial acetic acid to bring the pH to U.7 per liter* The fractionation procedure is as follows: a* 25 ml* of A*C.D. plasma are placed in the master tube in the -5°C bath* After the plasma has cooled to 0°C, but before appreciable freezing has occurred, 100 ml* of Solution I are added with stirring at a rate not exceeding 20 ml* per minute* Stirring is continued ! ' i i for an additional fifteen minutes, after which time the mixture is centrifuged for 30 minutes at 1*000 R*P*M* and -5^C* The precipitate remaining after centrifugation is designated as fractions I + II + i III, and the supernatant as fractions IV + V + VI* | ; b* Fraction IV + V * VI is transferred to a new tube, and 10 ml* ; of Solution II are added with stirring at -5°C* The mixture is ! allowed to stand at least 15? minutes before centrifuging 30 minutes j at 1*000 E*P*M* and -5°C* The precipitate remaining after centri- | fugation is designated as fractions IV + V and the supernatant as j ! ' ! fraction VI* c* The serum albumins in fraction IV + V are extracted by stirr ing for one hour with 175 ml. of Solution III. The mixture is then centrifuged for 30 minutes at 1*000 R.P.M* and -5°C. The precipitate remaining after centrifugation is designated as fraction TV and the supernatant as fraction V* The alcohol is removed from the fraction by lyophilization. The 25 resulting preparation is dissolved in a minimum volume of 0*2 M pH U.5 acetate buffer and placed in Visking tubing* The preparation is then dialyzed against U changes of 0*2 H pH U*5 acetate buffer, approximately 1000 volumes for each buffer change* Following this treatment the tubes are dialyzed against six changes of 1000 volumes each of distilled water* The resulting protein solution is passed through a column of 50 mesh Dowex 50 resin in the hydrogen cycle in order to remove the last traces of sine* The eluate is checked i ' for zinc content by dithizone extraction as previously described* The final albumin product represents a yield of 87 per cent i of the total rat plasma albumins* The composition as judged by i i electrophoresis is 90-92 per cent albumins and 8-10 per cent j /S -globulin (23)* lit* Equilibrium Dialysis Procedure The technique of equilibrium dialysis was employed for the | stTKfcr of the interaction of zinc ion and rat plasma aXbuain. A | modification of the method described by Gurd and Goodman (15) was employed* The procedure was as follows s i 200 ml* of solution containing varying concentrations of zinc as ZnClg and enough sodium nitrate to give an equilibrium concentra tion of 0*15 M in the total system were placed in a 250 ml* Erlenmeyer flask, together with a bag of Visking cellulose casing (8/32" inflated diameter) containing 2 ml* of a 1 per cent solution 26 of protein* The flasks were maintained in a OOC cold room for 1|8 hours with occasional agitation* After the equilibration period the bag was removed from the flask and the zinc concentration inside and outside the bag determined by dithizone extraction as described previously* The concentration of bound zinc ion was determined as the difference in total concentration of zinc inside and outside the bag* The Donnan effect was considered to be negligible in the presence of 0*15 M NaNO^. l£* Partial Specific Volume The partial specific volume of protein solutions was determined by density measurement with a pycnometer of 8*929k ml# capacity, according to the relation* V * apparent specific volume v ■ volume of the pycnometer in cm3 mo • net weight in gnu of pycnometer when filled with protein solution m • net weight in gm* of pycnometer filled with solvent Wp • weight fraction of protein in solution 27 The protein solution used contained 1 gnu per 100 ml* The partial specific volume proved to be 0*73* 1 16* Nitrogen Determination i The protein samples were digested in 2 ml* of a 1 si solution of sulfuric acid and distilled water, saturated with potassium sulfate* One to two drops of superoxol were added during the course of digestion to aid in clearing* Digestion was continued for one hour after clearing* The digest was then transferred to a Pregl still and distilled into 5 ml* of 2 per cent boric acid solution containing 10 ml* of 0*1 per cent brcsacresol green and 2 ml* of 0*1 per cent methyl red per liter* Levels of nitrogen from 100 to 200 pg* were determined by- titration with 0*01 N sulfuric acid* ! 17* Determination of Free Sulfhydryl Groups i The free sulfhydryl groups of the protein molecule were | determined by a modification of the method described by Boyer (3)* ; i P-chloromercuribenzoate combines strongly and quite specifically with sulfhydryl groups, and the absorption spectrum of the compoTand differ^ from that of p-chloromercuribenzoate. The amount combined is determined from the increase in absorbancy at 25>0 mju, assuming that the change is the same as that reported for simple compounds with cystine or glutathione* 28 III* EXPERIMENTAL 1* Removal of Zinc from Fraction V a* Dialysis Techniques Fraction V, following lyophylization to remove alcohol, proved to be quite insoluble in distilled water since it exists as the zinc salt* Solubilization may be effected immediately upon addition of an acid buffer* In these studies acetate buffers of pH U*6 and V/2 0*1 were used* Solution of the protein in distilled water was usually assured after dialyzing against 0 to U changes of 1000 volumes each of the acid buffer, followed by dialysis against running j tap water for 12 hours and finally by dialysis against h changes of 1000 volumes each of distilled water* lyophylization of the solution obtained following this treatment resulted in a white fluffy prepara- , i tion quite soluble in distilled water, but still containing some | zinc as judged by qualitative dithizone extraction* In order to i insure an equal or greater removal of zinc, versene (ethylene- diaminetetraacetate) was added to the acetate buffer used to a final concentration of approximately 0*01 M. The product obtained after this treatment was used without further treatment in electrophoretic studies of Fraction V* I b* Use of Ion Exchange Resins Since the product obtained after dialysis still contained appreciable amounts of zinc, passage through ion exchange resins 29 in order to effect a more quantitative removal of that metal ion was resorted to. In view of the established efficiency of Dowex 50 in the sodium cycle for the removal of calcium ion, this resin was tided first. It was observed, however, that zinc, being more tightly bound to the protein, was more difficult to remove than calcium. It was found that the exchange of hydrogen for zinc pro ceeds considerably more effectively than the exchange with sodium, because of the high affinity of the protein for protons. Since, j S with the use of the hydrogen cycle resin all salts present would be ; converted to the corresponding acids, it is important that a minimum amount of these salts be present. It is evident that, ideally, the process could be considered as the conversion of the metal proteinate to the isoelectric protein. The reduction in pH during this process does not appear to be deleterious. Since a column of coarse (50 mesh) resin was used for the zinc removal and correspondingly the rate of flow of protein solution through the column is rapid, several passages of the solutions through the column were necessary to produce a preparation which gave no color change with dithizone. A glass column 75 cm. high with a bore of 11 mm. was used, filled with a suspension of resin in water to the 50 cm. mark. 2. Refractionation of Fraction V It is possible, as with the fraction V obtained from human plasma, to effect a small degree of purification of fraction V from rat plasma by reprocessing with Solution III (20). The resulting product contains approximately 9U per cent albumin and 6 per cent fi- globulin as judged by electrophoresis. A continuation of such reprocessing, however, is obviously one of continuously diminishing returns. Thus it was clear that some more fruitful solution of the problem of purification needed to be sought. a. Solubility in Alcohol Solutions It was felt that studies of the solubility behavior of the albumin-rich fraction V might throw some light on possible conditions for the separation of the albumin from the yff-globulin contaminant. Thus a system was set up in which the alcohol concentration and the pH were varied, but the ionic strength was kept nearly constant at 0. 01. It was found that the minimum solubility under these conditions was, as expected, in the vicinity of the isoelectric point of the albumin, i.e.3 at pH U.8 - U.9# Under the conditions listed no appreciable precipitation occurred below an alcohol concentration of 25 per cent by volume. Maximum differences in solubility appeared to occur at 30 per cent alcohol by volume• Values of pH below U.O were not used since it was feared that too high a concentration of protons might lead to denaturation of the protein. A typical pH- solubility curve is shown in Figure 7* Above a pH of 7 little, if any, detectable precipitation occurred. The insoluble and soluble preparations were freed of alcohol by lyophylization, dissolved, and the compositions of the fractions so obtained approximated qualita- I £ 10- PRECIPITA TE SUPERNATANT I ^inii u i l " 70 60 6 . 0 40 5.0 F / G 7 * t h e S O U J & IL /T Y b e h a v i o r o f t r a c t / O N I T F RO M RAT PLASMA IAI 30 °/o E T H A N O L , T/2.0.0H tively by zone electrophoresis on filter paper at pH 8*6* None of the fractions so obtained proved to be free of theJS -globulin contaminant* Since it was felt that differential salting in of protein might be occurring, the ionic strength of the buffers used was reduced to as little as 0*001* However, no improvement occurred* All the fractions obtained contained both albumins and-globulins • 3* The Addition of Zinc to the Refractionating System Although the results obtained by precipitating fraction V with alcohol at low ionic strengths were far from satisfactory in provid ing a homogeneous preparation, the results at pH showed some possibility of separating a more homogeneous preparation in the vicinity of the isoelectric point* Since the attempts made in systems containing buffer salts failed, a system was devised in which it was felt that the protein might act as its own buffering agent* When 1 ml* of a 10 per cent solution of protein was placed in the cold room at -5°C and a 23*5 per cent cold alcohol solution was added slowly with stirring, turbidity immediately developed* After an hour the suspension was centrifuged and the precipitate removed for analysis* The product was dissolved in water so that the concentration was approximately 1 per cent (l gm# per 100 ml*) and was then subjected to electrophoresis* Ihe results showed the fraction so obtained to be quite homogeneous at pH 8.6* 33 Upon checking the fraction described above qualitatively for zinc by dithizone extraction it was found that considerable zinc was present* These findings led to the development of the following system for the refractionation of fraction V. To 1 ml* of a 10 per cent solution of fraction V are added, with stirring and at -5°C, 5 ml* of 23 per cent ethyl alcohol adjusted to pH 5*3 - with CO2 and 0*1 ml* of Solution II (the composition of Solution II is described in Section II, pp* 23)* The resulting suspension is allowed to stand for one hour, after which time it is centrifuged for 30 minutes at J 4OOO R*P*M* and -5°C* The resulting precipitate is dissolved in a minimum quantity of distilled water and treated to remove zinc as described in Section IH, pp* 28* The fraction will hereinafter be referred to as fraction V^* The result ing solution is lyophylized and stored* Originally the distilled water used in these experiments was provided by the Fresho Puro Water Company* Measurement of the pH of the alcohol solutions made with this water showed the pH values to be between 5*3 and 5*lw When distilled water from other sources became available the refractionation was not reproducible because of the increase in pH of the alcohol solution to about 6*2* It was assumed that the lower pH previously obtained was due to the presence of considerable amounts of dissolved CO2* Hence, CO2 was bubbled into the solutions to give a pH of 5*3 - 5*14 and solutions prepared in this manner provided the necessary requisite for the reproducibility: of the system* The preparations obtained in this manner, although showing a fair degree of electrophoretic homogeneity at a pH of 8*6, resolved into two (or more) distinct components at a pH of U*6* Furthermore, a fair degree of reversible boundary spreading was observed* It is true j that such behavior is common in preparations of albumins of other j species such as the horse and bovine albumins* However, it was felt j j desirable to produce a product which would, at least, display a more j advanced state of electrophoretic homogeneity, and if possible provide a means of separation of the several species of albumins present in i the preparation* The author can claim but little credit for the further advance in the refractionation of fraction V. The results are, in the main, j due to the failure to remove, as completely as possible, all of the | zinc frcm fraction V* Attempting to repeat the refractionation of j fraction V, as previously described, it was observed that a precipi tate formed immediately when the 23 per cent alcohol solution was added* When this precipitate was removed and subjected to electro phoretic analysis, it showed electrophoretic homogeneity at pH U*6 as well as at pH 8*6* Determination of the zinc present by dithizone extraction showed a quantity of zinc equal to one equivalent of the metal per mole of protein. 35 ■When proper precautions were taken for the removal of zinc from fraction V, and the refractionation repeated as before with the inclusion of an amount of zinc equal to one equivalent per mole the same results as above were obtained# The precipitate contained 66 per cent of the initial protein, the remainder was retained in solution# The solution was found to contain an albumin component migrating with a lower mobility than that of the precipitated albumin at values below the isolectrie point of the albumin# The fraction obtained as described above will be referred to as fraction V^# The material remaining in solution after centrifugation will be referred to as fraction k. Equilibrium Dialysis of Zinc and Rat Plasma Albumin These experiments were carried out as described in Section XI, pp. 25# The results are given in Table II# The change in the zinc binding behavior when zinc is added in | excess of 1 equivalent of zinc per mole of protein is most striking# Wo appreciable amount of zinc can be detected in the dialysate when the total amount of zinc in the system is less than 1 equivalent per mole of protein. For the amount of protein used, this represents a total of 12.8 y of zinc in the system# The lower limit of the analytical method is about 0.2 ^ of zinc# Taking this as the lower limit of the totail amount of zinc in the 200 ml# of dialysis fluid, the maximum concentration of free zinc is 3*1 x 10-8^* Calculation 36 TABLE II THE BINEECNG OF ZINC TO RAT PLASMA ALBUMIN Total Protein Total Zn. Bound Zn.++ Free Zn.++ Free Zn.++ /M pM/K pM/M pM pM/ml. 2 .125 Ca .125 — — — 2 .25 .25 — 2 .375 .375 — — 2 .5 •5 — — Z .75 .5 .25 1.25 x 10-3 2 1 .65 .U3 2.15 X 10-3 2 1.5 .76 .72 3.6 x 10-3 2 2 1.0 1.0 5.0 x 10-3 2 2.5 l.l l.U 7.0 x 10-3 2 h i.U 2.U 1.2 x 10-2 37 or the association constant for the first binding site from (P Zn) * k 0?) (Zn) leads to a minimum value of k to equal 107. V Treating the data for the second binding site in the same manner, assuming no interaction between binding sites, we arrive at a value for k2 of approximately 1*66 x 10^ for the association of zinc* The relationship is visualized below: Since (P Zn) « ki (p ) ( S ) " Then (P Zn2 ) , ko (P Zn) (Z n ) Assuming that the binding behavior between zinc and rat serum albumin follows that described by Gurd and Goodman (15) for human mercaptalbumin, that is, that the zinc ion is bound to the 16 imida zole groups of the protein, we may calculate the statistical value of ki on the basis of the value of the average association constant of 1<>3# This yields a value of approximately 16,000* While this is of the same magnitude as the value of k£ for rat plasma albumin, k^ is strikingly greater, obviously indicating a different binding mechanism* When the data shown in Table II are plotted according to the method of Klotz (27) the curve shown in Figure 8 results* Extra polation of the straight line portion of the curve to the Y-axis L I ■ . ! » ■ . . . I , . I . . . . . . . - . 1 . . . . mm- r m mfm. . . . . . . .mmn m m r m . . . . . . . . . . . . . . n . 1 2 3 4 5 Yin** *10^ F*SGQ . T H E E /N O fN G O F E '/N C TO P A T PLASM A ALBUMIN. R EQUAL S MOLES OF Z /N C BO UNO P E R MOLE OF PROTE/N. w CD 39 yields a value of 1.1 moles of zinc per mole of albumin. In view of the analytical difficulties this may be considered to indicate the binding of 1 mole of zinc with an affinity constant of 3.2? x 10^. The sharply changing slope at higher zinc concentrations indicates a much weaker binding of additional zinc ions, more in accord with the reported behavior of human albumin (1?). It should be noted, however, that the conditions under which zinc binding has previously been studied would not make it possible to detect the very strong binding of the first mole of zinc. Because only 1 equivalent (§ mole) of zinc ion was required % for the precipitation of fraction V2, the possibility of dimer formation, similar to that taking place with human mercaptalbumin and mercuric ion (18, 19), could not be overlooked. However, in aqueous solutions, no evidence of such an effect could be obtained in the ultracentrifuge. The sedimentation behavior in alcohol solutions, as influenced by zinc ion, has not been studied, and'it is quite possible that the zinc salt which precipitates is a dimer of the form: Albumin - Zinc - Albumin ?. The Nitrogen Content of Rat Plasma Albumin Nitrogen determinations were carried out on samples of dried albumin preparations as described in Section II, pp. 27* The deter minations gave an average value for protein nitrogen of 1?.8 per cent. Uo This value is close to the reported value for human albumin of from l£*95 to 16 per cent (6)* The protein nitrogen value given above is an average of five values* The minimum value was lf>*7 per cent and the maximum value was 15>*92 per cent* 6. Electrophoretic Studies with Hat Plasma Albumin Electrophoretic studies have been used to follow the progress of refractionation in the isolation of a purified albumin from rat plasma* Electrophoretic studies have demonstrated in the past that fraction V obtained from rat plasma was contaminated with 8 to 12 per cent yd -globulin as shown in Figure 9* Electrophoretic inhomogeneity was shown by the albumin component of fraction V at values at and below the apparent isoelectric point at pH h*8 - h*9* Examination of the width of the electrophoretic patterns at values of pH up to 7*0 also appeared to indicate a greater amount of boundaxy spreading than eould be accounted for on the basis of diffusion alone* The first refractionation of fraction V, as described in Section III, pp* 33, with 7-8 moles of zinc per mole of albumin and 21*8 per cent alcohol yielded a product with good apparent electrophoretic homogeneity at a pH of 8*6* At more acid values of pH, as demon strated in Figure 10, this product was still inhomogeneous* Electrophoretic patterns of the material from the second refractiona tion with 1 equivalent of zinc per mole of albumin and 21*8 per cent ethanol indicated the isolation of an albumin fraction which was 9 . B A B C 7V? O /=>/L/0*>£~T/C BA 'TTBRA/S OB BRACT/OATST A T 3. 6 ^ /2 . O ./ v b r o m a l b l/ b b b r . k2 t>FSC&A/D/A/G A SCFND/MG F /G . /O . FL etc 7-ROPHOR&T/C J & A T TFR M S OF FRACT/ONTT, A T O ./ A C S TA TF &UFFFJ^+ homogeneous over a pH range of k to 9* The electrophoretic patterns for fraction V£ at pH U*5 and pH 8*6 are shown in Figure 11# Data showing the variation of mobility with pH for fraction V2 and V3 are shown in a graphical form in Figure 12# This albumin isolated from rat plasma has a greater mobility in the basic pH range than human albumin* Comparative figures for rat and human i albumin at several values of pH are given in Table III. Some possible! * * 1 i conclusions regarding the ratio of charged groupings operating at J various pH values may be drawn from the pH-mobility curve* It may | be concluded that In rat plasma there is a higher ratio of carboxyl j to guanidine residues that exists in human albumin as evidenced by the faster mobility at pH 8.6* i 7* The Determination of Molecular Weight by Light Scattering As has been noted in Section II* page 13, the method of Brice, Halwer and Speiser (1*) was used* The data may be presented most j clearly in tabular forn as in Table IV* is calculated according to the relation 'I* « * l6n2TD Fa x Rwx Gs * 28#3 x 0*866 x Gs 3 ( 1 . 0 Rc Gw Gw where ^ » Absolute turbidity of the solution* D E S C E N D IN G A S C E N D / N G p h f 8.6 *r /2. Q . / V S P O N A L S T A R T S T A R T D E S C E N D I N G a s c e n d / n g p H *?.& 72 a ./ a/a A c e t a t e P / G - i f E L E C T * o p h o r e t / c h a t t e r n s o r RAT PLASMA ALBUM /N ERA CT/OA/ V z - C ONC EN TRA T/O N =- / GM PER /OO ML . -M TRACT/O N Vz SUPERNATANT FRACT/ON Q 4 5 6 7 />" E /G ./Il T H E p H M O & JL JT Y TSURVE R O R R A T P L A S M A ALBUM/Nm 0% U6 TABLE III COMPARISON OF THE ELECTROPHORETIC MOBILITIES OF RAT AND HOMAN ALBUMINS pH Buffer XL X Human Albumir^/ xo5 Rat Albumin 9.1 Veronal 6.8 9.0 Veronal 7.2 8.6 Veronal 5.8-6.2 6.8 7.7 Veronal 5.0 5.6 iw8 to li.9 Acetate 0 0 1/ Data of Armstrong et_ al^ (l, 2)* 1 * 7 TABLE IV LIGHT SCATTERING DATA FOR RAT PLASMA ALBUMIN Protein Concentration He Sample Ga/Gw Gm./ml. x 10~3 y'x 10“5 ^ 1 0.758 8.1 19 l.$9 2 0.39 U.3 9.8 1.62 3 0.205 2.5 5.15 1.79 U 0.105 1.2 2.68 1.65 5 0.052 0.6 1.31*2 1.65 U8 Gs * Average observed ratio of deflections Gw for the scattering solution at 90° to that for the working standard at 0°. P - Product of transmittances of the neutral filters used in determining the above ratio* a * Constant relating the reference standard to the opal glass reference standard* D * Diffusor correction fractor* T « Transmittance of the opal glass reference standard* h ■ Width of the diaphragm* H is calculated according to the relation H - 32 TC 3^2 (n-no)2 c2 3 X where H * Calculated constant for the solution used* c * Concentration in gnu/ml* nQ® Refractive index of the solvent* n * Refractive index of the solution* X* Wave length of the incident light in cm* N - 6.023 x H>23 Since a wave length of 5k6 m ji, was used, this reduced to k9 H » 6*18 x 105 n0 (n-no)2 c The refractive increment of the protein solution (n-no)2 was found to be 1.81* x 10~^, Plotting Hc/tf" against the concentration in | grams per ml* the plot shown in Figure 13 is obtained* Extrapolation s to infinite dilution leads to a value of 1.65 x 10-5 for Hc/7' • The ! plotting of the data follows the equation: He - 2Bc + 1 ! *f M which is in the form of the slope-intercept equation for a straight line, Y ■ mx + b. In this case b, the intercept on the Y-axis is equal to 1* The reciprocal of 1.65 x 10“ “ ^ yields a value of 6*1 x ; M ' loit or a molecular weight of 61,000. On the basis of the above relation the value for M is 61,000, j which is in good agreement with 63,1 * 00, the value for the molecular weight of rat albumin as determined by sedimentation and diffusion and with 61*, 000, the value determined from the binding of p-chloro- mercuribenzoate• 8. Ultracentrifugal Results Experiments with the ultracentrifuge were carried out as described in Section II, pp. 15-21. Experiments were conducted at concentrations of 1.5* 1*0 and 0.5 grams per cent. The data are shown in Table V. Extrapolation of the data to infinite dilution as shown in Figure ll* yields a value for S20 of U.35* / 2 5 4 5 6 7 8 $ C GMS./C C x /O'3 F /G./3 PLOT o r vs. C /R?/? 77/£~ p e t e r m / n a t / o n OF THE MOLECULAR WE/GH T O F R A T A L B U M IN B Y LJGHT SCATTERING • M EQUALS <Z/jOOO« 51 TABLE V ULTHACENTKIFUGAL BATA FOR RAT PLASMA ALBUMIN Protein Concentration gm./lOO ml* Buffer pH o CJ CO 1*5 0*1 M Na Acetate U.9 3*81 1*0 0*1 M Tris 7.0 U.03 1.0 0*1 M Tris 7.0 U.07 1*0 0.1 M Tris U.8 U.oU 1.0 0*1 M Tris U.8 It .08 1.0 0.1 M Tris U.8 U.d o*5 0.1 M Tris U.8 It.iU o*5 0*1 M Tris U.8 1*.22 o*5 0.1 M Tris U.8 3.77 6 5 4 20 3 2 /«o C QMS. F1G./+. P L O T O F T H F SFD/M FNTATiQN VALUFS F O R 'R A T ALBUMJN AT VARYING CQNCFNTRAT(ON $. FXTFAPOLA T/ON TO Z£R O CONCSNTRAT/ON yF/LOS AR Szo VAJLUF Oj.SS 53 9* Diffusion Results Determinations of the diffusion constant were carried out as described in Section II, page 21* Experiments were conducted at 1.0, 0*5 and 0*3 grams per cent rat plasma albumin* A typical diffusion concentration gradient pattern is shown for a 0*5 per cent rat plasma albumin sample in Figure 15* Bata are tabulated in Table VI for a 0*5 per eent albumin sample* Extrapolation of the data to infinite dilution, as shown in Figure 16, gives a D2Q value of 6*2* 10* Calculation of the Molecular Weight From Sedimentation and Diffusion Data I » The molecular weight of rat plasma alb train was estimated from Svedberg1s relation *%D " RTs where ! R « 8*3lU x 107 ergs per mole per degree s » Sedimentation velocity (iu35 x 10-3-3 sec*"3-) D * Diffusion constant (6*2 x 10-7 cm2 per sec*) V • » 0*730 cm3 per gram (20°) 0*9982 gm* per cm3 (water at 20°C) T - 293#2°K The molecular weight determined in this, way is believed to be dc/dx - X 57/4 AT F /G . / 5. D /F FUS i ON P A TTE R N O F F A 7" FLA SMA A L& U M /N A T SSjO SO SFC. C0/VCGA?TFAT/O/V = &.£■ QM PER /O O M F - 55 TABLE VI DIFFUSION DATA FOR RAT PLASMA ALBUMIN 1 j sec* H h2 A A2 a2/jj2 OJ 2 j^2 0 18.5 3U2.25 7.5 56.25 .1693 3.6U x 10-2 2160 12.3 151.29 8.2 67.2U •UU5 8.58 x 10-2 5700 10.lt 108.16 9.3 86.U9 .80 I5.1t3 x 10-2 22,320 6.9 lt7 .6 l 8.2 67.2U 1.U12 27.2 x 10-2 79,200 5.1 26.01 7.7 59.29 2.278 U3.9 x 10-2 96,960 lu7 22.09 7.3 53.29 2.U01 U6.lt x 10”2 56 7 s 4 3 t / / • 5 / . < 9 3“ C G M S . / / Q O Mi P /G ./6. PLO T OP T M P O/PPUS'/OA/ COA/STA/V7 POP R A T P LA S M A A L& U M /M A T VAPy/NC CONCPNTRA T/OAJ. 57 accurate within 5 to 10 per cent (29) and refers to the anhydrous protein# On the basis of the above relation the value of Mgj) is 63,UOO, which is in good agreement with 61,000, the value for the moiecular weight of rat plasma albumin as determined by light scattering and with 6U,000, the value determined from the binding of p-chloro- mercuribenzoate• 1 1 . The Shape and H y d ra tio n o f R a t Plasm a A lbum in The effect of shape and hydration of a molecule upon its sedimentation or diffusion is expressed as the molar frictional ratio of the solute. This ratio can be computed frcm the sedimenta tion and diffusion constants taken together (39)# The molar fric tional ratio, f/fo, is the ratio of the resistance of a molecule to motion through a viscous medium in sedimentation or diffusion to the resistance of a spherical anhydrous molecule of the same molecular weight# The deviation from unity, the frictional ratio of a spherical, unhydrated molecule, is due to the combined effects of solvation and shape# The frictional ratio may be found from the relation f/f0 - (i-Vp)V3 x io -8 (D2sV) Thus for rat albumin K .2 x io'02 x ii.35 x iW V o .? 3 (1-.730 x 0.9982) y3 * io-» 58 t/to - 1.3 The molar frictional ratio may be considered the product of two factors: f/fo - f/fe x fe/f© such that f/fe depends on solvation only and fe/fQ depends only on the molecular shape. The factor f/fe is given as a function of hydration by Kraeraer as f/fe - (rV2 + Vi) 1/3 Vi where r * number of grams of water bound to each gram of protein Vg a partial specific volume of water at 20°C ( - 1.00) » partial specific volume of the protein. Assuming that the protein molecule is essentially an ellipsoid of revolution Perrin (35) has shown that the relation for prolate ellipsoids in terms of the ratio for the long to the short axes, a/b is fe/fo ■ [l-(b/a)2J i_______________ (b/a)2/3 in 1* (l-(b/a)2)i b/a From the Kraemer relation the factor f/fe due to hydration is, for the lower limit of hydration (r * 0.2) f/fe - (0.2 x 1.00 + .730)V3 . i.oo5 .73 and for the upper limit of hydration (r * 0*8) $9 - (0*8 x 1*00 + 7.3) - 1*036 .730 Since f e / f o - f / f p W l then for the upper limit of hydration fe/fo - 1.25 and for the lower limit of hydration fe/fo » 1.29 R e fe rrin g th e s e to P e r r in ’ s r e la t io n we f in d an a x ia l r a t io f o r r a t serum alb u m in o f fro m U :1 to 6 : 1 . 1 2 * The D e te rm in a tio n o f F re e S u lfh y d r y l Groups The determination of free sulfhydryl groups in the rat albumin molecule was carried out as described in Section IX, pp* 27. Boyer gives, for the molar absorbance index for p-chloromercuribenzoate, the value of h.6 x 103 at 25>0 m ji* On this basis the concentration of p-chloromercuribenzoate is 1*U x 10~k moles per liter* The protein concentration used was 0*6 gm. per liter* Examination of Figure 17 shows that 3.7 x 10-£ moles of p-chloromercuribenzoate were bound to the albumin* This results in an equivalent weight for albumin with p-chloromercuribenzoate of 16,000* Thus U equivalents would yield a value of 6i|.,000 in good agreement with the molecular weight of albumin found by sedimentation and diffusion* The data thus indicate that rat plasma albumin contains four free O P T I C A L DENSITY 60 Z Q p-Cf-/LOROMEBCURfBENZOATE Q A L B U M (N < 2t p C M B ~h A L B U M /N Q zeo • 2.50 245 300 290 280 270 WAVELENGTH f~/G. /7\ OPT/CAL B£~ HA \//OR SHOW/NQ C O M PL BY FORMAT/ON OF A L B U M / N A N D p-C.Hi.ORO M F R C U R / SFA/ZOA T F . 61 sulfhydiyl groups per molecule, in contrast to human mercaptalbumin of Hughes, which contains one sulfhydryl group per mole* 62 IV. SUMMARY AND CONCLUSIONS A system has been described for the isolation of a fraction of rat plasma albumin which is electrophoretically homogeneous at both acid and alkaline values of pH. The data obtained with the ultra centrifuge and from measurements of free diffusion indicate a mole cular weight for this fraction of 63*1*00, a value in good agreement with that found from light scattering data. Data obtained from equilibrium dialysis of the albumin fraction with zinc indicate an affinity constant for the first binding site of 3*2$ x 10&. The reaction of the molecule with p-chloromercuribenzoate indicates the presence of four free sulfhydryl groups per molecule of albumin. Of the physical characteristics which have been investigated, the behavior of rat plasma albumin toward zinc is perhaps the most interesting. It has been assumed that the first binding site for zinc is an imidazole group. Since the affinity constant of imidazole alone is of the order of 103 and since the value of kg for the binding of the second zinc ion is of the order of 10^, a value close to that to be expected on a statistical basis for a protein molecule with approximately 16 imidazole groupings, one must argue the presence of an imidazole grouping so situated on the protein molecule that its affinity for zinc ion is much higher than that normally to be expected* The presence on the albumin molecule of four free sulfhydryl 63 groups might appear to offer some promise that the sulfhydiyl group may be the grouping through which the strong binding of zinc occurs. However, in an identical system, mercuric ion, assumed to have a much higher affinity for sulfhydryl groups than zinc showed essentially no binding to the albumin* It is interesting to note that rat plasma albumin has a molecular weight of the same order of magnitude as the albumins isolated from human, porcine, equine and bovine plasmas* It may be assumed that the shape and hydration of the molecules are also similar on the basis of similar values of the molar frictional coefficients. However, certain differences are apparent between rat and human albumins* While the mercaptalbumin of Hughes (18, 19) shows the presence of one sulfhydryl group per mole, it has been demonstrated that rat albumin contains four free sulfhydryl groups per mole* Thus one could not reasonably expect the dimer formation with rat albumin and mercury that obtains for human albumin* Although the isoelectric points for the albumins of all species studied appear to be similar, the differences in electrophoretic mobility at basic pH values indicate some differences in the relative proportions of charged groupings, particularly guanidine and carboxyl groups. This may be taken as indirect evidence of some differences in the amino acid composition of rat albumin and the albumins of other species. The method for isolation of rat plasma albumin shows promise of 6k extension to other members of the rodent family* Extension of the method to plasma of the guinea pig (26) has resulted in a fraction V of similar composition to that found for rat plasma* BIBLIOGMPHY 66 1* Armstrong, S.H., Jr., Budka, and Morrison, K.C., J . Am. Chem. S o c ., 69, Ulo (19U7). 2. Armstrong, S.H., Jr., Budka, M.J.E., Morrison, K.C., and Hasson, J., J. Am. Chera. Soc., 69, 17U7 (19U7). 3. Bqyer, P.D., Abstracts, Division of Biological Chemistry American Chemical Society, 123 rd. Meeting, Los Angeles, Mar. (1953). U. Brice, B.A., Halver, M., and Speiser, R.J., Opt. Soc. Am. ha, 768 (1950). 5. Cohn, E.J., Strong, L.E., Hughes, W.L., Jr., Mulford, S.J., Ashworth, J., Melin, M., and Taylor, H.L., J. Am. Chem. Soc., 68, 1*59 (191*6). 6. Cohn, E.J., Hughes, W.L., Jr., and Weare, J.H., J. Am. Chem. Soc. 69, 1751* (191*7). 7. Cohn, E.J., Leutscher, J.A., Oncley, J.L., Armstrong, S.H., Jr., and Davis, B.D., J. Am. Chem. Soc. 62, 3396 (1950). 8. Cohn, E.J., Gurd, F.R.N., Surgenor, D.M., Barnes, B.A., Brown, R.K., Derowaux, G., Gillespie, J.M., Kahnt, F.W., Lever, W.F., Lui, C.H., Mittleman, D., Mouton, R.F., Schmid, K., and Uroraa, E.J., J. Am. Chem. Soc., 72, U6£, (1950). 9* Cohn, E.J., ^Interactions of Proteins with Each Other1 1 , in Blood Cells and Plasma Proteins, Academic Press, New York, New York, (1953). 10. DuVigneaud, V., nStudies on the Hormones of the Posterior Pituitary” in Chemical Specificity in Biological Interactions, Vol. II of the Memoirs of the University Laboratory of Physical Chemistry Related to Medicine and Public Health, Harvard University, 1952, in preparation. 11. Ferry, R .M ., Cohn, E.J., and Newman, E.S., J. Am. Chem. Soc. 58, 2370, (1936). 12. Ferry, R.M., Cohn, E . J . , and Neuman, E .S ., J. Am. Chem. Soc. 60, 11*80, (1938). 67 13* Gjessing, E.C., Ludewig, S., and Chanutin, A., J. Biol* Chem. 170, 55, (19l*7). Ill* Gjessing, E.C., and Chanutin, A., Arch, of Biochem* 27, 191, (1950)* 15. Gurd, F.R.N.. and Goodman, D*S., J. Am. Chem. Soc. 7h, 670, (1952). 16. Gurd, F.R*N*, and Murray, G.R., Jr., J. Am. Chem. Soc. 76, 187 (195U)• 17. Hardy, W.B., and Gardner, S., J. Physiol*, 1*0. X7III, (1910)* 18. Hughes, W.L., Jr., J. Am. Chem. Soc., 69, 1836, (I9i*7). 19* Hughes, W.L., Jr., Cold Spring Ha it or Symposia Quant* Biol* U, 79, (191*9). 20* Hughes, W*L«, Jr., Personal Communication* 21* In General, Papers in J. Clin* Invest, 23» 1*17 to 606, (191*1*). 22* In General, Papers in J. din. Invest, 21*, 657, 662, 671, 698, 701*, 793, 802, (191*5). 23. Keltz, A., "The Isolation of Rat Plasma Proteins1 1 , Masters Thesis, University of Southern California, 1952* 2l*. Keltz, A., and Mehl, J. W., Federation Proc. 12, 2l*l*, (1953). 25. Keltz, A., and Mehl, J.W., J. Am* Chem* Soc., In Press. 26. Keltz, A., and Knowles, R.G., Unpublished Results* 27. Klotz, J.M., "Proteins Interactions" in "The Proteins", Vol* I part B, Edited by H. Neurath and K. Bailey, Academic Press, New York, N.Y., 1953. 28* Li, C.H., and Tarver, H., Personal Communication* 29. Lundgren, H.P., and Ward, W.H*, "Molecular Size of Proteins" in Amino Acids and Proteins, Edited by D.M* Greenberg, Charles C* Thomas, Springfield, 111*, 1951. 30. McFarlane, A*S., Biochem. J., 29, 1*07, 660, (1953). 68 31* Mehl, J.W., Humphrey, J*, and Winaler, R.J., Proc. Soc. Exptl. Biol, and Med. 72, 106, (I9i*9). 32. Mellariby, J., Proc. Royal Soc., London, B 80, 399, (1908). 33* Gncley, J.L., Scatchard, G., and Brown, A., J. Phys. and Colloid Chem. 51, 181+, (19U7). 3k♦ Pederson, K.O., Ultracentrifugal Studies on Serums and Serum Fractions,Uppsala, 178 pp., (19U5). 35* Perrin, F., J. Phys. Radium, 1, (1936). 36. Pickels, E.G., and Beams, J.W., Science 81, 3k2, (1935)* 37• Roberts, S., Personal Communication. 38. Schmid, K., J. Am. Chem* Soc. 75* 60, (1953)* 39. Svedberg, T.O., and Pederson, K.O., The Ultracentrifuge, Oxford* The Clarendon Press, (191*6). U0. Wu, H., Chinese J. Physiol., 7, 125, (1933). baivertitir or Cstttonb UMI Number: DP21562 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. Dissertation Publishing UMI DP21562 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

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Asset Metadata

Creator Keltz, Alan (author)

Core Title The isolation and characterization of rat plasma albumins

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1954-08

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-1601

Unique identifier UC11348043

Identifier DP21562.pdf (filename),usctheses-c17-1601 (legacy record id)

Legacy Identifier DP21562.pdf

Dmrecord 1601

Document Type Dissertation

Rights Keltz, Alan

Type texts

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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