University of Southern California Dissertations and Theses

Studies of human ɑ₂ macroglobin: physical and enzyme binding properties

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Studies of human ɑ₂ macroglobin: physical and enzyme binding properties

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Content STUDIES OF HUMAN MACROGLOBULIN: PHYSICAL AND ENZYME BINDING PROPERTIES by Russell Lee Saunders 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) August 1970 UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA S 0 0 0 7 This dissertation, written by .............. JRus.as.n..Xe.e...&auMer.s................. under the direction of h.I s . . . Dissertation Com mittee, and approved by all its members, has been presented to and accepted by The Gradu ate School, in partial fulfillment of require ments of the degree of D O C T O R OF P H IL O S O P H Y ...... Tf Dm * D ate Augustl97p DISSERTATION COMMITTEE ACKNOWLEDGEMENTS Day to day discussions concerning not only this dissertation but the fields of protein and iramunocheraistry is an example of the personal attention that I was so fortunate to receive. For this I am greatly indebted to Dr. Wilton E. Vannier, my advisor. I also wish to thank Dr. Samuel Allerton and Dr. Thomas Fife for their cooperation and assistance as members of my committee. Much assistance and a great in sight into the problems of my dissertation were supplied by Dr. Bernard Haverback and Barbara Dyce; and guidance and support was given to me in the early part of my gradu ate work by Dr. John Mehl, my first advisor. Acknowledgement is made to the Department of Bio chemistry, University of Southern California, for support from a U. S. Public Health Service Training Grant for four years. Special thanks goes to Mr. A1 Bender and Mr. Nicholas Saavedra for their excellent technical assistance. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................. ii LIST OF TABLES................................ vi LIST OF ILLUSTRATIONS............................ vli LIST OF ABBREVIATIONS AND SYMBOLS............... ix Chapter I. INTRODUCTION AND HISTORICAL BACKGROUND.......................... 1 II. EXPERIMENTAL PLAN........... 14 III. MATERIALS AND METHODS................ 19 Gel Filtration Enzyme Assays Standardization of Enzymes Trypsin Assay Chymotrypsin Assay Assay for Trypsin Binding Protein Amidase Activity Activity Units pH Stat Experiments Rates of a2 M Trypsin Complex Formation Enzyme Binding Studies Using Gel Filtration Iodinated Enzymes Enzymatic Activity Maximum Binding Studies Competition Experiments Centrifugation Methods Preparative Ultracentrifugation Sedimentation Velocity Ultracentri- figation Sedimentation Equilibrium Ultra centrifugation iii TABLE OF CONTENTS (Continued) Chapter Page III. MATERIALS AND METHODS (Continued) Electrophoretic Methods Analytical Acrylamide Gel Electrophoresis; pH 8.9 Analytical Acrylamide Gel Electrophoresis; pH 7.8 Preparative Acrylamide Gel Electrophoresis; pH 7.8 Immunological Methods Immunoelectrophoresis Immunodiffusion Antisera IV. RESULTS................................ 33 Purification of a, H from Plasma Purification of <x2 M from Serum Sedimentation Velocity Ultracentri fugation Analytical Acrylamide Gel Electro phoresis Serum and Plasma dp M a.? M Trypsin Complexes Effect of Methylamine, Chymotrypsin and Trypsinogen Sedimentation Equilibrium Ultracentri fugation a2 M from Serum and Plasma and io H Trypsin Complex Immunological Studies Immunoelectrophoresis of Purified a2 M from Plasma and Serum Immunodiffusion Studies of a2 M Trypsin Complex Preparative Acrylamide Gel Electro phoresis Sedimentation Equilibrium Studies of Fractions from Preparative Gel Electro phoresis iv TABLE OF CONTENTS (Continued) Chapter Page IV. RESULTS (Continued) Immunological Studies of Preparative Gel Fractions Immunodiffusion Analysis Immunoelectrophoresis Summary of Trypsin Binding and Amidase Activity Trypsin and Chymotrypsin Complexes with a2 M Investigation of Catalytic Properties of Chymotrypsin and Trypsin Complexed with a2 M Binding Ratio of Chymotrypsin to a2 M Determined by Titration Binaing Ratios Determined Using Gel Filtration, Absorbancy and Enzyme Activity Binding Ratios Determined Using Gel Filtration, 131i Labeled Enzymes and Enzyme Activity Competition Between Chymotrypsin and Trypsin; Time of Addition Effect Trypsin Exchange Experiment V. DISCUSSION...............................80 VI. SUMMARY................................101 APPENDIX....................................103 LIST OF REFERENCES..........................113 v LIST OF TABLES Table Page 1. Summary of Molecular Weight Determinations of a2 M from Gel Filtration Fractions. • *+6 2. Molecular Weight Determinations of Proteins from Preparative Gel Fractions......................... 57 3. Summary of a2 Macroglobulin Trypsin Binding Data....................... 62 4. Summary of Amidase Activity........... 63 5. Binding Ratios of a, M Chymotrypsin Complexes Determined by Gel Filtration, Absorbancy and Enzyme Activity ........ 69 6. Maximum Binding of Trypsin and Chymo trypsin to a2 M ................... 71 7. Binding of Trypsin and Chymotrypsin When Added Simultaneously to <»2 M. . . • 73 8. Binding of Trypsin to an <x2 M Chymo trypsin Complex and Chymotrypsin to an a2 M Trypsin Complex When Complexes Formed with Excess Enzyme........... 7^ 9. Binding of Trypsin to a2 M Chymotrypsin Complex and Chymotrypsin to a2 M Trypsin Complex When Complexes Formed with Equimolar Amounts of Enzyme .... 76 10. Binding of Trypsin to an a2 M Trypsin Complex Formed by Incubation with Equimolar Amounts of Trypsin .......... 77 11. Exchange of Labeled Trypsin with Unlabeled «2 M Trypsin Complex............... 79 vi LIST OF ILLUSTRATIONS Figure Page 1. Elution Diagrams for Purification of &2 M from Plasma........................ 34 2. Elution Diagrams for Fractionation of <*2 M from Serum......................... 36 3. Sedimentation Velocity Schlieren Patterns for Plasma a2 M ............. 39 4. Analytical Acrylamide Gel Electrophoresis Patterns for pH 8.9 Gels............. 41 5. Analytical Acrylamide Gel Electrophoresis Patterns for pH 7.8 Gels............. 42 p 6. Plot of In y. vs. r. for Leading Edge, Middle, ana Trailing Edge of Bio-Gel A5m a2 M P e a k ........................ 44 7. Plot of M app. for a, H vs. Protein Concentration ........................ 45 8. Immunoelectrophoresis of a M from the Second Bio-Gel A5m Fractionation of Plasma................................ 48 9. Reaction of Rabbit Anti-Trypsin (x-Trypsin) with an ag M Trypsin Complex......... 49 10. Preparative Acrylamide Gel Electro phoresis in pH 7.8 Gels of A, Plasma <»2 M; B, a2 M and a2 M Trypsin Complex and C, Serum a 2 M .................... 51 11. Analytical Acrylamide Gel Electrophoresis of Fractions Obtained by Preparative Gel Electrophoresis of A, Plasma a2 M and B, a2 M and a 2 M Trypsin Complex . . 53 vii Figure Page 12. Analytical Acrylamide Gel Electrophoresis at pH 7.8 of Serum a _ M Preparative Gel Fractions .....................5^ 13. Diagrams of Analytical Gel Staining Patterns from Figure 1 3 ...................55 14. Plot of In yA vs. r 2 for Plasma a2 M from Preparative Gel Fraction 4 .......... 56 15. Ouchterlony Immunodiffusion Plates Comparing Antigenic Properties of Serum a? M Preparative Gel Fractions 2 to 6 ...................................59 16. Immunoelectrophoresis of Fraction 4 Obtained by Preparative Gel Electro phoresis of Serum a2 M ...................61 17. A, Hydrolysis of BAPNA Catalyzed by Trypsin and Trypsin a9 M Complex; B, Hydrolysis of GPNA Catalyzed by Chymotrypsin and Chymotrypsin a2 M Complex...................................66 18. Titration of Chymotrypsin witha2 M . . . . 67 viii LIST OF ABBREVIATIONS AND SYMBOLS BAPNA STI TAME Amidase a2 M AA/hr/ml A280 GPNA - benzoyl-DL-arginine p-nitroanilide - soybean trypsin inhibitor - p-toluenesulfonyl-L-arginine methyl ester - Refers to physiological enzyme bound to o>2 macroglobulin that catalyzes BAPNA hydrolysis - a2 macroglobulin - Change in absorbancy at 410 m/i due to BAPNA hydrolysis catalyzed by amidase - Absorbancy at 280 mit - glutaryl-L-phenylalanine p-nitroanilide; a chymotrypsin substrate - Fringe displacement from base line in sedi mentation equilibrium experiments - Distance in cell from center of rotor in sedimentation equilibrium experiments ix CHAPTER I INTRODUCTION AND HISTORICAL BACKGROUND Purification of a» Macroglobulin (a2 M) A new method was needed for the purification of M that retained all the trypsin binding activity. Methods in the past were not designed to maintain near physiologi cal conditions during the a2 M purification. Pure a2 macroglobulin ( a2 M) was prepared by Schultz, et al. (26) by ammonium sulfate fractionation of Cohn III-O fraction and found to be pure by immunological and ultracentrifugal analysis; having a value for S° 20 of 19S. An extension of this method was used by Rasafimahaleo and Bourrillon (20) employing ammonium sulfate fractionation, preparative starch gel electrophoresis and Sephadex G-200 gel filtra tion. Dunn and Spiro (3) in order to prepare a highly pur ified a2 M for amino acid analysis used a Cohn III-O powder which they subjected to ultracentrifugation (to remove low density lipoproteins), ammonium sulfate fractionation, and repeated gel filtration with Sephadex G-200. The material purified by these workers had a value for S° 2Q of 20.6 S 1 2 with a 6% contamination by a 30.8 S component. Methods employing Cohn III-O fraction but not relying upon ammonium sulfate fractionation include a procedure by Brown and workers (1) which consists of repeated ultracen trifugation at several densities. A modification of this method by Mehl, et al. (18) depends on centrifugation of dialyzed Cohn III-O fraction and gel filtration of the pellet material on Sephadex G-200. Similar procedures (19,14) involve the repeated zone centrifugation of Cohn III-O fraction in a sucrose density gradient. A prepara tive method which yields material of high purity was de scribed by Mehl and Howard (10). Cohn III-O fraction was dialyzed and low density lipoprotein removed by centrifuga tion in a high density solvent. This was followed by fractional precipitation by polyethylene glycol, column chromotography on DEAE cellulose, fractional precipitation at low ionic strength and pH and adsorption of inactive protein on betonite. These authors found a2 M to have an sw,20 value 17*0 and using a diffusion coefficient de termined by height-area analysis of the schlieren pattern at one concentration estimated the molecular weight to be 820,000. This value has been found by other workers (25)* The desirability of purifying a2 macroglobulin for determination of proteolytic enzyme binding properties has led to methods which involve mild conditions during the 3 fractionation of the protein. Whole plasma or serum is used as the starting material rather than the alcohol frac tionated Cohn III-O material. Crude preparations of a 2 macroglobulin in which the macroglobulin is separated from low molecular weight proteins (including the low molecular weight trypsin inhibitors) have been obtained by Ganrot (7) and James (14). A method which enables one to obtain a2 macroglobulin of high purity from serum or plasma was developed by Knight and Dray (15) using rabbit serum. Serum was centrifuged in KBr-NaCl to remove low density lipoproteins and pelleted material was fractionated on Sephadex G-200. Three peaks were observed with a2 macro globulin occuring in the first peak. This material was dialyzed at pH 5.1 and after a precipitate had formed the supernatant was chromatographed on DEA£ cellulose with the <Xg macroglobulin emerging essentially unadsorbed. Pooled materials from acrylamide gel disc electrophoresis experi ments were used to obtain rabbit a2 M of high purity. The sedimentation coefficient S2Q w of this material was found to be 18.6 S. (at a concentration of 2 mg/ml). By repeated ultracentrifugation into high density salt solutions and dialysis of solutions of varying pH Fillitti-Wurmser was able to obtain a high molecular weight protein which migrated electrophoretically in the a2 globulin region on starch block electrophoresis and gave a single 19 S peak upon sedimentation velocity ultra centrifugation. The pH of the salt solutions ranged from 6.2 to 7.3. The lower pH precipitated high molecular weight a and 0 globulins. All of the above methods have one point during the purification of a2 M that may not be favorable for its trypsin binding activity; such as the low pH (pH 5.0) required for ion exchange chromatography, the ammonium ion in ammonium sulfate fractionation (which is known to de stroy trypsin binding activity) and ethanol in Cohn III-O fractions. The methods used for this research involved gel filtration in 0.1 M Tris-HCl buffer at pH 7.6-7.7 and preparative acrylamide gel electrophoresis at pH 7*8. These are conditions that favor the preservation of high trypsin binding activity of a2 macroglobulins. In addition to the more gentle conditions for isolation the high pur ity that can be achieved with acrylamide gel electrophore sis should permit determination of a more accurate molecu lar weight by sedimentation equilibrium ultracentrifuga tion. Enzyme Binding Properties Haverback and Dyce (9) found a serum protein migrat ing in the a2 macroglobulin region that combined with trypsin. The enzymatic activity towards Benzoyl-DL- arginine p-nitroanilide (BAPNA) was retained; however, the trypsin was no longer inhibited by soybean trypsin inhibit or. Mehl, et al. (1?) purified (X£ macroglobulin from Cohn III-O fraction and found the trypsin binding properties described by Haverback. The <Xg macroblobulin purified by Howard and Mehl (10) had a trypsin binding capacity of 50Ji g of trypsin per mg of a2 macroglobulin. The concen trations of a2 M were determined by absorbancy measurements at 280 m# and a value for E^q of 8.70. BAPNA hydrolysis rates were used for determination of bound trypsin. Ganrot (8) performed binding studies using ‘ L^1I label ed trypsin and a radial diffusion immunoassay for a2 macro globulin concentrations. The antisera employed were ob tained by immunization with a highly purified macro globulin isolated by ammonium sulfate fractionation. Using this method he found binding ratios similar to those of Howard and Mehl. Ganrot (7) and James (14) found that when enough trypsin was added to serum to complex with all the a2 macroglobulin, the enzyme activity toward BAPNA did not markedly decrease. Additional amounts of added enzyme lost activity owing to the inhibition by a1 trypsin inhib itor. These experiments led the authors to believe that (Xg macroglobulin had a higher affinity for trypsin them trypsin inhibitor which is known to totally inhibit trypsin 6 activity towards BAPNA. These authors also purified a2 M but the enzyme binding capacity was estimated to be only 7 »5m g of trypsin per mg a2 M. Other proteolytic enzymes are known to bind to <*2 macroglobulin. Haverback and Dyce (9) found that a-chymo- trypsin activity was also found in the a2 macroglobulin region when chymotrypsin and serum were mixed and subjected to electrophoresis in starch gel. Several workers (13,19) reported a2 macroglobulin fractions that were able to in hibit plasmin. Steinbuch, et. al.. (28) reported that when a2 macroglobulin combines with plasmin the fibrinolytic activity was inhibited but esterase activity was little affected. Ganrot (8) found that plasmin was bound to the a2 macroglobulin fractions after Sephadex G-200 gel filtra tion of a mixture of a2 M and plasmin and that this enzyme competed with trypsin for sites on macroglobulin. Lanchantin and co-workers (16) found that their a2 macroglobulin preparations combined with thrombin in rough ly the same molar ratio as trypsin suggesting that the same binding sites on a2 macroglobulin might be involved. In addition, they found the esterase activity of a2 macro globulin-bound thrombin was essentially unchanged but con version of fibrinogen to fibrin was inhibited. Szceklik (29) reported that on starch block electro phoresis of serum the natural enzyme activity as measured 7 by hydrolysis of CBz diglycyl-L-arginine-2-napthyl amide was found in the a2 macroglobulin and lipoprotein re gions. The enzymatic activity in the macroglobulin re gion decreased with increasing time of contact between the serum and the clot. There was no such effect on the Bj lipoprotein esterase. For this reason it was assumed that the enzyme bound to a2 macroglobulin in serum was thrombin and that it was released or inhibited by the clot during clot formation. The author believed this phenomenon was the reason for the discrepancies in levels of natural ser um amidases found by various workers. Mehl, et al. (18) found that macroglobulin isolat ed from Cohn III-O fraction by DEAE cellulose chromato graphy had esterase activity as measured by the substrate tosyl arginine methyl ester (TAME). When this material was subjected to dialysis at low ionic strength a precipi tate formed. Haverback and Dyce (4) found that this ma terial had relatively higher amidase and esterase activity than the supernatant protein, and this natural enzyme ac tivity was not inhibited by STI. There has been much speculation as to the nature of this bound enzyme. The possibility that it may be throm bin or plasmin (29) has been discussed. Haverback and Dyce (4) observed that natural enzyme bound to a2 H had kallikrein activity (conversion of kininogen to kinin) and 8 this activity appeared to be inhibited by STI and the kallekrein inhibitors. The ability of a2 M to bind trypsin was found by Mehl, et al. to be destroyed by 0.2 M ammonium ion (17) and they suggested that a2 M prepared by ammonium sulfate fractionation was unsatisfactory if binding activity was to be maintained. The trypsin binding activity was not restored by dialysis. Other workers (28) found that tryp sin, thrombin, and plasmin inhibition by a2 M was destroy ed by 0.25 M methylamine or hydrazine. These authors claimed that 70-80# of the activity was recovered when the methylamine was dialyzed away. M of high purity was employed in our research so that absorbancy values of a2 m could be used to calculate the binding ratios. It was believed that competition be tween two different enzymes might help elucidate the na ture of the binding mechanism. Trypsin and chymotrypsin were the enzymes of choice since these enzymes were read ily available in high purity and more was known about their mechanisms of catalysis than was known for plasmin or thrombin. The binding ratio of chymotrypsin had to be determined before competition experiments were initiated. Ganrot (8) labeled only one enzyme, trypsin, in his exper iments with trypsin and plasmin. In our experiments, both enzymes were labeled to more accurately measure competi- 9 tive binding ratios. In addition, the affect of the order of enzyme addition was determined. Electrophoretic Properties Changes in the starch gel electrophoretic patterns were observed by James and co-workers (14) when trypsin was added to a2 macroglobulin. A splitting of the a2 macroglobulin band was observed on the addition of 200MS of trypsin (200^ g) to 2 mg of M and one of the bands disappeared when greater amounts of enzyme were added. Steinbuch (28) indicated that whereas purified a2 macro globulin shows the characteristic narrow band of slow a2 globulin in starch gel electrophoresis, methylamine treat ed material reveals a fringe at the anodic side of the original band or splitting to form two closely associated bands. After dialysis this pattern remains unchanged al though, as previously mentioned, the binding activity was reported to be restored. This electrophoretic alteration was not seen on cellulose acetate or agar. A change in the electrophoretic pattern resulting in a faster moving band also occurs on the binding of plasmin to a2 macro globulin (28). Howard and Mehl (10) found that fractions of macro globulin enriched in the faster sedimenting 19 S esterase produced by low ionic strength precipitation had a higher 10 concentration of the faster moving material observed on starch gel electrophoresis. Although it was not discussed, Dunn and Spiro presented photographs showing that more fast band material appeared after the ammonium sulfate fractionation step. The electrophoretic characteristics appear not to be governed by the carbohydrate moiety since Ganrot (6) found that <*2 M treated with neuraminidase showed no change in electrophoretic properties. Howard (10) discussed the possibility that forms of M differing somewhat in chemical composition (amino acid, carbohydrate or both) were present originally in plasma or that partial degradation could occur during puri fication giving rise to the appearance of more than one species on electrophoretic analysis. Acrylamide gel electrophoresis affords better electro phoretic resolution of protein species than starch gel electrophoresis. Using this method, we hoped to investi gate in more detail changes in the electrophoretic proper ties of a2 M. In addition, different pH conditions were expected to give even better resolution of the bands. The effect of not only trypsin but chymotrypsin on the electro phoretic properties of a2 M were investigated. Further more, titration of a2 M with the enzymes was performed to determine if the electrophoretic changes were related stoichiometrically to the amount of added enzyme. 11 It remained to be determined whether or not the fast band had bound trypsin and whether or not there was a change In the a2 M molecular weight or structure on enzyme binding. Since a2 M has a natural enzyme "amidase", it was believed that the slow band may represent binding pro tein and the fast band the a2 M-amidase complex. Prepara tive acrylamide gel electrophoresis should separate the binding protein from the a2 M with enzyme activity. This assumes that the natural enzyme binds by the same mechan ism as trypsin and forms a faster moving a2 M complex. Preparative electrophoresis, in addition, yields a more pure protein. This allows the determination of a more ac curate molecular weight and measurement of the maximum binding ratio. Immunological Properties With their highly purified a2 macroglobulin prepared by ammonium sulfate fractionation, Schultze (27), et al. prepared antibody to a£ macroglobulin which they made com mercially available. It is this ammonium sulfate fraction ated material that most commercial firms use to make anti body to a2 macroglobulin. Ganrot (6) noticed that different components of macroglobulin appeared on starch gel electrophoresis but found these materials to have identical specificity toward 12 antibody to Schultze's highly purified a2 macroglobulin. The author implied that a2 macroglobulin may be homogene ous immunologically but heterogeneous in electrophoretic, sedimentation, and enzyme binding properties. Dunn and Spiro (3) prepared a2 macroglobulin from Cohn III-O fraction by ammonium sulfate precipitation and density gradient ultracentrifugation. This material show ed two arcs on Immunoelectrophoresis with anti-whole human serum. After repeated gel filtration on Sephadex G-200 the slower moving arc was eliminated. James (14) performed Immunoelectrophoresis of a2 macroglobulin complexed with trypsin and reported that the a2 macroglobulin component was still present but with a slightly increased mobility. Steinbuch (28) found that when a2 macroglobulin was allowed to react with methyl amine a slight distortion of the precipitin line occured on Immunoelectrophoresis but there was no observable change in mobility of the a2 M. Rabbit a2 macroglobulin was prepared by Knight and Dray by gel filtration and preparative acrylamide gel elec trophoresis and found to be highly purified by analysis on analytical acrylamide gel electrophoresis. Antibody pro duced when goats were immunized with this protein gave two arcs when tested by immunoelectrophoresis with whole normal rabbit serum. The faster less dense arc was attributed to 13 an macroglobulin while the slower more intense line was due to a2 macroglobulin. These workers detected two a2 macroglobulin allotypes by immunizing rabbits with a crude a2 macroglobulin pool from rabbit serum and found by double diffusion analysis that antisera with two different specificities were pro duced. We performed experiments to determine whether the slow or fast a2 M had different antigenic properties. There was the possibility that the two species observed on starch gel electrophoresis represent two distinctly differ ent proteins. CHAPTER II EXPERIMENTAL PLAN macroglobulin has been prepared by ammonium sul fate fractionation by various workers and was found to have different sedimentation coefficients depending on the methods used. Preparations of macroglobulin from Cohn III-O fraction showed a variation in S values as well as enzyme binding properties. The only method mentioned in the introduction giving a product of high purity that did not involve the purification of a macroglobulin by am- monium sulfate fractionation or from a Cohn fraction was the preparation of a2 macroglobulin from rabbit serum by Knight and Dray (15). In all previously reported methods there was some step in the purification procedure where the conditions used might not be suitable for maintaining the enzyme bind ing activity of a2 macroglobulin. This activity might be sensitive to changes in ionic strength, pH, and the polar ity of the solvent. Ammonium ion destroys trypsin binding activity. Cohn fractionation requires ethanol precipitation and in some 14 15 cases the protein was lyophillized, frozen or DEAE cellu lose chromatography performed in pH 5.1 buffers. A pH of 5.1 is far removed from pH 7.0-8.0 where maximum enzyme binding activity occured and approaches a pH range where dissociation might be observed. From the variation in the sedimentation coefficients and the trypsin binding properties of a2 macroglobulins prepared by various workers it appeared that this molecule was sensitive to the conditions used for purification. For this reason a new purification method was designed that would isolate a2 macroglobulin under conditions of ionic strength, pH, and polarity of solvent (and in some cases concentration) that approximated physiological con ditions. The method involved ultracentrifugation into a concentrated sucrose solution as a preliminary step for large scale preparative runs. Bio-Gel P-300 and Bio-Gel A5m gel filtration was used for both preparative and small scale purification procedures. Preparative acrylamide gel electrophoresis was performed as a final step in the puri fication. The physical properties of the purified protein were studied by sedimentation velocity ultracentrifugation and sedimentation equilibrium ultracentrifugation in order to assess homogeneity, sedimentation coefficients, and appar ent weight average molecular weights. The immunological 16 properties of the a2 macroglobulins were also studied. Trypsin binding properties have been measured by other workers with variable results depending on the a2 macroglobulin preparation used in the studies. The most 131 accurate experiments were performed by Ganrot using J I labeled trypsin to determine an enzyme binding capacity of a2 macroglobulin; however, an immunoassay was used to de termine the a2 macroglobulin concentration. Immunoassay methods for a2 M concentration measurements do not achieve the accuracy required for determining enzyme binding ratios. Since we obtained a highly purified protein, the a- mount of a2 macroglobulin could be determined by a spec- trophotometric measurement based on its extinction coef ficient. It is believed that these measurements give a more accurate measurement of the a2 macroglobulin concen tration than the immunological assay. Experiments to study the competition between two pro teolytic enzymes for the enzyme binding sites on <*2 macro globulin were performed by Ganrot (8). These studies in volved only kinetic measurements to determine the enzyme concentrations. The enzymes selected were plasmin and trypsin; however, the maximum binding of plasmin was not determined separately. An objective of this thesis was to study competition between two proteolytic enzymes when the 17 maximum binding capacity of a2 macroglobulin was known for both enzymes. Labeled trypsin and chymotrypsin were used to determine the binding capacity for each enzyme alone. Then competition experiments were performed in which one enzyme was labeled and the effect of the presence of the other enzyme determined. In all cases enzyme binding was measured by enzyme assay and compared, with the data from labeling studies. When a highly purified a2 macroglobulin prepared from Cohn III-O fraction (10) was subjected to disc electro phoresis by the Davis and Ornstein method (2) two bands appeared, A modification of the electrophoretic properties was observed (two bands merging into one) when stoichio metric amounts of trypsin were added to a M. For this reason a thorough study of the electrophoretic properties of a2 macroglobulin and a2 macroglobulin trypsin complexes was initiated. These experiments were necessary to see if all the species observed on analytical acrylamide gel elec trophoresis were involved in enzyme binding. The results of such experiments, it was believed, would add much to the interpretation of the direct enzyme binding experi ments. In addition, when a pH 7*8 system was used rather them the pH 8.9 gels of Davis and Ornstein, five bemds ap peared when electrophoresis of purified <*2 M was performed. 18 The questions which arose with this observation were the following: 1) Did all the bands represent a2 M? 2) Did they all have the same molecular weight? 3) Were the dif ferent mobilities due to charge variations as a result of different amino acid composition or a conformational change that resulted in different amino acids or sialic acid becoming exposed on the surface of the molecule? 4) Did all species bind trypsin and chymotrypsin? Did some bind one enzyme and not the other? 5) Could amidase ac tivity be found associated with all or only some of the bands? Did those which have amidase activity fail to bind trypsin and vice versa? 6) How did the binding of trypsin and chymotrypsin affect the five bands? 7) How did methyl- amine effect the bands? 8) How did increasing amounts of trypsin up to approximately equimolar amounts affect the bands? 9) Did the bands represent proteins with different antigenic characteristics? To answer many of these questions, preparative acryl- amide gel electrophoresis was performed in an attempt to separate the different a2 M electrophoretic species. Com parisons of the bands were made with regard to trypsin binding, amidase activity, antigenic properties and molecu lar weight. Analytical gels were used to determine the ef fect of trypsin, chymotrypsin and methylamine. CHAPTER III MATERIALS AND METHODS Gel Filtration Bio-Gel P-300, 100-200 mesh, a polyacrylamide gel with an exclusion limit of 300,000 molecular weight was used in a column of 120 cm length and 2.5 cm diameter, containing a volume of 580 cc of gel. The flow rates ranged from *4-8 ml/hr with the fractions being collected every 30 minutes. Bio-Gel A5m (agarose) with an exclusion limit of 5 x 10^ molecular weight was employed in a column similar to the Bio-Gel P-300 column described above and under sim ilar conditions. Sephadex G-100, equilibrated with the buffer used for enzyme assays (0.05 M Tris-HCl-0.03 M CaClg), was used for binding experiments in a column of 90 cm length, 1.5 cm diameter and having a gel volume of 150 cc. Enzyme Assays Standardization of Enzymes Worthington 3 x crystallized bovine trypsin was used. 19 20 Trypsin concentrations were determined by the weight of 1# enzyme used or spectrophotometrically using an E2qq of 15.2 (10) to convert absorbancy at 280 mjt to mg/ml. Ac tive trypsin was determined by titration with STI which can be obtained as a more uniform stable crystalline pro tein and the trypsin found to be 80# active. The concentration of Worthington 3 x crystallized bovine a-chymotrypsin was measured by weight and for more 1 < j L accurate assays, spectrophotometrically using an E ^ q q value of 20.3 (2k). Active enzyme was determined by the method of Bender, et al. (24) which relies upon a spectral change due to reaction of cinnamoyl imidazole with the reactive site of chymotrypsin. By this method the chymotrypsin was found to be 80# active. Trypsin Assay Benzoyl-DL-arginine p-nitroanilide (BAPNA) (Mann Re search Laboratories) was the substrate selected for trypsin (17). The trypsin activity was measured by the rate of re lease over a period of 30 minutes of p-nitroaniline which could be followed spectrophotometrically at 410 npc. A stock solution of 1 mg/ml (2.5 x 10~^ M) BAPNA was prepared by dissolving the solid in distilled water by heating for 15 minutes at 70°C. The reagent was then cooled and filter ed. In the assay 1.0 ml of the BAPNA stock solution was 21 used with 3»0 ml of enzyme and assay buffer (0.05 M Tris- HC1-0.03 M CaClg, pH 7.65) to make the final reaction vol ume 4.0 ml. The stock trypsin solution consisted of a 1 mg/ml trypsin solution in 0.001 N HC1. This was diluted 1-20 with assay buffer and 0.3 ml (15/* g) of enzyme was used in the assay. Trypsin complexed to a2 macroglobulin was assayed by this method. The points determined for rate plots are averages of 5 runs. Chymotrypsin Assay The substrate used for chymotrypsin was glutaryl-L- phenylalanine p-nitroanilide (GPNA) (Mann Research Labora tories). Chymotrypsin activity was measured by the rate of release of p-nitroaniline as determined by the absorb- ancy change observed at 410 m/e . A stock solution of 6.7 mg/ml was prepared by dissolving 40 mg of GPNA in two ml of methanol and stirred slowly into assay buffer and made up to 50 ml. Stock a-chymotrypsin was prepared by dissolving a- chymotrypsin in 0.001 M HC1 to give a concentration of 1 mg/ml, and 0.27 ml (270> / t g) was used in the assay. One ml of GPNA solution was added to 3 ml of assay buffer con taining 270/*g of a-chymotrypsin to start the reaction. Chymotrypsin bound to < » 2 macroglobulin was assayed by this 22 method. Assay for Trypsin Binding Protein To determine the amount of trypsin binding protein in fractions from gel filtration and preparative electrophor esis experiments; 0.4 ml of solutions of the fractions; (estimated to contain not more than 600>< g of a2 M) 1.0 ml of pH 7.65 assay buffer and 0.04 ml of 1 mg/ml trypsin solution were mixed and 15 minutes allowed for complex formation between a2 macroglobulin and trypsin. To this mixture was added 0.06 ml of a 1 mg/ml solution of STI in distilled water for a 15 minute incubation period. To start the reaction 0.5 ml of BAPNA was added. The final reaction volume was 2.0 ml and the final concentrations were 0.047 M Tris, 0.015 M CaCl2 and 0.25 x 10"3 M BAPNA. The protein concentrations in the fractions varied from 50 to 800>u g. Each assay included a trypsin standard containing no sample or STI. This gave the absorbancy change at 410 nu< due to BAPNA hydrolysis by 15>u g of free trypsin. The blank contained both trypsin and STI as well as substrate and allowed correction for non-enzymatic hydrolysis of substrate. STI binds 0.8 to 1.0 moles of trypsin per mole of a2 M (10) and free trypsin was found to be totally inhib- 23 ited by this ratio of STI to trypsin in the BAPNA assay. Therefore the only trypsin activity remaining was due to trypsin which was bound to the M in the sample and thus protected from STI. To determine the activity of bound trypsin relative to free trypsin, trypsin was preincubated for 15 minutes with graduated amounts of <Xg macroglobulin before addition of substrate. Decrease in activity was proportional to the amount of macroglobulin added at low concentrations but leveled off to about 65% of the catalytic activity of free trypsin at the equivalence point. Amidase Activity To a reaction vessel, 0.5 ml of solutions of the fractions, 0.5 ml of 0.1 M pH 7*65 Tris buffer and 0.5 ml of assay buffer were mixed and 0.5 ml of BAPNA solution added to start the reaction. The final concentrations -3 were O.O63 M Tris, 0.0075 M CaClg, 0.25 x 10 M BAPNA. The amount of protein from samples varied from 50 to 3000M g. The tubes were stoppered, stored in temperature blocks at 25 or 37° C for 60 hours with readings at 410 mu . taken every 20 hours to follow the release of p-nitro- aniline. Appropriate controls were run. Activity Units In the assay to determine the amount of trypsin bind- 24 ing activity in a fraction the residual trypsin activity not inhibited by STI was measured by the hydrolsis rates of BAPNA and compared to trypsin standards. This was re corded as the/c g trypsin per ml of fraction or per mg of protein. Amidase activity was a measure of enzymes al ready present which were capable of hydrolyzing BAPNA and the activity was recorded as. absorbancy change per hour per ml due to the release of p-nitroaniline (AA/hr/ml). For specific activity, the M g trypsin/ml or a A/hr/ml is divided by the absorbancy at 280 x r j l of a sample in 1% which an ^Z80 of 10 was considered to be approximately 1 mg/ml of protein. This approximate extinction coeffici ent was only used for relative estimates of protein con centrations of gel filtration fractions and not for pre cise measurements of trypsin binding activity. pH Stat Experiments Experiments to estimate the rate of enzyme-macro- globulin complex formation were performed with p-toluene- sulfony-L-arginine methyl ester (TAME) using a pH Stat method. A radiometer automatic titrator (model TTT-1) with a recorder and motor driven syringe (0.5 ml capacity) for delivering standard base into the reaction vessel was used for these studies. The reaction volumes could be varied from 4 ml to 650 ml by using different electrodes 25 (Radiometer) and vessels. For smaller volumes (10 ml) a temperature coil was used. Larger volumes (200-650 ml) were brought to the desired reaction temperature and re quired no regulation. Large vessels consisted of wide mouth reagent bottles sealed by a rubber stopper with holes for electrodes, the syringe outlet tube, a nitrogen line and thermometer. Stirring was performed with an in sulated magnetic stirring motor. The reaction solvent was 0.04 M KC1 - 0.01 M CaClg unbuffered. The substrate was p-toluenesulfonyl-L-argin- ine methyl ester (TAME). Stock trypsin solution was a 1 mg/ml solution in 0.001 N HC1. This was diluted to lOOXg/ml with a solution of 0.04 M KC1 - 0.05 M CaClg and adjusted to pH 7.65 thirty minutes prior to use. A few seconds after the start of the reactions the titra tion plots were linear which allowed the calculation of the initial rates. The initial substrate concentrations were calculated from the weight of crystalline reagent used in making stock solutions and were occasionally checked by allowing the reaction to go to completion. A- greement was within 5#. Rates of M Trypsin Complex Formation Ten ml of reaction solvent and 0.05 ml of <Xg macro- globulin solution (1 mg/ml) in 0.005 M Tris and reaction 26 solvent were mixed and the pH adjusted to 7.65. A volume range of 0.02 - 0.05 ml of a 100>v g/ml trypsin solution was added, the pH adjusted to 7.65 and the reaction initi ated after various Incubation times by the addition of 1.0 ml of a 0.1 M TAME solution. The titrant was 0.005 N NaOH added with a syringe of 0.5 ml capacity equal to 100# on the recorder chart. Since 1 0 0 moles of TAME were added, the chart widths corresponding to 10M moles of base represented 10# of the reaction. This gave ample range for the determination of initial rates. Enzvme Binding Studies Using Gel Filtration Iodinated Enzymes For binding studies, trypsin and chymotrypsin were labeled with 1-^1Iodine by the method of Hunter and Green wood (11). For these experiments, 5 mg of crystalline en zyme was dissolved in 1 ml of 0.05 M sodium phosphate buf fer, pH 7.65, to give 5 mg/ml of protein. A volume of 131 0.1 ml was used for iodination with 1 me of ^ Iodine. The specific activity of the enzymes was approximately 1 x 10^ cpm perxrg of enzyme. For the binding studies, labeled enzyme was mixed with unlabeled enzyme (90 to 300 >wg) to give approximately 100,000 cpm. 131 Results for the J Iodine experiments are the averages of the cpm found in two peak fractions containing M from 27 three gel filtration experiments. Enzymatic Activity The assay methods described previously were used to measure chymotrypsin or trypsin bound to a2 M. The rates of BAPNA hydrolysis were corrected for the k0% inhibition of trypsin by a2 M and the rates of GPNA hydrolysis cor rected for the 30# activation of chymotrypsin by a2 M. Maximum Binding Studies Excess (300/eg) of trypsin or chymotrypsin was added to 3 mg of a2 M and free enzyme was separated from a2 M by gel filtration on a Sephadex G-100 column equilibrated with 0.05 M Tris-HCl-0.03 M CaCl2 assay buffer. The 131 amount of enzyme bound to a2 M was measured by the Io dine radioactivity or enzymatic activity. The determina tion of the amount of enzyme bound was based on averages of values from two fractions from the macroglobulin peak. Each such binding experiment was repeated three times. Competition Experiments Three types of competition experiments were performed as follows: 1) 300^ g of both enzymes were added simultaneously to 3 mg of a2 M and incubated for 15 minutes. 2) 300m g of one enzyme was incubated with 3 mg of a2 M 28 for 15 minutes before incubation with ^OO^tg of the other enzyme. 3) 90xg of one enzyme (half saturation of a2 M) was in cubated with 3 mg of a2 M for 15 minutes before in cubation with 300x g of the other enzyme. All complexes were separated from free enzyme by Sephadex G-100 gel filtration. The amount of bound enzyme 111 was determined by J Iodone activity or enzymatic activity corrected for the effect of a2 M on catalytic activity. The results were averaged as described for the maximum binding studies. Centrifugation Methods Preparative Ultracentrifugation A modification of the method of Fillitti-Wormser (5) was used to fractionate ACD plasma. A volume of 15 ml of plasma was layered on 20 ml of 20# sucrose and centrifuged for 26 hours at 27,000 rpm in a Beckman-Spinco No. 30 rotor. The pellet and 5 ml of the bottom fraction were used for gel filtration on Bio-Gel P-300. Sedimentation Velocity Ultracentrifugation Sedimentation velocity ultracentrifugation experi ments were performed in a Beckman-Spinco Model E analyti cal ultracentrifuge according to Schachman (22). The sol- 29 vent was 0.1 M Tris-HCl buffer pH 7.6. Sedimentation Equilibrium Ultracentrifugation Molecular weight determinations were made using the high speed meniscus depletion method of Yphantis (30). Fluorocarbon was not used in the cell since preliminary experiments indicated <*g M appears to dissociate in the presence of fluorocarbon. The partial specific volume, i7 , was assumed to be 0.733 as reported by Dunn and Spiro (3) and the density, S , was determined to be 1.00313 for 0.1 M Tris-HCl buffer. Speeds in rpm ranged from 8,225 to 16,200. All photographs were taken after running for 24 hours. This time was found to be sufficient to reach equilibrium. Electrophoretic Methods Analytical Acrylamlde Gel Electrophoresis: pH 8.9 The Buchler analytical acrylamlde gel electrophoresis apparatus was used with the standard tubes being replaced by ones with an internal diameter of 6 mm and a length of 7.7 cm. The method of Davis (2) was used with the stack ing gel omitted and the gel composition changed to % acrylamlde. 30 Analytical Acrylamlde Gel Electrophoresis: pH 7.8 The buffer solution consisted of 1.0 ml N,N,N',N'“ tetraethylmethylenediamine (TEMED), 150 ml 1 N HC1, 10 g Tris all adjusted to pH 7.8 with 1 N HC1. All other rea gents to make the gels were the same as with the Dayis sys tem and the electrode solutions (pH 8.3) were identical. The conditions of electrophoresis were the same with the run being continued 20 minutes beyond elution of the track ing dye. Preparative Acrylamlde Gel Electrophoresis: pH 7.8 The reagents used for the preparative gel were the same as those used for the analytical gels at pH 7.8. The electrode solutions uced were also identical. The internal diameter of the gel tube was 1-3/4 inch es and the length 3.0 inches. The gel length was 5.3 cm and it was 4.5 cm in diameter. The 5% acrylamlde gel had a volume of 80 cc with the following composition: 10 ml of pH 7.8 (Tris-HCl-TEMED) solution, 10 ml of distilled water, 20 ml of 5% acrylamlde solution and 40 ml of a 1 mg/100 ml potassium persulfate. The gel was allowed 90 minutes to polymerize and placed between two reservoirs containing the pH 8.3 elec trode solutions. Two to 10 mg of sample in 15# sucrose was layered over the gel. Electrophoresis was performed for 6 31 hours at 80 - 90 ma. The gel was sliced In 0.4 cm sections and eluted in 0.1 M Tris buffer, pH 7.65. The approximate location of protein could be detected by staining a longi tudinal section of the gel and matching it with the un stained section. Generally, the protein could be located by a faint yellow band visible 2.0 cm from the top of the gel. The species of proteins in each slice were later de termined by analytical gel electrophoresis under the same pH conditions. The absorbancy at 280 mjjl , amidase activity of each fraction were also determined. Immunological Methods Immunoelectrophoresis Immunoelectrophoresis was performed by the method of Scheidigger (23) using 1% agarose (Calbiochem) gel in bar bital buffer at pH 8.2 and an ionic strength of 0.028. Immunodiffusion Immunodiffusion was carried out according to Ouchterlony (20). The Ionagar gel (CoLab) was 0.8# in 1# saline buffered at pH 8.4 with 10# v/v borate buffer. Antisera Hyland Laboratories goat anti-whole human serum was used for the detection of impurities in (Xg M preparations and studying antigenic properties of the electrophoretic 32 species of a2 M. Antisera were prepared by injecting rabbits with 5 mg of our purified a2 M in complete Freund's adjuvant (Vitco) followed by 5 mg of a2 M in Freund's incomplete adjuvant (Difco) after two weeks, animals were bled two weeks after the last injection. The CHAPTER IV RESULTS Purification of cu M from Plasma Following centrifugation into 20% sucrose, the pellet ed protein was applied to a column of Bio-Gel P-300. The elution diagram in Figure 1A shows the separation of plasma macroglobulins from the lower molecular weight proteins. Fractions in the macroglobulin peak (2?0 ml to 3^0 ml) were pooled, concentrated to 15 ml and applied to a column of Bio-Gel A5m and eluted with 0.1 M Tris-HCl buffer at pH 7.65. The elution diagram in Figure IB illustrates the separation of the macroglobulin fraction into three peaks. Fractions high in trypsin binding activity, 290-3^0 ml, were pooled, concentrated to 15 ml and rechromatographed on the same Bio-Gel A5m column. Figure 1C illustrates an elution diagram that indicates a single protein peak with trypsin binding activity as well as natural amidase activ ity. The binding activity was located by the assay dis cussed in the methods section and employs STI as a means of inhibiting unbound enzyme. Naturally occurring bound 33 3.000 2.000 A280 1.000 0. 000 270 350 440 500 550 o o 2.000 o oo o o 1.000 0.000 £2 l . O 330 350 380 230 290 420 440 8 1. 000 0. 500 to 7 , ‘ \ \ o CM 280 0. 000 390 300 330 360 420 Eluant Volume Fig. 1.— Elution diagrams for purification of a9 M from plasma. A, Bio-Gel P-300; B, Bio-Gel A5m and c C, repeat A5m. Fractions selected for repeated gel filtration are indicated by the bracket, (| ■ ■ — I). ______ Absorbancy at 280 m , « g of trypsin bound/ml, a Amidase activity as A A/hr/ml. A A /hr/m l A A /h r/m l 35 enzyme in the fraction was measured by the rate of BAPNA hydrolysis. Purification of cto M from Serum Bio-Gel P-300 gel filtration was performed on 27 ml of human serum from a normal individual. The elution dia gram is shown in Figure 2A. Although the column was over loaded with protein, separation of macroglobulins from pro teins of lower molecular weight was achieved. Fractions from the trypsin binding peak, 200-280 ml elution volume, were pooled, concentrated, and 12 ml of the 15 ml of the concentrate was rechromatographed on Bio-Gel P-300. The elution diagram is shown in Figure 2B and illustrates a separation of trypsin binding macromolecules from lower molecular weight serum proteins. Assays were performed to measure both natural enzyme and trypsin binding activity in various fractions. Fractions in the 200-300 ml range from the second Bio-Gel P-300 chromatography were concen trated and the total amount chromatographed on a column of Bio-Gel A5m. The elution diagram is shown in Figure 2C. The macroglobulins which gave a single peak on P-300 re solve into three peaks on Bio-Gel A5m. Trypsin binding and natural enzyme activity were measured as before and the results plotted with the elution diagram in Figure 2C. It can be seen that trypsin binding activity occurred in 36 3.000 2.000 A 280 1 — H 1.000 A 0. 000 i 200 2.000 1.000 0. 000 0 300 400 500 Eluant Volume 200 300 400 Eluant Volume 0.300 0.800 0.150 0.400 • ^ 280 W 0.000 0.000 200 300 400 $00 300 400 500 I < < Eluant Volume Eluant Volume Fig* 2.— Elution diagrams for fractionation of <&2 M from human serum. A, Whole serum on Bio-Gel P-300; B, First peak fractions from A rerun on Bio-Gel P-300; C, First peak fractions from B rerun on Bio-Gel A5m and D, Second peak fractions from C rerun on Bio-Gel A5m. Fractions selected for repeated gel filtration are indica ted by the bracket, ( | ------ 1 ). ______Absorbancy 280, » M g trypsin bound/ml, A Amidase (A A/hr/ml) 37 the second peak and amidase activity in all three peaks. Turbidity in fractions from the first peak necessitated centrifugation of the assay solution when amidase activity was measured. Variation of rates with time occurred with this material and also material from the third peak so readings at 120 hours were used for the values of amidase activity only as a qualitative measurement. For a discus sion of the variation of these rates with time see Ap pendix A. Fractions in the 320-370 ml range were pooled, con centrated and rechromatographed on Bio-Gel A5m. The elu tion patterns showing a single peak with slight skewing at the heavier edge of the peak is seen in Figure 2D. Trypsin binding activity and natural amidase activity were measured as above. It was observed that trypsin binding of this material from serum had a higher specific trypsin binding activity (see Table III, page 62) them the material from plasma. Sedimentation Velocity Ultracentrifugation The purified cl^ M from plasma was characterized by sedimentation velocity ultracentrifugation. The experi ments were performed on pooled fractions in the cl^ M peaks from the second Bio-Gel P-300 and first Bio-Gel A5m gel filtration. The schlieren patterns are illustrated in J 38 Figures 3A and B. Analytical Acrylamlde Gel Electrophoresis Serum and Plasma ou M Two bands were observed when analytical acrylamlde gel electrophoresis at pH 8.9 was performed with a2 M from serum and plasma, Figure k. The slower band of a2 M from serum appeared to be more intense them the slower band of plasma a2 M. When larger amounts of a2 M were applied to the gels, traces of contaminemts appeared. The use of the pH 7*8 gel permitted better separation of the electrophoretic species of M and five bands ap peared for plasma and serum M, Figure 5* As in the pH 8.9 gels, the faster moving material was relatively more prominent in the plasma M. The serum M showed more of the slowest moving material, with four less prominent faster moving bands. a 2 w Trypsin Complexes To determine which of the five species of protein in the purified a2 macroglobulin were involved in trypsin binding, increasing amounts of trypsin were added to plasma a 2 macroglobulin preparations and analytical acrylamlde gel electrophoresis performed at pH 8.9* When increasing amounts of enzyme were added up to a 58.5ag/mg a2 M ratio, 39 Fig. 3.— Sedimentation velocity schlieren patterns for plasma a2 M. A, pooled fractions 60 to 90 (Fig. 1) from Bio-Gel P-300 gel filtration. B, fractions 160 to 180 of second Bio-Gel A5m gel filtration. Samples were run in 0.1 M Tris-HCl buffer at pH 7.65. The phase plate angle was 55°C and the photographs were taken 19 and 15 minutes after reaching full speed for A and B, respective ly. 40 the intensity of the slower band diminished. The band was nearly absent when the ratio was 29.3/ttg trypsin per mg a2 M. When l4.6>w g trypsin/mg a2 N was run all the bands had not yet been converted to a single fast compon ent. The area of the fastest species did stain more in tensely. The slowest bands still seemed to be present in the same relative amount as the intermediate bands although both were decreased considerably. This may indicate that the enzyme bound to all the slow species equally. Effect of Methylamine. Chymotrypsln and Trypsinogen The effect of 0.2 M methylamine on the <*2 M electro phoretic pattern was determined and the results in Figure 5 show the convergence of the five bands into a single fast band. This is interesting since 0.2 M methylamine is known to destroy enzyme binding activity (18). Trypsinogen when A B 12 3 4 Fig. — Acrylamide gel electrophoresis patterns for pH 8.9 gels. A, a2 M from plasma; B, a? M from sez*um; 1-^, a2 M with increasing amounts of added, trypsin; 0, 1^.6, 29.3 and 58.5Xg trypsin per mg a2 M, respectively. A B I 2 3 4 Fig. 5.— Acrylamide gel electrophoresis patterns for pH 7.8 gels. A, ou H from serum; B, a? M from plasma; 1-4, ap M from plasma, a2 M with 58.6.* g trypsin, a2 M with 5°.6>«g chymotrypsin, and a2 M with 0.2 M methyl amine, respectively. 43 added to a2 M did not alter the electrophoretic pattern. Alpha-2-macroglobulins which were found to bind one-half the amount of chymotrypsin as trypsin, were also converted to a single fast band by addition of 60m g of chymotrypsin per mg of a2 M. Sedimentation Equilibrium Ultracentrifugation a 2 M from Serum and Plasma and a. H Trypsin Complex The molecular weights as presented in Table I fall in the range of 620,000 to 680,000. A pool of a2 M from serum gave a molecular weight of 635,000 and a plasma a2 M trypsin complex a value of 630,000. 2 The In yj^ vs. r^ plots for fractions from the fast edge, middle and trailing edge of the Bio-Gel A5m column run of a2 M from plasma are given in Figure 6. The linear plot8 indicate that the preparations are fairly homogeneous by weight. To test for aggregation or dissociation which may occur when the concentration is varied, <x2 M from the sec ond Bio-Gel A5m gel filtration was run at different concen trations ranging from 0.2 mg/ml to 5 mg/ml. The plot of concentration vs. apparent molecular weight is illustrated in Figure 7 and the lack of concentration dependence indi cates that aggregation or dissociation does not occur over the concentration range studied. 2*4 2.000 1.000 0. 000 -1. 000 51.0 50.4 52.0 51.5 2 Pig. 6.— Plot of In y* vs. r. for leading edge, middle and trailing edge or Bio-Gel A5m a2 M peak. The concentrations were 0.5 mg/ml, speed-7,928 rpm and the temperature 20°C. The calculated molecular weights are 680,000, 632,000 and 629,000. #___ Leading edge r Middle A Trailing edge *5 680 660 CO I o 640 £< o. a ) 11 620 600 0 1.0 2 .0 3.0 Concentration (m g/m l) 4.0 Fig. 7.— Plot of app. for a2 M vs. protein concen tration. All runs were performed at 7,928 rpm in 0.1 M Tris-HCl buffer, pH 7.65 and at 20°C. 46 TABLE I SUMMARY OF MOLECULAR WEIGHT DETERMINATIONS OF <*« M FROM GEL FILTRATION FRACTIONS c Cone. Description of Sample mg/ml Mw app. Plasma M Pool, Bio-Gel A5ma 0.5 636,000 Leading edge, Bio-Gel A5mb 0.5 680,000 Middle, Bio-Gel A5m 0.8 632,000 Trailing edge, Bio-Gel A5m 0.6 629*000 Pool, Bio-Gel A5m 0.2 630,000 Pool, Bio-Gel A5m 0.5 628,000 Pool, Bio-Gel A5m 1.0 635,000 Pool, Bio-Gel A5m 2.2 620,000 Pool, Bio-Gel A5m 3.8 636,000 Serum M -------< C -- Pool, Bio-Gel A5m 0.5 635,000 a, M Trypsin Plasma a2 M + Trypsin 0.5 635,000 a. Pool from second Bio-Gel A5m fractionation. b. Fractions obtained during course of second Bio-Gel A5® gel filtration. 47 Immunological Studies Immunoelectrophoresis of Purified a» M from Plasma and Serum The preparations from the second Bio-Gel A5m fraction ation of plasma a2 M were analyzed by Immunoelectrophoresis with Hyland goat anti-human serum. A single precipitin arc with some splitting was observed when a concentration of 3 mg/ml was used, Figure 8B. A similar arc was seen with a2 M from serum and a2 M trypsin complex. The samples displayed a trace of a slower moving component when high concentrations (6 mg/ml) of antigen were used, Figure 8A, Immunodiffusion Studies of M Trypsin Complex When a2 M trypsin complex, prepared by gel filtration of <*2 M incubated with excess trypsin, was tested with Hyland anti-a2 M serum one precipitin line resulted on immunodiffusion as seen in Figure 9. A more crude prepara tion of «2 M (no trypsin) gave two lines when tested with the Hyland antisera. Rabbit anti-trypsin antisera formed two precipitin lines when tested with bovine trypsin. The antisera did not form a line when tested with a 2 mg/ml solution of a2 M trypsin complex. The amount of bound trypsin was 110>ug and the lowest level of sensitivity of the antisera in this test was 25>ug of trypsin. This sug gested that the anti-trypsin-trypsin interaction might be Pig. 8.— Immunoelectrophoresis of a? M from the second Bio-Gel A5m fraction of plasma. Electrophoresis was performed by the method of Scheidigger (21). The antiserum in the trough was Hyland goat anti-human serum. A, Plasma a£ M, 6 mg/ml; B, Plasma M 3 mg/ml. ^9 Fig. 9.— Reaction of rabbit anti-trypsin (x-trypsin) with an a 2 M trypsin complex. Anti-human <*2 m ( x - a M) with a2 M trypsin and crude 311(1 anti-trypsin with trypsin are also shown. 50 inhibited when the trypsin was bound to a2 M and that the trypsin may be buried inside the a2 M structure. The pos sibility that there was an antibody-antigen reaction with no resulting precipitation could not be excluded by these experiments. Preparative Acrylamide Gel Electrophoresis Preparative acrylamide gel electrophoresis was per formed ona2 H from plasma, from serum and a mixture of 9 parts of plasma H and 1 part <x2 M trypsin complex. The plots showing the absorbancy at 280 mx, the trypsin binding activity and amidase activity are illustrated in Figure 10. The plots for serum and plasma a2 M show a skewing of trypsin binding activity toward the slower fractions while the amidase activity closely follows the absorbancy plot. The results for the mixture of M and a2 M trypsin com plex show the bound trypsin in only the faster fractions. As with the serum and plasma a2 M, trypsin binding activ ity was found only in slower migrating fractions. In sum mary, when trypsin binds to a2 M a fast moving species of a2 M is formed but amidase can be present with both slow and rapidly migrating m. Analytical acrylamide gel electrophoresis was per formed on each fraction from the plasma a2 M, plasma a2 M- o oo CJ 300 200 1 0 0 000 3 4 6 0 1 2 5 7 o oo CO o 00 C O 51 20 15 1 0 5 i « Q ) W l < 5 Fractions 1 0 0 0 .4 050 0.2 0.0 000 O' 3 4 6 2 7 0 1 5 r 0 £ w ft ft T> O m Fractions 200 1 0 0 000 8 2 3 4 5 6 7 1 20 1 0 ( D U 1 ■3 Fractions Fig. 10.— Preparative acrylamide gel electrophoresis in pH 7.8 gels of A, plasma a2 M; B, a_ M trypsin complex and C, serum a2 M. a280» "■"trypsin binding activity asMg trypsin bound/ml, and — ▲ — amidase activity (AA/hr/ml). 52 a2 M-trypsin complex and serum a2 M experiments and the stained gels shown in Figures 11 and 12. A schematic presentation of these results is given in Figure 13. The gels show the separation of the slow and fast species a- chievod by preparative electrophoresis. In all cases the fractions showing the presence of slower bands were high in trypsin binding activity and had amidase activity. The fractions with fast material were low in trypsin binding activity but maintained relatively similar specific ami dase activity. Sedimentation Equilibrium Studies of Fractions from Preparative Gel Electrophoresis The studies were carried out with the fractions ob tained by preparative gel electrophoresis of plasma <*2 M, serum a2 M and the mixture of plasma a2 M and a2 M trypsin complex. In each case the various fractions were analyzed by sedimentation equilibrium ultracentrifugation. The summary of the molecular weights of the various fractions is given in Table II. When only one value is given the 2 In y^ vs. r^ plots were linear and indicate that the material is relatively homogeneous by weight. An example of such plots is given in Figure I** for fraction k of the preparative gel run of plasma M. When the fractions were not homogeneous the slope of the contaminating lighter material was also used to give an estimate of its molecular 53 12 3 4 5 Fig. 11.— Analytical acrylamide gel electrophoresis of fractions obtained by preparative gel electrophroesis of A, Plasma a» M and B, «2 M and a? M trypsin complex. Numbers 1-5 represent fractions 1-5. Fig. 12.— Analytical acrylamide gel electrophoresis at pH 7.8 of serum a£ M preparative gel fractions. A, starting material; 2-6, preparative gel fractions 2-6. Fig. 13.--Diagrams of analytical gel staining patterns from Figure 13. Illustrated for the purpose of clarifying the results shown in the photographs. The actual gels were used to identify the band numbers since the photographs do not indicate them clearly. The millimeter distances of migration can be estimated from the scale on the left. Numbers are assigned to bands in a way that each number represents a possibly different species. Vj\ V/i 56 3.000 2.000 In y. 1.000 0. 000 37. 5 37.0 36.2 36.5 2 Pig. 1**-.— Plot of In y, vs. for plasma a, M preparative gel fraction k. The speed was 7*928 rpm the temperature 20°C. The calculated was 620,000 from and r H c m rv^- r H c m m' wo « h c m rv* ^ 57 TABLE II MOLECULAR WEIGHT DETERMINATIONS OF PROTEINS FROM PREPARATIVE GEL FRACTIONS Plasma ou M Fraction No. V V1 400.000 340.000 585,000 605.000 620,000 340.000 580,000 Serum ou M 630,000 650,000 685,000 650,000 640,000 Plasma a_ M: a, M-Trypsln 300,000 595,000 620,000 630,000 200,000 560,000 58 weight. These values are listed in Table II. It can be seen that the molecular weights ranged from 605,000 to 685,000 in the homogeneous preparations. The higher val ues for the a2 M from serum are believed to be due to a better separation of the M from contamination by lower weight material. These values also better illustrate the fact that there is no appreciable molecular weight differ ence among the slow and faster moving species. immunological Studies of Preparative Gel Fractions Immunodiffusion Analysis Analysis by immunodiffusion of the various fractions of M from preparative gel electrophoresis was performed. Fractions 2-6 (see Figure 13) were placed in adjacent wells and in the center well a rabbit antiserum prepared against a crude preparation of a2 macroglobulins was used. The arcs illustrated in Figure 15 indicated that species labeled 1, 2, and 3 in Figure 13 and found in fractions 1-5 all form an arc that coalesces with all of the other spec ies. The coalescense of arcs suggests antigenic identity or only undetected minor antigenic differences among the fractions. Fraction 6 shows an extra line which is not believed to be due to an macroglobulin but a contamin ating protein. Fig. 15.--Ouchterlony immunodiffusion plates comparing antigenic properties of serum cu N preparative gel fractions 2 to 6. The vari ous fraction numbers are indicated over the wells. The center well contained anti-macroglobulin serum prepared against a P-300 macro globulin pool. The arcs appear to coalesce indicating that the species are similar immunologically. The concentrations of antigen in the well were in the range of 80 to 200>cg/ml. 60 Immunoelectrophoresis Immunoelectrophoresis was performed with fraction 4 from one of the high trypsin binding fractions the prepar ative gel electrophoresis of serum <*2 M using Hyland anti human serum and showed a single precipitin arc as illus trated in Figure 16. Summary of Trypsin Binding Activity and Amidase Activity Trypsin binding data for <*2 M from plasma or serum at different stages of purification are summarized in Table III. Binding activity increased with purification and serum <x2 M at all stages appeared to have higher bind ing activity than a2 M from plasma. The highest activity was found in fraction 3 from preparative gel electrophore sis of serum <*2 M. Amidase activity at various stages of purification indicated that the amidase activity increased with purifi cation and that M from plasma and serum had similar amidase activities. Table IV. 61 Fig. 16.— Immunoelectrophoresis of fraction 4 obtained by preparative gel electrophoresis of serum ag M. Antiserum in the trough was Hyland goat anti human serum, and the antigen concentration was 2 mg/ml. 62 TABLE III SUMMARY OF a MACROGLOBULIN TRYPSIN BINDING * DATA Description Me Trypsln/ml/A«Q0 Plasma a. M following first Bio-Gel A5m fractionation 32.8 Plasma a2 M following second Bio-Gel A5m fractionation 37.0 Plasma a2 M preparative gel electrophoresis fraction 3 57.5 Serum a2 M following first Bio-Gel P-300 fractionation 15.6 Serum a2 M following second Bio-Gel P-300 fractionation 30.0 Serum a2 m following first Bio-Gel A5m fractionation 43.0 Serum a2 M following second Bio-Gel A5m fractionation 57.8 Serum a2 M preparative gel electrophoresis fraction 3 75.0 Serum < *2 M preparative gel electrophoresis fraction k 67.7 63 TABLE IV SUMMARY OF AMIDASE ACTIVITY Description a A /hr/ml/AogQ Plasma au M following first Bio-Gel A5m fractionation 27.7 Plasma a2 M following second Bio-Gel A5m fractionation 33.8 Plasma a2 M preparative gel electrophoresis fraction 4 45.5 Serum a. m following first Bio-Gel A5m fractionation 26.7 Serum a2 M following second Bio-Gel A5m fractionation 37.2 Serum o-2 M preparative gel electrophoresis fraction 4 48.2 64 Trypsin and Chymotrypsln Complexes with M Investigation of Catalytic Properties of Chymotrypsln and Trypsin Complexed with M The rate of BAPNA hydrolysis was followed for 15>ttg w2 of trypsin and 6,3 x 10“ M BAPNA. Rates were compared to the rates found using 15>ag of trypsin that had been al lowed to react for 15 minutes with 830xg of M. This was an amount estimated to give a molar ratio of <*2 M to trypsin of 2.3 to 1 based on a molecular weight of 820,000 for the a2 M. The results, indicated an inhibition of trypsin catalysis by 35# due to complex formation with a2 M, Figure 17A. To determine the effect of a2 M on GPNA hydrolysis by chymotrypsln, 268x g of chymotrypsin was com pared with 268xg of chymotrypsin which had been allowed to react with 14.8 mg of M. The rate of absorbancy change with time due to GPNA hydrolysis catalyzed by chymo trypsin and its <&2 M complex are shown in Figure 17B. In stead of inhibition, 30# activation of chymotrypsin was observed when complexed with M. Binding Ratio of Chymotrypsln to cu M Determined by Titration Although the maximum binding ratio of trypsin to <*2 M had been determined (8) the binding ratio for chymotrypsin to a2 M had to be measured before experiments could be 65 performed to study competition between chymotrypsin and trypsin for M binding. To determine the maximum amount of chymotrypsin that would bind to M, a constant amount of chymotrypsin, 268xg, was allowed to react for 15 minutes at 25°C with increasing amounts of M in the range of 1-15 mg and the rates of hydrolysis measured. The relative velocity of the reaction with increasing amounts of a2 M as compared to the ratio otjuE chymotryp- sin/mg a2 M was plotted in Figure 18. The activation ef fect plateaued when the ratio of chymotrypsin to a2 M was between 24 and 30xg of chymotrypsin per mg of a2 M. Binding Ratios Determined Using Gel Filtration. Absorbancy and Enzyme Activity Another approach to determine the binding ratio of chymotrypsin to a2 M was to form the complex with excess enzyme and then separate the complex from the free enzyme by gel filtration. One mg of chymotrypsin was added to 12.0 mg of <x2 M and Sephadex G-100 gel filtration per formed. From the absorbancy under the free enzyme peak, the concentration of free enzyme was determined. Subtract ing this value from the amount of chymotrypsin added gave the amount of enzyme complexed to M. The concentration of a2 M was determined by absorbancy measurements at 280 m/t of fractions from the first peak. The small ab sorbancy contribution by chymotrypsin was neglected since 66 800 600 400 200 000 1 0 15 5 0 Minutes 400 300 200 1 0 0 000 1 0 15 0 5 Minutes Fig. 17.— A, Hydrolysis of BAPNA catalyzed by trypsin and trypsin m complex. B, Hydrolysis of GPNA catalyzed by chymotrypsin and chymotrypsin M complex. A Free enzyme m a 2 M enzyme complex Relative Velocity GPNA Hydrolysis • a a H- TO I-* 00 • 1 i ►0 C l " 3 < + H" o 3 O o a r *< a o c+ 3 •o t n H* 3 X H - e l s' CO a \ 3 cQ » c-r K - * * 0 1 Q »-3 CO CO o o o CO o oo o o ON -0 68 the molar extinction coefficient was approximately 20 times that of chymotrypsin. The results presented in Table V show a binding ratio of 29.6xg of chymotrypsin per mg of <*2 M. The chymotrypsin bound to a2 M was also determined by measuring the rate of hydrolysis of GPNA and correcting for the <*2 M activation effect. The results indicated in Table V show 32.2>ag of chymotrypsin bound per mg of a2 M. 69 mg_a2. 12 12 TABLE V BINDING RATIOS OF «2 N CHYMOTRYPSIN COMPLEXES DETERMINED BY GEL FILTRATION, ABSORBANCY AND ENZYME ACTIVITY M x g bound CT 355 (absorbancy) 390 (GPNA hydrolysis) MR CT/mg *z M 29.6 32.2 70 Binding Ratios Determined Using Gel Filtration. Labeled Enzymes and Enzyme Activity. Gel filtration of a2 M with excess ^^1 labeled chymO' trypsin was performed and the amount of chymotrypsin bound to m in the early fractions was determined by measuring gamma emission of the fractions and enzymatic activ ity by measurement of the rates of GPNA hydrolysis. A similar experiment was performed using 1- ^ 1I labeled tryp sin and BAPNA for the enzymatic assay. A comparison of these results is given in Table VI and illustrates that the binding ratio is 53m g of trypsin per mg of <*2 M and 25m E of chymotrypsin per mg of ag M. Competition Between Chymotrypsin and Trypsin; Time of Addition Effect To determine if there was competition between trypsin and chymotrypsin for binding to M. Table VII illus trates that by adding the enzymes simultaneously, a2 N was found to have 12.6^4 g of chymotrypsin per mg M and 41,5m B trypsin per mg of a2 M indicating competition at the chymotrypsin site. The next experiments were performed to determine if different binding ratios would be found if the enzymes were added in sequence. Table VIII shows that even though only one half as much chymotrypsin bound to a2 N as does trypsin, nearly all trypsin binding was prevented by pre- TABLE VI a MAXIMUM BINDING OF TRYPSIN AND CHYMOTRYPSIN TO M Enzyme added to form complex Bound Trypsin Bound Chymotrypsin Eb Mg E/mg a2 M M g/mg M -tfg/mg M T-131! b 90 53.6 ° T 90 53.0 CT-131I 90 - 25.5 d CT 90 - 25.5 a) The complexes were formed by incubation for 15 minutes with enzyme and isolated by gel filtration. b) T-^^i CT-131Iodine labeled trypsin or chymotrypsin; enzyme bound measured by radioactivity. T, CT - Unlabeled trypsin or chymotrypsin; enzyme bound measured by enzymatic activity corrected for M inhibition or activation. E-Enzyme. •>3 TABLE VI (Continued) c) Highly purified a? m from preparative gel electrophoresis gave a trypsin binding ratio of 75m .S of trypsin per mg of a2 M. Using a M of 650,000 this gave a molar ratio for trypsin to a2 M of approximately 2:1. Thewpreparations used above were not as pure but it can be assumed that trypsin bound in a 2:1 ratio. d) This was assumed to be a 1:1 molar ratio. N> TABLE VII BINDING OF TRYPSIN AND CHYMOTRYPSIN WHEN ADDED SIMULTANEOUSLY TO ag M a Enzymes added to form complex Bound Trypsin Bound Chymotrypsin Eb M g of each E/mg M m g/mg M Mg/mg a2 M T-131I and CT 90 and 90 41.5 12.6 T and CT-131I 90 and 90 43.0 12.0 T and CT 90 and 90 41.5 13.2 CT and T 900 and 90 25.5 26.0 a) The complexes were formed by incubation of the a? M for 15 minutes with excess trypsin and chymotrypsin and isolated by gel filtration. b) T-^^I, CT-^li _ 131 iodine labeled trypsin or chymotrypsin enzyme bound measured by radioactivity. T, CT - Unlabeled trypsin or chymotrypsin; enzyme bound measured by enzymatic activity corrected for <X£ M inhibition or activation effect. E - Enzyme u> TABLE VIII BINDING OF THYPSIN TO AN a? M CHYMOTRYPSIN COMPLEX AND CHYMOTRYPSIN TO AN a, M TRYPSIN COMPLEX WHEN COMPLEXES FORMED WITH EXCESS ENZYME Enzyme added to form complex Second Enzyme Bound Trypsin Bound Chymotrypsin Eb M g E/mg a2 M E Mg E/mg ag M M g / m g a2 M ^ilg/mg a2 Tb 90 ct_131i 90 52.0 1.2 T 90 CT 90 51.8 1.8 CT 90 T-131i 90 *.5 25.3 CT 90 T 90 2.1 27.0 a) The first complex was formed by incubation of ap N with an excess of one enzyme. This was followed by addition of an excess of tne other enzyme and the free enzymes separated from the complex by gel filtration. 131, b) T-~-'“I, CT- I - ^ Iodine labeled trypsin or chymotrypsin; enzyme bound measured by radioactivity T, CT - Unlabeled trypsin or chymotrypsin; enzyme bound measured by enzymatic activity corrected for a2 M inhibition or activation effect. E - Enzyme -o 75 incubation with chymotrypsin. A similar experiment was performed with trypsin added first and followed by chymo trypsin. The data for these experiments in Table VIII in dicated that once the trypsin filled the sites chymotryp sin could no longer bind. A series of binding studies were performed to deter mine if an equimolar amount of an enzyme preincubated with M f°r 15 minutes could affect the binding of another enzyme to M. The results in Table IX suggested that sites which should be available were unable to bind enzyme and that the addition of excess enzyme in the second step failed to fill the sites that should be available if the <*2 M had not been inactivated. In both cases, preincuba tion with trypsin or chymotrypsin, the results indicated that the enzyme binding in the first step was not complete; suggesting the importance of enzyme concentration in com plex formation. The -^Ij labeling of trypsin allowed another experi ment of this type to be performed in which (Xg M was pre incubated with cold trypsin and followed by 1^1I trypsin. The results of these experiments presented in Table X re vealed incomplete trypsin binding in the first addition as well as considerable inactivation of remaining sites. An experiment was run to determine the effect of a TABLE IX BINDING OF TRYPSIN TO a9 M CHYMOTRYPSIN COMPLEX AND CHYMOTRYPSIN TO a? M TRYPSIN COMPLEX WHEN COMPLEXES FORMED BY INCUBATION WITH EQUI- MOLAR AMOUNTS OF ENZYME Enzyme added to form complex Second Enzyme Bound Trypsin Bound Chymotrypsin Eb X g E /m g <x2 M E x g E /m g a 2 M X g /m g a 2 M X g /m g a 2 Tb 30 ct-131i 90 19.0 3.9 T 30 CT 90 19.8 6.0 CT 30 t-131i 90 8.1 - CT 30 T 90 9.7 17.8 a) The first complex was formed by incubation of a, K with slightly less than equi- molar amounts of one enzyme. This was followed by addition of an excess of the other enzyme and gel filtration to separate free enzymes from the complex. b) T-1^1!, CT-1^1! _ 1^1iodine labeled trypsin or chymotrypsin; enzyme bound measured by radioactivity T, CT - Unlabeled trypsin or chymotrypsin; enzyme bound measured by enzymatic - > 3 activity corrected for a_ M inhibition or activation effect. E - Enzyme TABLE X BINDING OF TRYPSIN TO AN a„ TRYPSIN COMPLEX & FORMED BY INCUBATION WITH EQUIMOLAR AMOUNTS OF TRYPSIN Enzyme added to Bound Unlabeled Bound Labeled form complex Second Enzyme Trypsin Trypsin Eb x g E/mg a2 M E xg E/mg a2 M Xg/mg *2 M Xg/mg <*2 M T 30 T-131I 90 22.0 3.3 T 30 T 90 21.8 a) The first complex was formed by incubation of <x2 M with slightly less than equi- molar amounts of unlabeled trypsin. This was followed by addition of excess labeled trypsin and separation of the free enzyme from the complex by gel filtra tion. b) T-^-^I - -^Iodine labeled trypsin; enzyme bound measured by radioactivity T - Unlabeled trypsin.; amount bound measured by enzymatic activity corrected for inhibition or activation by a. M. E - Enzyme -o 78 ten-fold excess of chymotrypsin over that used in previous experiments. The chymotrypsin was added simultaneously with a three-fold excess of trypsin over the half satura tion amount for trypsin binding to a2 M. The results in Table VII showed that when the enzymes were added simultan eously that the concentration of enzyme regulates the com petition for the sites of M. In addition, it can be seen that chymotrypsin competes only for one site. Trypsin Exchange Experiment It seemed that the dissociation rate constants for the dg N enzyme complexes were quite low since the gel filtration results were in close agreement with the data obtained by titration of enzyme activity and indicated that dissociation did not occur to any appreciable extent during gel filtration. An indication of the irreversibil ity of complex formation was provided by an isotope ex change experiment. Unlabeled trypsin in 1.5 times the trypsin saturation amount was added to a2 M and incubated for 15 minutes. Trypsin labeled with 1^1i was added to the mixture and allowed to equilibrate for ^8 hours. The amount of ^^1 trypsin found in the a2 M peak after gel filtration was measured and the values presented in Table XI indicate that exchange was negligible between free and bound trypsin. TABLE XI EXCHANGE OF LABELED TRYPSIN WITH UNLABELED TRYPSIN COMPLEXED WITH a£ M Enzyme added to form complex M g E/rag a2 M 90 Second Enzyme E >rg E/mg a2 M T-131I 90 Bound Unlabeled Trypsin xg/mg a2 M 53 Bound Labeled Trypsin Xg/mg a M 0.006 a) The first complex was formed by a 15 minute incubation of a2 M with excess unlabeled trypsin followed by an excess of labeled trypsin. Twenty-four hours were allowed for exchange of labeled with unlabeled trypsin. -o vO CHAPTER V DISCUSSION Isolation and Purification of Macroglobulin Isolation of o . M by centrifugation and gel filtra tion was found to be a suitable method for large scale fractionation since the bulk of serum proteins could be eliminated in the first centrifugation step. This allowed material with a high a2 M concentration to be applied to moderate size laboratory columns. The yields for the large scale preparative method were only 35# due to the fact that only the pellet material from the preparative ultracentrifugation was used and that narrow cuts were made from the peak fractions containing a2 M obtained by gel filtration. The main advantage of the preparative technique used in our work over most previous methods was that changes in ionic strength and pH from physiological conditions were held at a minimum and that ammonium sul fate which was known to destroy enzyme binding activity was not used. The reason for the high trypsin binding activity achieved on preparative acrylamide gel electrophoresis was 80 81 believed to be due to the separation of the nonbinding from the binding macroglobulins. The method involving the use of gel filtration alone was employed to isolate a2 M from serum when only small volumes were available. The release of proteolytic en zymes during clotting may affect the amount of enzyme binding protein; however, the high binding activity associ ated with the M prepared from serum indicates that this may not be a major problem. The fact that the binding ac tivity of a2 M prepared from serum was higher after gel filtration than material prepared from plasma was most likely due to the fact that the plasma had been stored prior to fractionation; while serum was processed the same day the blood was drawn. Another advantage of the new method was that small amounts of serum could be used when the a2 M from patients with various diseases was to be investigated. Although 30-40 ml of serum was used in the method described, as low a quantity as 3 of serum has been used on smaller col umns to successfully purify <*2 M. The smaller volume al lowed the centrifugation step to be eliminated and a gel filtration step on P-300 substituted. This permitted the enzyme binding and amidase activity to be monitored in a way which more quantitatively reflects the activities in whole serum or plasma. Such studies may prove to be valu- 82 able for determining variations in proteolytic enzyme levels associated with macroglobulins in various diseases and also for the comparison of M enzyme binding levels of serum and plasma. Sedimentation Velocity Ultracentrifugation The sedimentation coefficient for the <x2 M prepared by the new method was 18.2 S in 0.1 M Tris-HCl buffer at pH 7.65 for a protein concentration of 3 mg/ml while that for the material of Mehl and Howard prepared from Cohn fraction III-O was 18.3 S which indicated that the two preparations had similar sedimentation properties. These authors determined a diffusion coefficient by a height area analysis of the schlieren peak from the sedimentation velocity experiment at a single concentration, and using this value found a molecular weight of 820,000. For our molecular weight determinations, it was decided that a sedimentation equilibrium method would be used. This method did not require the use of the diffusion coeffici ent. Also, less sample was required and concentrations closer to ideal could be used. Sedimentation Equilibrium Ultracentrifugation The Mw of the <*2 M further purified by a second frac tionation on Bio-Gel A5m was found to be in the range of 630,000 to 680,000 and varied little in fractions from the 83 leading edge, middle and trailing edge of the agarose gel 2 filtration peak and gave linear In y^ vs. r^ plots. The obvious disagreement between these values and those found by sedimentation velocity ultracentrifugation may be due to an incorrect estimate of D by peak area- height measurements of plates from the velocity ultra centrifugation experiments. This method was not accurate and extrapolations to infinite dilution were not obtained. The more accurate methods using low speed diffusion meas urements in the ultracentrifuge or measurements in a free boundary electrophoresis cell were not performed. The presence of lighter materials which would lower the molecular weight so drastically that such a reduced weight average molecular weight would be obtained should be reflected in differences in the molecular weight of material from the leading edge and trailing edge of the Bio-Gel A 5m elution peak. The data indicated the molecular weight does not change noticeably across the peak. Pre liminary experiments using a Bio-Gel A5m column and marker proteins show M to be eluted at a volume significantly larger than the elution volume for V N (molecular weight, 920,000). These results favor the of 650,000 over the established value of 8^0,000 ( 28). In addition, the protein seemed to be pure on Immuno electrophoresis with anti-human serum and Tf M and 3 -lipo- 84 proteins were not observed in the more pure fractions that were used for the molecular weight determinations. The multiple bands seen on acrylamide gel electro phoresis indicate heterogeneity. That this was charge and not weight heterogeneity was shown by molecular weight measurements with isolated bands and is discussed in the next section on acrylamide gel electrophoresis. Acrylamide Gel Electrophoresis Analytical acrylamide gel electrophoresis at pH 8.9 of the material prepared by Howard and Mehl (10) and the a£ macroglobulin prepared by the new method revealed two bands, a slow band and a faster more diffuse band. At this point it was realized that the macroglobulin prep arations were electrophoretically heterogeneous although they displayed a single symmetrical peak on sedimentation velocity ultracentrifugation. A question that had to be answered was whether or not both species were capable of binding proteolytic enzymes. When approximately equimolar amounts of trypsin were added the slower band appeared to acquire the same mobility as the fastest band. This sug gested that only the slower species was the binding pro tein and that the faster material contained the bound en zyme. Support for this hypothesis came when the results from the pH 8.9 analytical acrylamide gel electrophoresis 85 were compared with a2 macroglobulins prepared from serum and plasma. The material prepared from fresh serum was found to have higher binding activity and showed more of the slow moving species than the a2 macroglobulin prepared from plasma. When a pH 7*8 gel system was developed and used it became apparent that the problem was even more complex. Five bands appeared with the slowest band be lieved to be the same as the slowest band seen on the pH 8.9 runs and the four faster bands representing the dif fuse fast band seen at pH 8.9* When increasing amounts of trypsin or chymotrypsin were added there was uniform dis appearance of the slower four bands. All four bands ap peared to migrate as the fastest material when enzyme was added. Support for this came when an a2 M trypsin complex was mixed with uncomplexed M and electrophoresis per formed at pH 7.8. The intense band from the complex mi grated with the fastest of the five bands of the uncom plexed material. Preparative acrylamide gel electrophoresis of the M from serum or plasma and the re-running of the various fractions on analytical gels revealed a separation of the slower from the faster moving materials. This indicated that the patterns seen were not artifacts but different species of proteins with varying electrophoretic mobili ties. 86 It was also observed that methylamine could cause the conversion of all the slow bands Into a single fast band* This important observation indicated that proteolysis was not necessary to cause the unique mobility change. Whether or not methylamine binds to a2 M is not known* When preparative acrylamide gel electrophoresis was performed the trypsin binding protein could not be sepa rated from the <*2 M with amidase activity. It appeared that even the slow species had natural enzyme activity and that this activity tended to be only slightly higher in the faster a2 M fractions. Preparative electrophoresis allowed the isolation of a highly purified protein rela tively free of faster moving species. The trypsin binding activity of this material was the highest found for any of the a2 M materials tested. This verified the suggestion that the binding protein was associated with the slower bands. The fact that material of higher binding activity was obtained from serum was probably due to the fact that serum had a higher percentage of the slower moving trypsin binding species which permitted isolation of this material from the faster nonbinding proteins. The experiment in which one part of a2 M trypsin com plex and 9 parts of «2 M were subjected to preparative acrylamide gel electrophoresis was performed to be certain that the enzyme was still bound after electrophoresis and 87 that the faster moving species was the one complexed with trypsin. The results indicated that both assumptions were correct. In addition, in this experiment partial separa tion of the M trypsin complex from <*2 M still able to bind trypsin was quite obvious. The fact that <*2 M ami- dase complexes could not be separated from a2 M binding protein as was the M trypsin complex suggested a differ ent mechanism for the binding of natural enzyme and that small amounts of amidase were bound to a2 M without chang ing the mobility. Sedimentation equilibrium ultracentrifugation studies of the various species of different electrophoretic mobil ities showed little variation in the molecular weights of the proteins from slower to faster moving bands. Immuno diffusion experiments revealed that the proteins of differ ent electrophoretic mobility showed no major immunological differences. Proteins which show charge heterogeneity but which appear to be immunologically identical and homogene ous by weight may be species with amino acid substitutions, with different amide content or in the case of glycopro teins, a different sialic acid content. It is felt that there may be another possibility in the case of a2 M. Naturally occurring proteolytic enzymes may be bound to o2 M in different enzyme macroglobulin ratios, and differ ent enzymes with varying isoelectric points may be bound 88 giving ag M enzyme complexes with different net charge* The electrophoretic studies that showed the merging of the five bands into the one fastest species when excess enzyme was added are difficult to explain. Why should the binding of a more positively charged molecule such as trypsin make the complex more negative? A masking of the highly electropositive region on the molecule by the en zyme may occur. This seems unlikely since the enzyme it self is more electropositive than <x2 M. Conformational changes could take place that would cause buried negative charges to become exposed on the surface, cause positive charges to be buried, or alter ion binding properties of ag M. Another way that the mobility could be changed is that the enzymes could cleave and remove highly electro positive fragments from the <*2 M but the fra8ments would have to be small since significant changes in the molecular weight were not observed. Trypsin or chymotrypsin could displace electropositive molecules bound to a 2 M and change the mobility; however, these molecules would have to be highly electropositive since the trypsin itself has an isoelectric point near 9*0. Mobility changes due to siev ing effects could occur by differences in shape and fric tional properties. 89 Binding Studies When the amount of trypsin that could be bound to the isolated slow species of a2 M from preparative acrylamide gel electrophoresis was determined by measurement of the rl catalytic activity of the bound enzyme, it was found to be » approximately 75>< g of trypsin per mg of a2 M. This gave a molar ratio of trypsin to <x2 M of 2,50 if a molecular weight of 820,000 was used for ®2 M and 2*02* a ®olecu- lar weight of 650,000 was selected. Therefore, it appeared that the maximum number of enzyme binding sites on a2 M was two if 650,000 was close to the correct molecular weight. The binding ratio determinations and competition ex periments for trypsin and chymotrypsin binding to ®2 M sites were performed with a less purified preparation of ®2 M from plasma and the molar ratio was found to be 1,42 assuming a molecular weight of 650,000. From these studies M appears to have only one binding site for chymotrypsin. When the two enzymes in equal concentrations (three times the amount required for saturation of <x2 M with chymotrypsin) were added simul taneously there was a reduction in the binding for both enzymes; trypsin from 53 to kZjt g per mg of a2 M and chymotrypsin from 25 to 1ZA g per mg of a^ M. One explan ation of these results is that trypsin and chymotrypsin 90 compete for one site on the M molecule with equal af finity. The possibility that chymotrypsin competes with a lower affinity at the two trypsin binding sites seems unlikely since the M was found to bind only one mole of chymotrypsin per mole of a2 M. The results observed when a ten fold excess of chymo trypsin over trypsin was utilized show 25m g of trypsin bound and 26M g of chymotrypsin bound per mg of H in dicated again that competition occurred at only one site. Chymotrypsin filled one site completely due to its excess concentration and trypsin filled the remaining site. Puz zling results occurred when chymotrypsin (which as indi cated above binds only one site) was incubated first with ag M before the addition of trypsin. In this case, all the trypsin binding was prevented. This suggested a time dependent modification of the <Xg M molecule when enzyme was bound to one site that inactivated or prevented bind ing at the other site. j j Experiments involving a 15 minute pre-incubation with one half the amount of trypsin needed to completely com plex M, before the addition of more trypsin, indicated that inactivation of the second site had occurred since little if any additional trypsin binding was observed. These experiments were quantitatively inconclusive, how 91 ever, since the amount of trypsin bound was not equal to the half saturation amount. This was not expected since the otg M trypsin complex was believed to have an extremely high association constant. This will be discussed later in this section. It is not certain at this point whether the rate of complex formation or the equilibrium constant played the important role in these experiments. In fact, the mechanism may be more complicated; involving a slow conformational change induced by the enzyme or a proteoly sis step by the enzyme before the formation of the final complex• The data from electrophoretic studies seemed to sup port the binding studies. When a 1:1 molar ratio of tryp sin to <Xg N was reached the electrophoretic mobility of what appears to be the majority of the slower species was modified so that all the material would move as a single fast band. This suggested that the filling of one site may be sufficient to modify the M so as to prevent binding at the second site. A possible explanation may be that the second site was sterically hindered when one site was complexed to chymotrypsin. Data from experiments where half saturation amounts of enzyme were added to a2 failed to support the steric hindrance hypothesis. When chymotrypsin was added to a2 M in a 1:1 molar ratio only 17.8Mg of chymotrypsin was bound to a2 M; Table IX, 92 row rather than the expected 25Mg. When the 17»&JUg of chymotrypsin was bound to M, one might have expected 17.8Mg/ml of a2 M trypsin sites adjacent to the chymotryp sin sites to have been hindered. This left 53«6>#g (total sites for trypsin) less 2 x 17.8 or 35»6Mg (sites of chymotrypsin + sites hindered) or 18.0xg of trypsin that was expected to be bound per mg of H, As indicated in Table IX, row k, only 9.7Mg of trypsin/mg a2 M was bound. These results suggested that more of the a2 M was Inacti vated than just the < * 2 M that was bound to trypsin. An alternative to the steric hindrance hypothesis would be a hypothesis involving a conformational change caused by chymotrypsin binding that would prohibit trypsin binding. The true situation is most likely more compli cated since the above results with half saturation amounts of enzyme suggest that some M that does not bind enzyme appears to be inactivated. A simple model for this reac tion is indicated in Appendix B. In this model, the a2 M is changed to an activated binding form by proteolysis by the enzyme and then becomes converted to the same nonbind ing form by a conformational change or by enzyme binding. The enzyme molecules compete for the <»2 M sites before the conversion to an inactive form of a2 M. In this model the enzyme concentration would be important in two steps, the proteolysis step and the binding reaction. 93 Slgnlficance of Results The intention on initiating this research was to study the subunit and chain structure of <*2 M and develop a structural model and define enzyme binding in terms of such a model. In addition, it was anticipated that natural enzym .s could be liberated from dissociated M or identi fied with certain subunits. Analytical acrylamide gel electrophoresis at pH 8.9 of a purified preparation of <*2 M revealed two bands which indicated charge or possibly size heterogeneity. When run on analytical gels at pH 7.8, these same preparations revealed five bands. It was de cided that this heterogeneity should be studied prior to subunit investigation. Several questions had to be an swered. Are all five species related to dg M? Is the electrophoretic mobility variation due to charge or size differences? Are all or only certain species involved in enzyme binding? Do all or only certain species have the natural enzyme activity? Can the species with the amidase activity be separated from trypsin binding protein? Ideal ly, it seemed that the five species should be separated be fore beginning an investigation of subunit and chain struc ture since this would reduce the possible number of differ ently charged subunit species. During the course of the research these questions 9^ were answered. All the slow electrophoretic species ap peared to be macroglobulins and bound trypsin since all five components formed a single band on trypsin binding. The mobility differences seemed to be due to charge heter ogeneity differences since all the species had similar molecular weights. Just the slow moving components ap peared to be involved in binding additional enzyme and a fraction with maximal enzyme binding activity could be ob tained by isolating the slow moving species. All five components were found to carry about the same amount of natural amidase enzyme activity. The slow material with high binding activity still had amidase activity; however, the faster moving components had only amidase activity. Thus, it was possible to isolate M with natural enzyme activity and without trypsin binding activity; however, a separation of trypsin binding protein from natural enzyme was not achieved. Since some of the M species were separated by preparative gel electrophoresis sufficiently so that they gave only one band on analytical acrylamide gel electro phoresis they may be better suited for subunit studies them the original mixture of a2 macroglobulins. It should also be possible to separate each of the five bands by re peated preparative gel electrophoresis fractionations. 95 A crucial question which remains to be resolved is whether or not the natural enzyme binds at the same sites and by the same mechanism as trypsin and chymotrypsin. First indications from the electrophoretic separation of the different species of a2 M suggest that the sites and possibly the binding mechanisms may differ. More informa tion is needed about the natural enzyme before such specu lations can be supported. The activity of the amidase en zyme carried by a2 m for the catalysis of BAPNA hydrolysis was l/400th the activity found when a2 M was saturated with trypsin. The question of whether or not this enzyme is trypsin, similar to trypsin or has strikingly different behavior towards BAPNA must be answered. In addition, whether or not the enzyme is inhibited only by 35 to as with trypsin bound to a2 M or whether it is almost com pletely inhibited must be determined. Answers to these questions are necessary before the amount of natural en zyme bound to a2 m can be estimated. One approach would be to isolate and characterize the amidase enzyme or en zymes . The enzyme binding studies indicated that a2 M could bind at least two moles of trypsin and one mole of chymo trypsin per mole of a2 M. It was also found that trypsin and chymotrypsin competed for a site on the <*2 M, when add- 96 ed simultaneously but when chymotrypsin which bound only one site was added first the free trypsin site was inactiv ated. Experiments using half saturation amounts of enzyme gave results that favored a conformational change over a steric hindrance hypothesis. The physiological importance of the trypsin binding activity of H remains unknown. It is known that the serum concentration of this protein is considerable; 2-3 mg/ml, and since it is known to bind to thrombin and plasmin it may play an important role in the blood clotting or clot lysis mechanisms. The natural enzymes bound to <*2 M may be enzymes that required inhibition by a2 M or may be that M was a carrier for these enzymes. If the latter is true and the natural enzymes are bound to M in the same manner as trypsin then a mechanism which al lows the enzymes to be released must be postulated if the enzymes are to hydrolyze high molecular weight substrates. This is necessary since it is known that trypsin bound to a2 M is greatly inhibited in its ability to catalyze the hydrolysis of high molecular weight proteins such as ca sein and hemoglobin. The release of vasoactive substances which require a proteolytic enzyme presents a different situation since an M enzyme complex may readily hydro lyze smaller proteins and peptides. For example, it may 97 readily hydrolyze the N-terminal lysine of the decapeptide precursor of bradykinin to form the active vasodilator, bradykinin and in this fashion act as an aminopeptidase. Another role would be for this enzyme to inactivate the bradykinin by hydrolyzing the N-terminal arginine. A Postulated Mechanism for Enzyme Binding and the Function of a Macroglobulin The amino acid sequences of trypsin and chymotrypsin are quite similar and one could imagine that the two en zymes could bind to M at a site which was oriented to react with a high affinity for these enzymes. Thrombin and plasmin have been reported to bind to M and this indicates a rather broad specificity for the M site. Methylamine may react at the binding sites which may have an affinity for basic molecules. The fact that trypsinogen does not appear to bind to M or alter the electrophoret ic properties of a m indicates that proteolysis or a liberated end group due to zymogen to enzyme conversion may be important. It is known that these zymogens undergo a conformational change when activated and the new conform ation may favor binding. Liberated end groups may also create a center that has a high affinity for the a2 M sites. Even if we consider proteolysis to be essential for 98 binding the question must be answered as to how the enzyme binds to ag M in what appears to be an irreversible reac tion. One explanation would be that the enzyme forms an enzyme substrate complex with M and the deacylation step is blocked. It is known, however, that the enzyme retains its activity. This fact tends to exclude the pos sibility of an enzyme-a2 m acyl complex. One of the best ways to explain an irreversible com plex is to postulate a conformational change in the M which traps the enzyme after it has reached its binding site on the M. This conformation change could occur in several ways. The enzyme could form an enzyme substrate complex with M at a site where the affinity of °2 M for enzyme is quite high. The enzyme could then catalyze splitting of the M and before the enzyme could move away from the site of hydrolysis it would become trapped by electrostatic forces or held within a hydrophobic environ ment. Steric hindrance may also prevent the enzyme from being released from its site of action. A possible mechanism for the irreversible complex is one that involves a conformational change but in this case one induced by the hydrolysis product of the enzyme. A new N-terminal amino acid can be created by proteolysis of the (X2 M. If the enzyme acts at more than one site, a pep- 99 tide could be released and bind specifically to the M. The new N-terminal amino group or the basic peptide re leased could induce structural changes in the M that result in the trapping of the enzyme at its site of action before being released from the acyl enzyme complex. The reaction may go on to form the free enzyme but now the en zyme is held within the ®2 M structure. This change in M conformation would be the same as the one induced by methylamine and other basic amines. Once M reacts with methylamine the hydrolytic site is buried within the Struc ture of the N and no longer accessible to enzyme. From our experiments, it appears that the amidase associated with <x2 M is not bound by the same mechanism as trypsin and chymotrypsin and in fact may be an integral part of the »2 M structure. I feel that «2 M "“W itself be an enzyme or enzyme carrier designed for the hydrolysis of specific low molecular weight substrates such as basic peptides. Another possibility is that a M may hydrolyze specific proteins while being regulated by basic physio logical amines. The fact that M amidase was found to have kallekrein activity ( 4 ) indicates that a2 M either induces formation of a kallekrein or has the potential it self to convert kininogen to kinin. Both steps are be lieved to involve a proteolytic enzyme. 100 Binding of the arginyl or lysyl groups of &na2 M substrate to the active site of a2 M could act as an enzyme substrate complex. It is a well accepted hypothesis that formation of an enzyme substrate complex induces a conform ational change in enzymes. It may even be that the tryp sin and chymotrypsin binding and the effect of methylamine, all believed to induce a conformational change in M, are a consequence of the naturally occurring enzymatic activity associated with a2 M. CHAPTER VI SUMMARY Human a macroglobulins were shown to resolve into five distinct electrophoretic species on acrylamide gel electrophoresis at pH 7*8. The components have similar antigenic characteristics, specific amidase activity and molecular weights (650,000); however, only the more slowly moving species were able to bind trypsin. The five bands were converted to a single fast nonenzyme binding compon ent by complexing with trypsin or chymotrypsin in a 1:1 ratio or reacting with 0.2 M methylamine. Direct a2 m binding studies with labeled trypsin and chymotrypsin gave 2:1 and 1:1, respectively for the maximal molar binding ratios and indicated a competition between the enzymes for one site on the a2 M. Binding of one mole of chymotrypsin by one mole of a2 M prevents the further binding of a second mole of trypsin. Preincubation with amounts of trypsin insufficient to produce maximal binding will prevent subsequent additional trypsin binding. The unique changes in the electrophoretic mobility of 101 102 a M when complexed with one mole of enzyme favor the hy- 2 pothesis that a structural change in the M and not a steric hindrance prevents further trypsin binding. APPENDIX APPENDIX A Variation of Amidase Activity with Time To determine if amidase activity varied with time, 0.2 ml of fractions from the second Bio-Gel A5m gel fil tration, Figure 1, were assayed and the absorbancy change measured with time in hours. Turbidity occurred when BAPNA was added to fractions from the first peak. These were not centrifuged or filtered in these studies to elim inate the possibility of removal of active material with filtration or centrifugation. As the rate of absorbancy change due to increasing turbidity created initially by BAPNA addition, leveled off, the rates of absorbancy change became linear. These rates were found to be similar to those obtained with filtered samples. The kinetic data from the assay of these fractions are shown in Figure 19* Fractions taken from the M peak displayed linear rate plots for BAPNA hydrolysis as is seen in Figure 20. Filtration was not necessary in this case. Fractions from the third peak showed a change in catalytic activity with time. Due to this variability an arbitrary time of 120 hours was selected for the readings of natural enzyme ac tivity. These are the values used in Figure 20. 104 105 800 § 600 o 200 000 192 144 96 48 0 Hours Fig. 19*— Amidase activity of fractions selected from the center of the three peaks from the Bio-Gel A5m gel filtration experiment shown In Figure 1. Amidase activity Is given as A A/hr/ml of fraction. A Fractions from the first peak m Fractions from the second peak m Fractions from the third peak APPENDIX B A Hypothetical Model for the Mechanism of Enzyme Binding to QU M. (See Figure 20) The binding studies with trypsin and chymotrypsin have the following characteristics: 1) Trypsin binds to a2 M in a molar ratio of 2:1. 2) Chymotrypsin binds to m in a molar ratio of 1:1. 3) When a three-fold excess of trypsin and chymotrypsin are added simultaneously to H, there appears to be competition between the enzymes for the sites on the a£ M* Since chymotrypsin binds to only one site, this competition most likely occurs at only one of the trypsin sites. k) When chymotrypsin is added to fill one site and then trypsin added there is a loss of trypsin binding ac tivity even though there should be a site available for trypsin binding. 5) When chymotrypsin is added in equimolar amounts, be fore trypsin is added, incomplete chymotrypsin binding is found. On the subsequent addition of trypsin, less trypsin is bound than would be expected if there were 106 10? steric hindrance by the bound chymotrypsin. 6) When an equimolar amount of trypsin is added before the addition of more trypsin, binding is incomplete and when additional trypsin is added, less is bound than would be expected by steric hindrance. 7) Both sites are filled; one completely with chymotryp sin and one with trypsin when a ten-fold molar excess of chymotrypsin and a three-fold excess of trypsin are added simultaneously. 8) All bands seen on acrylamide gel electrophoresis are converted to a single fast band when the molar ratio of trypsin to M approaches 1:1. 9) All the bands convert to a single fast band when chymotrypsin is added to M. These properties suggest that the mechanism of enzyme binding may be more complicated than direct complex forma tion between enzymes and a2 M. There appears to be a time dependent alteration of o2 M to a nonbinding form in the presence of enzyme and this is not always explained by en zyme occupying all the available sites. Therefore, the model must account for an alteration in H to an inac tive form as well as enzyme binding. The evidence is not sufficient to propose a unique mechanism. The one illus trated in Figure 20 is only a hypothetical model. This 108 o(2M-E( 1 / > " J ° ( 2M + ( ) + ( ) E t oCgM1 + ( ) E -/— ► B I I I Fig. 20.— Possible mechanism for enzyme binding to a2 M. a M° - Unreacted a2 M enzyme binding protein 2 E - Proteolytic enzyme a 2 Ma - a2 M activated by proteolysis a2 M1 - Inactive a2M enzyme binding protein | | - Acrylamide gel electrophoresis pattern ( ) - Represents the number of moles of enzyme 109 mechanism requires that the enzyme activate the N to a binding form, M , (by proteolysis) and at this stage 1) the enzyme cam bind to M or 2) the a2 M passes to an inactive form, M*. Both the rate of conversion to Ma in step I and the competition between enzyme binding and conversion to a2 M* in step II would be dependent upon enzyme concentration. This may explain why higher enzyme concentrations (3 times equimolar amounts) will give maxi mum binding while lower enzyme concentrations show incom plete binding but considerable conversion of the H to a nonbinding form. APPENDIX C Rate of a~ M Enzyme Complex Formation Before the investigation of M enzyme complexes was initiated, experiments were performed to determine the best conditions for the study of complex formation. The concentration of M, and enzyme and the time allowed for complex formation were investigated. To perform such studies a substrate was needed which was readily hydro lyzed in the presence of enzyme and permitted a low con centration of enzyme to be used. In addition a substrate that could be used in the automatic titrator was desired since this permitted the study of the catalysis of low en zyme concentrations. As shown in Figure 21, various concentrations of trypsin were employed from 0.5 to 2.0/«ug/ml). The concen tration range selected included the lowest trypsin concen tration expected for routine binding studies. The purpose of this study was to determine the concentrations needed to insure adequate conditions for binding. The figure illustrates that when the ct^ M concentration was as low as 110 Ill 40 30 20 10 0 Minutes of Reaction Fig. 21.— Determination of trypsin concentration and the required time for maximum binding with the lowest a2 M concentration to be used in binding studies. In all cases 50<4g/ml was the a2 M concentration. A, 0.5Ag/ml trypsin B, 1.0^tg/ml trypsin C, 2.0^(g/ml trypsin. The substrate used to detect inhibition of trypsin by M was 0.1 M TAME. 112 5&«g/ml a concentration of at least 2^

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Asset Metadata

Creator Saunders, Russell Lee, 1938- (author)

Core Title Studies of human ɑ₂ macroglobin: physical and enzyme binding properties

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1970-08

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)

Advisor Vannier, Wilton E. (committee chair), Allerton, Samuel E. (committee member), Fife, Thonas H. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-461564

Unique identifier UC11363233

Identifier 7112416.pdf (filename),usctheses-c18-461564 (legacy record id)

Legacy Identifier 7112416.pdf

Dmrecord 461564

Document Type Dissertation

Rights Saunders, Russell Lee

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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...

Repository Name University of Southern California Digital Library

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