AQAQg Arr. (av-1'" I'f! xymrn, ‘l \ \ ,.\ M... T{ ‘lOUI . \..~ . “ I, I . '..’I\ y .-.l\\.| EAST LANSING, MICHIGAN THE ISOLATION AND PHYSICAL-CHEMICAL CHARACTERIZATION OF KAPPA-CASEIN FROM COW'S MILK By Harold E. Swaisgood A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1963 L33|~11 2,]I8 Wok{ ABSTRACT THE ISOLATION AND PHYSICAL-CHEMICAL CHARACTERIZATION OF KAPPA-CASEIN FROM COW'S MILK by Harold E. Swaisgood The casein micelle of milk represents a complex of many inter- acting proteins, one of which - kappa-casein - stabilizes the other members in their ionic environment. However, the strong interactions between the micellar proteins impedes the isolation and characteriza- tion of the individual constituents. The purpose of this study was to develop a procedure for the isolation of kappa-casein and to inves- tigate its chemical and physical properties. These studies employed sedimentation-velocity and sedimentation- equilibrium ultracentrifugation extensively, as well as electrphoresis, viscosity, and various chemical analytical techniques. A procedure was developed for obtaining good yields of crude kappa-casein of about 90% purity. The crude kappa-casein was readily purified to approximately 97% purity for physical-chemical studies. The characteristics of the preparation were found to be similar to those previously reported for kappa-casein. Association of kappa-casein molecules to form large aggregates of relatively uniform size in inorganic salt solutions near neutrality precludes the study of the basic unit in these buffers. Therefore, the physical properties of kappa-casein were determined in strong dissociat- ing solvents. The weight-average molecular weight of the protein was approximately 125,000 in 677. acetic acid - 0.15 g NaCl and 5.0 fl guanidine-HC1 solutions. A molecular weight of approximately 56,000 Harold E. Swaisgood was obtained for the low-molecular weight component in 7.0‘§;urea, 33% acetic acid - 0.15'§.NaC1, and at low protein concentrations in 5. 0 g guanid ineoHCl. Reduction of the disulfide bonds caused the molecular weight to be lowered to 28,000 in.5.0 g guanidine°HCl and 67% acetic acid - 0.15'§;NaC1. Physical-chemical studies showed that the disulfide bonds were also destroyed in phosphate buffer at pH 12. Determination of the sulfhydryl groups by several chemical methods indicated 2-3 -SH groups per 28,000 g. An odd number of -SH groups per 28,000 g suggested at least one inter-molecular disulfide bond since there were no free ~SH groups. iMinimum molecular weights calculated from the chemical analyses supported the physical studies. These data led to the conclusion that the basic unit of kappa- casein was composed of two 28,000 molecular weight sub-units joined by disulfide bond(s). ACKNOWLEDGEMENTS The author expresses his sincere appreciation to Dr. J. Robert Brunner, Professor of Food Science, for his encouragement and counsel during this study. His philosophy of life as well as his scientific ability will be a constant source of inspiration. The writer also expresses his gratitude to Dr. Hans A. Lillevik, Associate Professor of Biochemistry, for many helpful discussions and for arranging for the amino acid analyses. The author wishes to acknowledge the financial assistance of a Predoctoral Fellowship from the Division of General Medical Sciences, United States Public Health Service. Grateful acknowledgement is also due to Dr. B. S. Schweigert, Professor and Chairman of the Department of Food Science, for providing research facilities and financial support. The author is also indebted to Mr. C. W. Kolar for performing the zonal electrophoresis experiments and to Mr. E. M. McCabe and other graduate students who have contributed to this study. And lastly, but never least, to my beloved wife, Janet, go my thanks for her patience and understanding during my entire graduate study program. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . o . o . . . . . . . o o o o . o . . . 1 HISTORICAL........................... 3 General . . . . . . . . . . . . . . . . . . . . . . . . . . 3 iMethods Employed in the Preparation of k-Casein . . . . . . 4 Method of Waugh and von Hippel (1956) . . . . . . . . 4 Method of Long (1958) . . . . . . . . . . . . . . . . 4 Method of Fox (1958) as Modified by Morr (1959) . .. 5 Method of Pilson, Henneberry, and Baker (1960) for Fraction A . . . . . . . . . . . . . . . . . . . 5 Met-110d Of MCKenzie and Wake (1961) o o o o o o o o o o 6 Method of Cheeseman (1962) .. . . . . . . . . . . . . 7 HathOd Of Hill (1963) o o e o o o o o o o o o o o o o 7 Method of Hipp, Groves, and'McMeekin (1961) for Preparinga3-C8831n 00.00.000.000... 8 Chemical Properties of k-Casein . . . . . . . . . . . . . 8 Composition . . . . . . . . . . . . . . . . . . . . . 8 Amino acids . . . . . . . . . . . . . . . . . . 8 Nitrogen . . . . . . . . . . . . . . . . . . . . 9 Phosphorus and NANA . . . . . . . . . . . . . . 9 Stabilization of 0%d6asein . . . . . . . . . . . . . . 10 Reaction with Rennin . . . . . . . . . . . . . . . . . 111 Physical Properties . . . . . . . . . . . . . . . . . . . . 14 Ultracentrifugal Characteristics . . . . . . . . . . . 14 Electrophoretic Characteristics . . . . . . . . . . . 16 iii PART I The Preparation and Preliminary Characterization of Kappa-Casein . . . . . . . . . . . . . . . . . . . . . . INTRODUCTION TO PART I . . . . . . . . . . . . . . . . . . Emmmummons Apparatus . . . . . . . . . . . . . . . . . . . . . . . Chemicals and Buffers . . . . . . . . . . . . . . . . . Preparatory Procedure . . . . . . . . . . . . . . . . Isolation Procedure for Preparation No. 1 . . . . Isolation Procedure for Preparation No. 2 . . . . Isolation Procedure for Preparation No. 3 and 3 A Additional Purification as Applied to Preparations N o O 3 and 3A 0 C O O O O O O O O O O O O O O 0 Isolation Procedure for Preparation No. 4 . . . . Analytical Methods . . . . . . . . . . . . . . . . . . Preparation and Yields . . . . . . . . . . . . . . . . Free-Boundary Electrophoretic Characteristics . . . . . pH of Minimum Solubility at 0.1 Ionic Strength . . . . Stabilization of ag-Casein . . . . . . . . . . . . . . Complex Formation with O%-Casein . . . . . . . . . . . Characteristics of the Isflasein Fraction (Preparation No. 4) . . . . . . . . . . . . . . . . . . . . . . . Action of Rennin on k-Casein Preparations . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . General . . . . . . . . . . ... . . . . . . . . . . . Electrophoretic and Ultracentrifugal Characterization . Stabilization and Interaction with 0%-Casein . . . . . iv 17 18 l9 19 20 20 21 22 22 23 24 24 29 29 32 32 32 33 33 34 53 53 54 56 Characteristics of the Preparation as a Primary Substrate for Rennin . . . . . . . . . . . . . Other Proteins Obtained During the Preparation of k-Casein or as a Result of a Slight Alteration in the Preparative Procedure . . . . . . . . . . . SW OF Pm I O O O O O O O O O O O O O O O O O O 0 Properties of Kappa-Casein . . . . . . . . . . . . . . . INTRODUCTION TO PART II . . . . . . . . . . . . . . . . EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . Apparatus . . . . . . . . . . . . . . . . . . . . . Chemicals . . . . . . . . ... . . . . . . . . . . . Chemical Methods . . . . . . . . . . . . . . . . . Amino Acid Analysis . . . . . . . . . . . . . Nitrogen . . . . . . . . . . . . . . . . . . Sulfur . . . . . . . . . . . . . . . . . . . . Phosphorus . . . . . . . . . . . . . . . . . . Nchetylneuraminic Acid . . . . . . . . . . . Disulfide Bond Reduction . . . . . . . . . . . Sulfhydryl Group Titration . . . . . . . . . Nitroprusside . . . . . . . . . . . . . . . . Physical Methods . . . . . . . . . . . ... . . . . Ultracentrifugation . . . . . . . . . . . . . Determination of protein concentration . Sedimentationaequilibrium . . . . . . . . Approach-to-equilibrium . . . . . . . . . Sedimentationdvelocity . . . . . . . . . Page 57 59 61 62 63 65 65 66 67 67 67 68 68 68 69 69 71 71 71 71 72 73 73 Densities and partial specific volume Intrinsic viscosity determinations . RESULTS ... . . . . . . . . . . . . . . . . . . . . Composition . . . . . . . . . . . . . . . . . . Physical Properties of k-Casein . . . . . . . . Properties in Non-Dissociating Solvents . Properties in Dissociating Solvents . . . Sedimentation characteristics in SDS selutionsooooooooooooo Sedimentation and viscosity characteristics in 5.0MGU solutions . . . . . . . . . Sedimentation and viscosity characteristics in concentrated acetic acid solutions adjusted to 0.15'1_![_NaCl . . . . . . . . . Sedimentation characteristics in 7.0 M urea solutions at pH 8.5 . . . . . . . . Sedimentation and viscosity characteristics in pH 12.2 phosphate buffer . . . . . . Physical Properties of Reduced k-Casein . . . . Sedimentation Characteristics in 5.0 1.1 GU Sedimentation Characteristics in 67% Acetic Acid Adjusted to 0.15 MNaCl . . . . . . Determination of Sulfhydryl Groups . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . Composition of k-Casein . . . . . . . . . . . 'Molecular Size and Interactions of k-Casein . Polymer Size of k-Casein in Neutral Salt Solutions ............... Dissociation of the k-Casein Polymers and the Properties of the Units Obtained . . . . . vi Page 74 75 76 76 77 77 77 77 79 80 81 81 82 82 83 84 103 103 105 105 106 Page Sedimentation, diffusion, and viscosity characteristics of k-casein in dissociating solvents . . . . . . . . . . . . 106 Sedimentation and diffusion characteristics of reduced k-casein in dissociating 801vents ‘ O C O C C O O O O O O O O O O O O C 109 Comparison of k-casein and reduced k-casein . . 110 Some other possible structures of k-casein . . 112 SUMMARYOF'PART‘II 120 LITERATURECITED 121 vii LIST OF TABLES TABLE Page 1. Comparison of the electrophoretic, ultracentrifugal, and yield characteristics of k-casein pre- paratims O O O O O O O O O O O O O O I O O O O O O 37 2. Quantitative characteristics of the sedimentation- velocity patterns of k-caseins shown in Figm 4 O O O O O O O O O O O O O O O O O O O O O 39 3. Electrophoretic properties of k-casein (Prepara- tion No. 3) in buffers from pH 2.3 to 8.6 . . . . . 40 4. The amino acid composition of k-caseins . . . . . . . 85 5. iMolecular weights of k-casein from chemical malysis I O O O O O O O O O O O O O O O I I O O O 87 6. Sedimentation coefficients of k-caseins at neutral pH 0 O O O O O O O O O O O O O O O O I O O O O O O 88 7. Sedimentation coefficients of purified Preparation No. 3 A in buffers containing SDS . . . . . . . . . 89 8. Equilibrium molecular weight data for k- and reduced k-casein in 5.0‘M36U, pH 4.8 . . . . . . . . . . . 90 9. Hydrodynamic properties of k-casein in 5.0 M GU, 67% acetic acid - O. 15 11 NaCl, and pH 12.2 phosphate buffer 0 O O O O O O C O O O O O O I O O O O O O O 91 10. Equilibrium molecular weight data for k- and reduced k-casein in 67% acetic acid - 0.15 M NaCl . . . . . 92 11. Determination of the total sulfhydryl groups . . . . . 93 12. Summary of the physical properties of k-casein and reducedk-casein................. 117 viii LIST OF FIGURES FIGURE Page 1. Procedure followed for obtaining k-casein from a concentrated urea-TCA solution of iso- electric casein or crude k-casein fraction ........ 27 2. Electrophoretic and sedimentation patterns of the material obtained from various steps of the isolation procedure . . . . . . . . . . . . 41 3. Electrophoretic and sedimentationdvelocity patterns of the k-casein preparations reported in Table 1 AQOQOQOQOOOOOODOO. O O O O O O O O O O O 43 4. Sedimentationdvelocity patterns of different k-casein preparations in pH 7.0 phosphate buffer . 44 5. Urea-starch gel electrophoretic patterns of o%-, k',mdx-Caseins oooooooooooooooo 4’6 6. Free-boundary electrophoretic patterns of purified k-caseins in pH 7.0 phosphate buffer, r/zs 0.1 O O O O O O O O O” C O O O O O O O O O O 47 7. Electrophoretic patterns of k-casein Preparation No. 3 in buffers ranging from pH 2.3 to 8.6, r/2 = 0.1 (see Table 3) O O O O O O O O C O O O O 48 8. A plot of the descending electrophoretic mobilities of k-casein Preparation No. 3 at various pH valm8(seeTable3)0.000.000.0000. 4’9 9. A plot of the nitrogen and 280 mp absorption for ~the k-casein remaining in solution at pH values ‘near‘the isoelectric point . . . . . . . . . . . . 50 10. The stabilization of ag-casein by various amounts of k-casein Preparation No. 3 . . . . . . . . . . . . 51 11. Electrophoretic and sedimentationdvelocity patterns of A) Hipp 95.31. (1952) a-casein fraction and k-Jah-casein complexes induced by B) precipita- tion from 3.0 M_ urea and by C) momentarily adjusting a solution of the mixture to pH 12 . ... 52 12. A plot of the reduced viscosity (ml/g) for k- casein in A) 67% acetic acid - 0.15 M. NaCl, B) 5.0.11 guanidine-HCI, and C) 0.1.MNa01 . . . . 94 FIGURE Page 13. Plot showing the concentration dependence of the apparent molecular weight of k-casein in 5.0 M guanidine-HCl and 67% acetic acid -0.15M_NaC1................... 95 14. Van Holds-Baldwin plots of short-column equilibriumdata for k-casein . . . . . . . . . . . 96 15. Trautman plot of approach-to-equilibrium data for k-casein in 5.0 l_i_ guanidine-HCl .......... . . 97 16. Trautman plot of approach-to-equilibrium data for k-casein in 33% acetic acid - 0.15 M NaCl . . . 98 17. Trautman plot of approach-to-equilibrium-data for k-casein in 7.0 M urea, pH 8.5, 25° C . . . . . 99 18. Trautman plot of approach-to-equilibrium data for k-casein in pH 12.2 phosphate buffer, 25° C O O O O O I O O C O O O O O O O O O O O O O O 100 19. Plot showing the concentration dependence of the apparent molecular weights in dissociating solvents for-reduced k-casein and k-casein exposed to PH 12 O O O O O C O O O O O O I O O O O O O O O O 101 20. Sedimentation-equilibrium patterns for k-casein and reduced k-casein in 67% acetic acid - 0.15M. NaCl and 5.0Mguanidine-HCl" . . . . . . . . . . . 118 LIST‘OF APPENDICES APPENDIX I Composition of Buffers . . . . . . . . . . . . . . . Properties of the Dissociating Solvents Used for or Correction of the Water....... Molecular Weight Calculations Sedimentation Coefficients to APPENDIX II . . . . . . . . . . . Introduction . . . . . . . . . Notation .. . . . . . . . . . . Sedimentation-Equilibrium . . . Theoretical. . . . . . . . Experimental Application . ‘Weight-average molecular weight entire cell contents ‘Weight-average molecular weight '(Van Holde-Baldwin plot). . . . . . . . for the atr Zquerage molecular weight for the ‘entire cell contents Example Calculation . . . . . . . . . . . . . Calculation of the Diffusion Coefficient . . Approach-to-Equilibrium . . . . . . . . . . . . . . Theoret 1 c a 1 O .4 O O O O O I O I O O O O O O 0 Experimental Application SedimentationdVelocity . . . . . . . . . . . . . . Calculation of the Coefficient The observed sedimentation coefficient . Correction of the_observed S to standard conditions . .f. . . . . . . . . . . . Correction for radial dilution xi Page 129 129 130 132 132 132 134 134 137 137 ‘ 139 140 140 143 144 144 146 148 148 149 149 Page The frictional ratia O O O O O O O O O O O O O 150 The Scheraga-Mandelkern constant . . . . . . . 151 APPENDIX III 0 O O O O O O O O O O O O O O O O O O O O O C O 154 xii INTRODUCTION The casein micelle of milk is a complex system of interacting proteins and calcium ions. The micelles are in equilibrium with polymer complexes of the individual components, the equilibrium being diaplaced towards the micellar form by calcium ion addition. Never- theless, the system is remarkably stable to rigorous industrial process- ing treatments such as pasteurization, sterilization, concentration, drying, reconstituting, freezing, and addition of sugar and/or inorganic salts. However, lowering the pH to 4.6 releases the calcium ions and the casein fraction precipitates. At least four individual proteins are present in the micelle, two of which (og- and B-casein) are individually insoluble at calcium ion concentrations normal to milk. There are undoubtedly more than four constituents since the I-casein fraction appears heterogeneous. The principle micellar system appears to be a complex of use and k-casein. The interactions leading initially to complexes and finally to micelles are strong and highly specific. This behavior was responsible for earlier conclusions that casein was a homogeneous substance. For example, the ahcasein fraction appears to be homogeneous by free-boundary electrophoresis and ultracentrifugation and, in fact, can be isolated intact from 4.6 M urea solutions. However, studies involving the addition of calcium ion or strong dissociating solvents show that abcasein is a complex of k-, og- and probably the I-casein fraction. Furthermore, these strong interactions make the isolation and purification of any one of the components difficult. Consequently the information concerning the properties of the individual components is incomplete. k-Casein has been shown to be the "protective colloid" of the mi- celle system. The interaction of this protein with the other components yields a stable micelle and in its absence the other caseins (with the exception of I-casein) will precipitate at low calcium ion concentrations. Also, the alteration of this single protein by the primary action of the enzyme rennin is sufficient to cause coagulation of the micellar proteins. Moreover, the interaction seems to be stoichiometric since a weight-ratio of 4 03-: l k-casein is preferred for complex formation. Relatively pure k-casein forms polymers of nearly uniform size in buffers not considered to be strong dissociating agents. In fact, this protein seems to interact with itself and other proteins more readily than any of the other micellar cdmponents. The polymers formed are relatively independent of pH, temperature, ionic strength, and calcium ion concentration suggesting that this is a rather unusual Specific interaction. In view of the above discussion, k-casein would appear to present a challenging system for the study of secondary bonding forces in proteins. However, before such a study can be pursued three objectives must be achieved: a) a method for preparing considerable quantities of pure protein must be developed, b) the chemical composition must be deter- mined, and c) elementary physical properties such as molecular weight, sedimentation coefficient, diffusion coefficient, intrinsic viscosity, and shape must be deScribed. The purpose of the research described @ herein was to achieve, insofar as possible, these three objectives. For the sake of organization, the thesis has been divided into two parts: Part I dealing with the method of preparation and preliminary characterization of k-casein and Part II describing the physical-chemical studies performed on the preparation. 1e di HISTORICAL General Linderstrdm-Lang (1925) first recognized the calcium-insensitivity of a casein fraction which he called Z-casein. Later he (Linderstrém- Lang, 1929) proposed the existence of a "protective colloid" in the casein fraction which was responsible for the stabilization of the casein micelle and was Specifically attacked by rennin. The classical electrophoretic separation of whole casein into a-, B- and y-casein, in order of decreasing mobility, was reported by Mellander (1939). Subse- quently, Warner (1944) suggested that (Dz-casein1 was heterogeneous and later, Nitschmann and Lehmann (1947) proposed that rennin did not act directly on abcasein, but rather, on a component thereof. The "protective colloid” theory has been the subject of considerable controversy. Recent experiments have supported this hypothesis so that at present it is universally accepted. The factor most responsible for the renewed impetus in casein research was the discovery of kappa-casein by Waugh and von Hippel (1956). While attempting to purify micellar casein, they obtained a fractionation of the casein into calcium-sensitive and calcium-insensitive fractions. Analysis of the calcium-insensitive fraction (Fraction S) revealed the presence of B-casein and a new component which they called k-casein. Furthermore, addition of Fraction S to the calcium-sensitive fraction in the correct proportions resulted in the formation of stable micelles in the presence of calcium ion. 1 This component has now been definitely shown to be heterogeneous and will be referred to as the abcasein fraction (Brunner, Ernstrom, Hollis, Larson, Whitney, and Zittle, 1960). C01 in 1‘82 Methods Employed in the Preparation of k-Casein Method of Waugh and von Hippel (1956) This method essentially consists of a physical separation at constant pH. Micelles were collected from skimmilk by adjusting the calcium ion concentration to 0.06 M_causing nearly all of the casein to concentrate in the micellar form, and centrifuging at 45,000 X G. Calcium ion was removed from the precipitate -- designated as first-cycle casein -- by using a chelating agent, e.g. sodium oxalate or citrate. First-cycle casein was made 0.25 M_with respect to CaCl2 and the calcium-sensitive fraction removed by centrifugation, first at 900 X C (37° C) and then at 90,000 X G (5° C). The supernatant, designated as Fraction S, contained about 70% k-casein. Later, Waugh (1958) reported a purifica- tion of Fraction S using centrifugation, salting-out, and isoelectric precipitation which yielded k-casein of approximately 90% purity. The contaminate conSisted largely of m-casein which is probably similar to the R-casein described by Long (1958), see Brunner g£_§l, (1960). Method of Long (1958) The starting material for this procedure was the a-casein fraction prepared by the method of Warner (1944). A 2 - 3% protein solution was adjusted to 0.2 M;CaCl concentration and the calcium-sensitive fraction 2 removed by centrifuging initially at 35° C and 9,000 RPM and finally at 25° C and 21,000 RPM. The k-casein obtained was contaminated with a slow-sedimenting component as shown by sedimentation-velocity patterns. The slow-sedimenting component was obtained by collecting the top layer from crude k-casein solutions centrifuged for 5 hours at 40,000 RPM. The protein recovered was shown to be different from previously described caseins and was designated as I-casein. It was stable to calcium ion, possessed an $20 of 22 1.3 S at pH 7, contained 1.18% phosphorus, and did not stabilize aé-casein or give a visible reaction with rennin. Method of Fox (1958) as Modified by Morr (1959) k-Casein was prepared from whole casein or from Warner's abcasein fraction. The preparative operations were performed at 0 - 5° C unless otherwise stated. The starting material was adjusted to pH 11.3 and held for 20 - 30 min. Calcium chloride was added to a final concentra- tion of 0.2 M, the pH lowered to 8.3 and the resulting precipitate removed. The temperature was raised to 30° C causing more calcium-sensitive material to precipitate. The supernatant was adjusted to pH 4.7, dialyzed against water, and the precipitate collected. These steps were repeated to further remove the calcium-sensitive fraction. The precipitate was dissolved in 6.6 M urea and crude k-casein obtained by diluting to 3.3 l_'1_ urea concentration. Crude k-casein was again dissolved in 6.6‘M;urea and the pH adjusted to 11.3 for a short time. After lowering the pH to 4.7 and diluting to 3.3 M_urea concentration, a brown precipitate was removed and k-casein was precipitated from the supernatant by salting-out,(1.5 y'ammonium sulfate). These steps were repeated twice. The k-casein obtained appeared homogeneous by sedimentation-velocity studies. ‘Method of Pilson, Henneberry, and Baker (1960) for Fraction A OFCasein fraction prepared by the method of Warner was adjusted to: pH 12 at 25° C for 45 min, then lowered to pH 7.0, followed by addition of CaCl2 to 0.25 M;concentration. Calcium sensitive arcasein,(Qg-casein) )5 was removed by centrifugation and Fraction A was precipitated by adjusting the pH to 4.5. The precipitate was redissolved and the steps repeated. The material obtained was homogeneous by paper electrophoresis. The preparation was considered similar to Fraction S on the basis of the stability to calcium ion, reaction with rennin, and ability to stabilize as-casein. Method of McKenzie and Wake (1961) Whole acid casein was treated with CaCl2 (gg_0.4 M) at pH 6.5 - 7.0. The calcium-sensitive fraction was removed by warming to 35° C and centri- fuging at room temperature, first at 900 X G followed by 40,000 X C. After removing the calcium ion with oxalate, the supernatant was warmed to 25° C and 25g/100m1 of anhydrous Na2804 was added. The precipitate (k- and B-casein) was removed by centrifugation (900 X G), dissolved in water and dialyzed against 0.005 M_NaCl. Following adjustment of the pH to 7.2, an equal volume of absolute ethanol was added, followed by 2‘M_ammonium acetate until a definite mucilaginous precipitate formed. The precipitate was dissolved in 6 M urea and dialyzed against 0.005 M NaCl. If the dissolved precipitate was dialyzed against water or too long against 0.005 M_NaCl, the k-casein precipitated and was only soluble with difficulty. The preceding steps with ethanol and ammonium acetate were repeated to assure complete removal of B-casein. Residual fat was removed from the final product by centrifuging at 90,000 X G. k-Casein was obtained in yields of £2_20% of theory and appeared homogeneous by velocity-ultracentrifugation and urea-starch gel electrophoresis. Method of Cheeseman (1962) a-Casein fraction prepared by the urea method of Hipp, Groves, Custer and McKeekin (1952) was treated with oxalate to remove residual calcium. The solution was made 3.3 M;with respect to urea at 4° C and the pH adjusted to 4.9, followed by dilution to 2 M urea concentration which precipitated aé-casein. The supernatant, containing k-casein, was dialyzed against water and adjusted to 0.2 M CaC12 concentration at pH 6.6 - 7.0. Calcium-sensitive material was removed by centrifugation at 37° C and 2500 RPM. The preparation was 90% pure by free-boundary electrophoresis at pH 7.3. About 25% recovery was obtained assuming 15% k-casein in whole casein. Method of Hill (1963) Whole acid casein was made 0.25 M with respect to CaCl2 at 3° C and pH 6.7 - 7.2. After removing the calcium-sensitive fraction by warming to 35° C and centrifuging at 5000 RPM, the supernatant was dialyzed against water and concentrated. The solution was again treated with CaClZ, but this time centrifuged at 50,000 X G. The crude k-casein was applied to a DEAE cellulose column in pH 6.25 acetate buffer. A gradient elution was obtained by mixing the previous buffer with various amounts of pH 4.5 acetate buffer adjusted to 0.5 M;with respect to CaClz. Under these conditions, B-casein was eluted first, whereas k- casein was not eluted until the CaClz molarity had increased and the pH was lowered. k-Casein was soluble at pH 4.7 in 0.5 M CaClz. The purity of the preparation was not determined by the common physical methods. Method of Hipp, Groves, and MCMeekin (1961) for Preparing a3-Casein Although the protein Was not designated as such, it is undoubtedly similar, if not identical, to k-casein and therefore will be described here. The calcium-insensitive casein was prepared from warner's oHommoux mo p unoooou sOHuooHHHuoopH mumposon Hommom wchooH t Q How mosHm> omuHooHo> moxoom owuononoouuooHo men we mofiuhomoum nooHuouoosHpom mcoHuouoeonm oHommoux mo moHumHHouoouoto cHoH% poo aHowomHuuooooHuHo eoHuouosmoHuooHo ozu mo somHHomEoo H MHmHuoHom m .ohouuom onpooomop scum venomous ones moHuHHHHQz o .oHomoo oHuuooHoomH Houou mo owouooouom osu mo commonexm p .o m on aH.o n N>r_ «o.m mo um summon ouotmmoto oH poHuHoo mos oHououm o .No.H mos oHououm mo sOHumHuoooooo H .ooHuomnw oHomooux otu on on rosemoum mos uoooomaoo anon o 82:28 .. H mamas. ..'I\'| I.. 1'. v- I .IIIII 1|..V . :l-’!a: ,L. .VK. .v\ Ar. \ As A AoAc 1.1a 33.4 fldful- Hah'si,if In. rant! If . . . I . I , . 1, a QIUa. II , l I l l l I al q 0 I I IMiall h ”VIII H J’ '4‘! (CU: fvfld UJAU? ' n 7 AH m H s F N o N a- L N, x 1 :3: 4:6..— .annukwarrvpnfl 39 TABLE 2 Quantitative characteristics of the sedimentation-velocity patterns of the k-caseins shown in Figure 4a % of total Concentration refractive Preparation (mg/ml) r72 820 X 1013 area No. 3 6 0.1 12.9 92 No. 3A 6 0.1 -- 90 Prepared by E. M. McCabe 10 0.2 13.2 90 No. 3 purified by ' ultracentrifugation 6 0.1 14.5 96 No. 3A, bottom layer obtained from ultracentrifugation 6 0.2 13.1 95 No. 3A, pellet obtained from ultracentrifugation 5 . 0.2 16.6 93 No. 3A, purified by ultra- centrifugation and elution from G-75 Sephadex 8.2 0.2 13.4 97 k-Casein prepared by the ‘McKenziedWake-method 10 0.1 13.4 97 Fraction S purified by ultracentrifugation and elution froth-75 Sephadex 6 0.2 15.0 - a Experiments were performed in pH 7.0 phosphate buffer. t3?! ([7 HCM U) Acete Acet; Aceta Aceta ACEta 40 TABLE 3 Electrophoretic properties of k-casein (Preparation No. 3) in buffers from pH 2.3 to 8.6 Electrophoretic characteristics ... Mobility Refractive areaa Protein 2 _lv-1 Buffer concen- cm sec System tration Descend- Ascend- Fast Slow ( r/ 2 .= 0. 1) pH (%) ing ing peak peak HC1:NaC1 2.3b 2.0 +2.4c +3.2 100° 0 +0.8d +2.2d Acetate 4.5b 0.6 -3.0 -3.1 ... ... Acetate 4.6 0.6 -4.4 -4.4 92.7 7.3 Acetate 4.7 0.6 -4.6 -4.7 ... ... Acetate 5.1 0.6 -6.3 -6.5 ... ... Acetate 5.3 0.6 -6.2 -6.6 ... ... Acetate 5.5b 0.6 -6.4 -6.4 96.2 3.8 -5 e 9e b d Phosphate 6.0 0.6 -6.8 -7.6 95.1 4.9 -7.4 Phosphate 6.5 0.6 -7.2 ~7.4 92.5 7.5 PhoSphate 6.7 0.6 -7.7 -8.1 93.7 6.3 Phosphate 7.0b 0.6 -s.2 -8.5 94.7 5.3 Phosphate 7.1 0.6 -7.6 -8.1 97.7 2.3 Phosphate 7.5 0.6 -8.6 -8.9 ... ... Veronal:HCl 8.3 0.6 -7.5 -7.6 95.0 5.0 Veronal 8.6b 1.0 -6.7 -6.8 99.0 1.0. EMeasured from the descending patterns. bElectrophoretic patterns for these analyses are shown in Figure 7. Higher concentrations of protein are required at pH 2.3 to give comparable refractive areas. 9Mobility of the leading portion of the divided peak. dMobility of the trailing portion of the divided peak. eCombined area of divided peak. 41 Figure 2. Electrophoretic and sedimentation patterns of the material obtained from various steps: of the isolation procedure. Only the ascending electrophoretic boundariesare shown. The studies were performed in veronal buffer (pH 8.6, 0.1r/2) except for D and G which were in phosphate (pH 7, 0.1 r72). A- a-casein fraction, Preparation No. 3. B-o- casein fraction, Preparation No. 3 A. C- TCA precipitate, Preparation No. 3. D- TCA precipitate, Preparation No. 3; sedimentation velocity, 59,780 RPM, 20° C. E- TCA precipitate, Preparation No. 1. F- Calcium ion precipitate, . Preparation No. 3. G- TCA soluble fraction, Preparation No. 1. H- TCAsoluble fraction, Preparation No. 3. A I \‘n‘zr:§- J k 43 nsscnmmc ssmmuxaI-mocru nae-1109mm mm DIAGRMB ‘— vnom. means 1- pa 8.6 pH 7.0 Iggy? 59,780 3.19.11. PREPARATION NO. 1 PIIPIIATIOI l0. 2 “A... PIIPIIATIOI IO. 3 A... Figure 3. Electrophoretic and sedimentation-velocity patterns of the k-casein preparations reported in Table l. Ultracentrifugation was conducted in phosphate buffer (pH 7.0) at 3° C. ‘28!— ' 44 Figure 4. Sedimentation-velocity patterns of different k-casein preparations in pH 7.0 phosphate buffer. The rotor speed was 59,780 REM. The first patterns in rows A and B and the middle two patterns in row C were obtained from a regular analytical cell. The other patterns were obtained from-experiments using the synthetic boundary cell. Row A (left to right) "f Preparation No. 3, 0.1 [72 buffer, 2° c, 36 min.; Preparation No. 3 A, on ['72 buffer, 5.1° c, 28 min.; Prepared by E. M. McCabe according to the procedure given in Figure 1, 0.2 ['72 buffer, 25° c, 16 min. , Row B (left to right) - Ultracentri- fugally purified preparation No. 3, 0.1 [72 buffer, 3° C, 36 min.; Preparation No. 3A, bottom layer from preparative ultracentrifugation, 0.2 ['72 buffer, 20° 0, 16 min.; Preparation No.» 3 A, pellet from preparative ultracentrifugation, 0.2 '72 buffer, 20° C, 16 min. Row C (left to right) - Preparation No. 3 A, purified by ultracentri- fugation and elution from Sephadex G-75, 0.2 /2 buffer, 25° C, 20 min.; same as previous protein, 0.2 [72 buffer, 25° C, 24 min.; Fraction S purified as above, 0.2 [2 buffer, 25° C, 16 min.; k-casein prepared. by the McKenzie ake method, 0.1 r7 2 buffer, 24° C, 16 min. 45 LII k 6 mg/ml s t 550 6 mg ml, =65° 10 mg/ml, o = 80° LI} IN 6 tag/ml, 0:600 6 ng/ml, o = 65° 5 «an, o = 65° C I i ." 10 mg/ml, e = 700 3.2Ing/ml, 6 = 70° 6. 2 mg n1, =70° 10 'ng ’ml 9 = 70° Figure 4 46 . 98 m8 kHOuouonoH touoomom HoCOHwom :Houmom mo ComeOLH .m .2 .HQ Eouw uwww o mums cHomounx oxozloHNCoMoz emu new m GESHoo CH chanson ozH ”ouoz .HuHmoomIEsHoHoo A< :OHuoouw oHomoonm Am eoHommo oHots A< "N caoHoo A< "H cESHoo .mowommolm poo Aux Almo mo monouuoa oHuoHotmouuooHo How nonoumnooub « 47 ASCENDING DESCENDING a e 4225 390.; 10.15 v. cm.“ ...JL A... 3800 sec.; 9.18 v. cm. ..IL .JL. 3500 sec. 9. 04 v. Cm Fi ing‘lre 6. Free-boundary electrophoretic patterns of purified k-caseins by fillit 7 .0 phosphate buffer, (72 = 0.1. Row A - Preparation No. 3, purified No, 3 tI‘acentrifugation. Row B - Preparation No. 3A. Row C - Preparation , purified by ultracentrifugation and elution from Sephadex G-75. 48 ELECTROPHORETIC PATTERNS DESCRIBING ASCENDING 4C. 5000 sec. - 6. 94 v. cm. ACETATE ”2 5000 sec. 8 13 v. cm. 5 l PHOSPHATE I =0. 24 v. ca. PIOBPIATE pl 6. 0 t 3600 lec.; =9. 41 V. cm. PIOBPIAT! pl 7. 0 3600 see. =10 00 V. cm. 7‘23 3600 sec. 10. 96 v. ca. i? Flgure ‘7 Electrophoretic patterns of k-casein Preparation No. 3 in buffers rangi11 g from pH 2.3 to 8.6, r'/2 = 0.1 (see Table 3). l\|l|| T: N 0...! I xttooE 020k0£fi°kso~im « 311re 8 a “mein P- I -8.0— >~ T E o “a 3: -6.o= ° T E >' .2 NE. E o 2 '1‘ -4.0~ q o 0 ~ .2 0 X 2 n L“ 3 -2.o—- O L 3 +2.0- —< +4.0 ph F- lgure 8. A plot of the descending electrophoretic mobilities of k- casein Preparation No. 3 at various pH values (see Table 3). ID" A 5 0 \ \E\°E\ CUUOkswz 0.0“ Flgure 9. casein reme fi T T I T l.0 — « I.O g E \ O O s 31 C 0 Q) U 00 C S B E 0.5 - ABSORBANCE - 0.5 g Q Q 0 NITROGEN l l I l J . oo o o 3.6 3.3 4.0 4.2 4.4 pH Flgure 9. A plot of the nitrogen and 280 mu absorption for the k- caseln remaining in solution at pH values near the isoelectric point. IOC C c c nnu nhv AH. °\O » CO.C3\0m Cm C..00°.U l 0.0 ~ 0 2 ‘13Ure 1C *EParati 51 '00 I I r 4g, 0, -Casein in Solution (%) l _ l 0 0.05 0.|0 0.l5 0.20 Ra’io Klas Figure 10. The stabilization of org-casein by various amounts of k-casein Preparation No. 3. ‘" iEUr Cf A) 9:7» “SD '} 52 ELECTROPHORETIC PATTERNS SEDIMENTATION—VELOCITY DIAGRAMS DESCENDING ASCENDING 52,640 R.P.M. HPHOSPHATE pH 7. O I g—es '— L’L. ..Jl. I: OJL-N—J Figure 11. Electrophoretic patterns and sedimentation-velocity diagrams b I 1 Of A) Hipp §__t _a___1. (1952) (Dz-casein and k-laS-casein complexes induced by ‘—-58'— Precipitation from 3. O M urea and by C) momentarily adjusting a Solution of the mixture to pH 12.U1tracentrifugation was conducted in phosphate buffer (pH 7. 5) at 20° C. DISCUSSION General The original goal was to obtain k-casein in good yields without requiring the use of a preparative ultracentrifuge. Large quantities oftxs- and B-casein could be removed from whole casein by adjusting solutions in 6.6 ygurea to 12% TCA (w/w) at 3° C. The contaminating B- casein was difficult to-remove, therefore, aficasein fraction prepared by a modification of the method of Hipp gE_§l, (1952) was used as the start- ing material. The aS-casein which remained with the k-casein following ureaJTCA fractionation was easily removed by low Speed centrifugation in the presence of 0.25 !;CaClz. Cyanate ion was 22£_operative in these concentrated urea solutions since isoelectric casein was used and the pH was lowered to <24.7 when TCA was added. Marier and Rose (preprint), using a spectrophotometric method of improved sensitivity to detect cyanate ion, have shown that cyanate is not present in acidic urea solutions, ie., lower than pH 5.5. 'k-Casein was obtained in quantities of 30 - 50% of the theoretical amount with a purity of 90 - 92%. Although the fraction did not have the desired purity for physical studies, it was relatively good compared to those preparations thus far reported. B-Casein (10%) was present in the k-casein prepared by Cheeseman (1962) and probably the k-casein fraction was also a contaminate, although ultracentrifugal studies were not performed to detect this component (this writer has experienced greater difficuity in removing.the l-casein fraction than the B-casein); The method of Fox (1958) as modified by Morr (1959) gave a preparation which was apparently hemogeneous by free-boundary electrophoresis and sedimentation- 53 54 velocity, however, a synthetic boundary cell was not used for the sedi- mentation analyses. This method involved repeated exposures to high pH, several times in the presence of 6.6 figurea. Waugh (1958) reported a purification step for Fraction S which increased the purity to 90%. k-Casein prepared by the method of Long (1958) (also see Morr, 1959), from a mixture similar to Fraction S was shown to contain the slow- sedimenting l-casein fraction in amounts of possibly more than 10%. Since the preparative ultracentrifuge was used initially for the constant pH methods, the volume of skimmilk was limited to one liter and thus the amount of k-casein obtained was also reduced. The method of McKenzie and Wake (1961) obtained good yields of apparently homogeneous k-casein, however, in some respects preparations by this method appeared to be "denatured" (Neelin, Rose and Tessier, 1962). Recently, Hill (1963) has published a method of preparing k-casein, but unfortunately the purity was not determined. The ability to obtain relatively pure k-casein in large amounts ‘was an asset when a preparative ultracentrifuge became available. Subsequent purification by ultracentrifugation and elution from G-75 Sephadex yielded a preparation which was 97% pure by analytical ultra- centrifugation using a synthetic boundary cell. Similar purity was indicated by urea-starch gel electrophoresis which showed only small quantities of several leading bands. With reSpect to these two criteria of purity, this preparation appeared identical to that prepared by the McKenzie-Wake method . Electrophoretic and Ultracentrifugal Characterizatigg “ The homogeneity of k-casein preparations was best determined by 55 sedimentation-velocity studies in phosphate buffer (pH 7, 0.1r7/Z) at 4° C in a synthetic boundary cell. Free-boundary electrophoresis could detect B-casein, however, the l-casein fraction moved with the k-casein (Long, 1958). At 4° C, both the B- and l-casein appeared in the slow- sedimenting boundary since B-casein has an $20 of 1.36 S under these conditions (Sullivan, Fithatrick, Stanton, Annino, Kissel, and Palermiti, 1955). The observed sedimentation coefficients for k-casein were in accord with those reported by others (McKenzie and Wake, 1961; Waugh and von Hippel, 1956; Long, 1958; Morr, 1959). Under certain conditions crude k-casein was observed to interact with B-casein. Free-boundary electrophoresis revealed a decrease in the relative area of the B-casein peak from 7.5% at pH 6.5 to 3.8% at pH 5.5. The relative area also decreased to 1% upon increasing the pH to 8.6. Furthermore, the mobility of k-casein reached a maximum at pH 7 to 7.5 and then decreased due to the incorporation of B-casein into the complex. Morr (1959) also observed a decrease in mobility at pH 8.4 as compared to 7. The work of McKenzie and Wake (19593) on Fraction S showed 80% of the area attributable to k-casein by free- boundary electrophoresis, but only 50% of the area by ultracentrifu- gation. Also, the mobility at pH 7 (phosphate, 0.1 rq/Z) was -4.88 Tiselius units which is much lower than the value reported herein, again indicating interaction with a-casein. These researchers also studied the "abcasein split" using both whole acid casein and first- cycle casein. They found the Split to occur more readily in acid casein and only when k-casein was present did it occur in other fractions, which led them to suggest that the split was due to the incorporation of k- casein into'fiifficultly reversible aggregates” with as-casein upon acid 56 precipitation. Their results are similar to the data reported here for the interaction of k-I and B-casein in that the relative area of the B- .. casein peak decreased on lowering the pH from 7.8 to 6.3 -- the decrease being most marked at 6.3 .. and on raising the pH from 7.8 to 8.2 (however, the area was nearly as large at pH 8.8 as at 7.8). Also, the electrophoretic split in the pattern for crude k-casein at pH 5.5 and 6.0 reported here appeared similar to the data reported by McKenzie and Wake for whole casein. Although these data are not in complete agreement, one must remember that k-casein occurs in whole casein as a complex with (XS-casein and therefore its properties will be influenced by the (ls-casein. On this basis, the "(x-split" in whole casein may be due to an interaction of the k-, Gig-complex with B-casein. This view is supported by the fact that B-casein is known to be present in the boundary ascribed to the Ol-Casein complex (Sullivan 3331. 1955). In alkaline buffers where k-casein is believed to be complexed With B-casein, the peak was not split, an observation attributed to the formation of the stable complexes. In acid buffers the casein complexes appeared to behave as association-dissociation systems re- sulting in the observed boundary Split. The isoelectric point of k-casein at 0.1 ionic strength lies batWeen pH 3.8 and 4.1 as determined by electrophoretic mobility and minimum solubility studies. This value agrees with that reported by Hipp 332;. (1961) for Qty-casein. Stabilization and Interaction with (XS-Casein The k-casein preparations were eXCellent stabilizers of (XS-casein. 57 At a k-flI-casein.ratio of l : 10 about 90% of the aS-casein was stabil- ized, a value similar to that reported by Zittle (1961). Complex formation was induced in a mixture of 1 : 4 (w/w) k-/as- casein by dilution in concentrated urea solutions and by short exposure to pH 12. In both cases, electrophoretic and ultracentrifugal analysis obtained results similar to that for an arcasein fraction prepared by the method of Hipp 25.31, (1952). The electrophoretic mobility of the complex obtained from urea solutions was greater than that for the<1~ casein fraction or the other complex, however, in View of the preceding discussion of B-casein interaction, this increase could be explained on the basis of removal of B-casein from the complex. In fact, the electro- phoretic pattern for the urea-formed complex showed less B-casein than that for the complex induced by pH adjustment. The sedimentation coefficient of the latter complex was larger than that for the abcasein fraction or the urea-formed complex, indicating either a different shape or a larger complex. One cannot conclude from these data that the complex which existed in the native micelle was formed, but only that a specific interaction occurred between k- and as-casein. ‘Moreover, since these proteins were not completely pure, the role of B- and l- caseins could not be determined. Characteristics of the Preparation as a Primary Substrate for Rennin Treatment of purified Preparations No. 3 and 3 A with rennin resulted in the formation of a precipitate in either redistilled water or buffered solution at pH 7. However, when a crude preparation containing large quantities (ES 30%) of I-casein fraction was treated with rennin in redistilled water, a precipitate did not form. Addition of NaCl to 0.1 58 3 concentration caused the solution to become opalescent, but still no precipitation occurred. Possibly an interaction had occurred between k- casein and the l-casein fraction resulting in greater stability of the para-k-casein. Long (1958) also observed an interaction between k- and l-casein. After repeated preparative centrifugation of crude k-casein in 0.2‘§_NaCl, analytical ultracentrifugation showed only a small amount of k-casein. However, a similar analysis conducted 48 hours later re- vealed a considerable amount of I-casein. Crude k-caseins, containing the l-casein fraction, served as good stabilizers of aS-casein, but studies were not performed to compare these with purer preparations. Nevertheless, the possibility exists that an interaction of the l-casein fraction with k-casein may increase the latter's ability to stabilize aS-casein. The soluble peptides cleaved from k-casein by the action of rennin in redistilled water at approximately neutral pH were obtained by ultra- centrifugation of the precipitated para-k-casein which yielded a hard pellet, easily separated from the supernatant. Not all of the soluble peptides, consisting of 30% by weight of the k-casein, were soluble in 12% TCA solution,as also evidenced by the amount of soluble nitrogen compared to the TCA-soluble nitrogen. Twenty-three per cent of the nitrogen was classified as soluble, a value in agreement with that reported by Wake (1959). The unpurified Preparation No. 3 A yielded a 7% increase in the TCA-soluble nitrogen. Probably this would have been greater for the purified material (Wake, 1959, reported 6.7% and Beeby, 1963; reported 8 - 9%). These results seem to indicate that more than one peptide is released by rennin. However, according to Wake (1959) some of the glyco-macropeptide -- 12% TCA-soluble material -- may be precipitated by 12% TCA. 59 A molecular weight of 8400 was calculated for the peptides using the sub-unit molecular weight of k-casein (see Part II of this thesis) and the fact that 30% of the original k-casein remained after removal of para-k-casein. This value was similar to the value obtained by short- column equilibrium -- gg_6000 -- and the range of values -- 6000 to 8000 -- reported by Nitschmann 25.21, (1957). Sedimentation-equilibrium experi- ments performed in this laboratory have indicated a molecular weight of SE 9000 for the glyco-macropeptide GMcCabe, unpublished data). Other Proteins Obtained Duringgthe Preparation of k-Casein or as a Result of a Slight Alteration in the Preparative Procedure By using a-casein fraction, free of electrophoretically discernible B-casein, as the starting material, ag-casein was obtained as a precipi- tate from 6.6 ELurea -- 12% TCA solutions. The preparation was homo- geneous when examined by free-boundary electrophoresis and sedimentation- velocity ultracentrifugation. However, a band which appeared to move slightly ahead of fi-casein was observed in urea-starch gel electrophoretic patterns (Figure 5). Nevertheless, ag-caseins prepared by other procedures have shown greater heterogeneity, e.g., Neelin gt a1. (1962), when exam- ined by this method. The value obtained for the sedimentation coefficient was in accord with those reported by Waugh and von Hippel (1956), Long (1958), and McKenzie and Wake (1959b); namely, 4.5 S, 4.6 S and 4.4 S, respectively. Also the electrophoretic mobility agreed with that given by'McKenzie and Wake (1959a); u =-7.17 Tiselius units in veronal buffer, pH 8.2. V Incorporation of 10.6 g/l NaCl into the 6.6 §_urea used to purify the Ohctsein fraction resulted in the complete absence of k-casein in the 60 material obtained in Step 6 of the preparation scheme (Figure 1). 0n the basis of the stability to calcium ion, sedimentation coefficient, non-reactivity with rennin, and inability to stabilize aé-casein, the material was considered similar to the l-casein fraction reported by Long (1958). There seems to be some inconsistancy in the product ob- tained (Neelin'gg‘gl., 1962). Three of the four preparations made in this laboratory were essentially identical. The fraction appears homo- geneous in neutral phosphate buffer, but ultracentrifugal studies in 5 E guanidine . HCl clearly indicated molecular heterogeneity. Urea- starch gel electrophoresis also revealed numerous bands (Figure 5), all of which moved ahead of the B-casein position. Particularly characteristic of the I-casein fraction was the band which moved with the front. Whether or not the band in the aS-casein region was as- casein remains to be determined. Since the fraction is homogeneous in non-dissociating solvents, a complex must exist between the con- stituent proteins under the conditions employed. SUMMARY OF PART I A method was developed for preparing k-casein of about 90% purity in yields approximating 30 - 50% of the ammmt theoretically present. The preparation was characterized by free-boundary electrophoresis, urea-starch gel electrophoresis, sedimentation-velocity ultracentrifu- gation, ability to stabilize (JCS-casein, and its reaction with rennin. The results of these characterizations confirmed that the protein possessed properties typical of k-casein as reported previously. Although the preparation was not as homogeneous as desired for physical studies, the nethod provided large quantities of the crude k-casein which could be readily purified, yielding a final product of 97% purity. The preparation scheme also afforded means of obtaining the as- and l-casein fractions. 61 PART II PROPERTIES OF KAPPA-CASEIN INTRODUCTION TO PART II The k-casein isolated from bovine milk forms relatively uniform aggregates in inorganic salt solutions below pH 11. To assess the properties of this protein more completely, the associated protein molecules must be dissociated prior to characterization. Unfortunately, the dissociation can be accomplished only by using strong dissociating solvents such as concentrated acetic acid, guanidine, urea or sodium dodecyl sulfate. These agents make the determination of themodynamic or hydrodynamic properties less accurate due to preferential interactions and the non-ideal behavior of protein in their presence. However, studies in recent years have shown that these errors probably were not greatly significant. To cite a few of the many examples; a) Harrup and Woods (1961) found that molecular weights of bovine serum albumin, egg albumin, B-lactoglobulin, lysozyme and insulin in anhydrous formic acid were in agreement with those detemined in other solvents; b) Kielley and Harrington (1960) found that the mole- cular weight of ribonuclease in 5 21 guanidine - HCl was in accord with the known value; c) Trautman and Crampton (1959) obtained the same re- stllt for ribonuclease in 6 1.4.. urea; d) Massey, Hofmann, and Palmer (19 62) obtained an Archibald molecular weight for lipoyl dehydrogenase in 6.5 If; urea which agreed with the chemical analyses of amino acids and FAD; e) Hofmann and Harrison (1963) found that the Archibald molecular weight of apoferritin in SDS solutions was in accord with X-ray diffraction studies; f) thantis and Waugh (1957) obtained a m°1ecular weight for insulin in 33% acetic acid - 0.15 1_’I_ NaCl which agreed with the chemical analysis; and g) Criddle, Bock, Green, and Tisd«Elle (1962) obtained the same molecular weight for the structural 63 prote chara prese repor 64 protein of mitochondrion in 67% acetic acid, 8'§,urea, and 0.1% SDS. Information relating to the chemical compositfimn and physical characterization of k-casein in neutral salt solutions and in the presence of dissociating agents were the objectives of the experiments reported in this section of the thesis. Sed'm petiomed 111C temp: Acapilla' studies a Optical a cell. Bc Of the v; PlaStic < Vindows 1 in an All served a The Plates 6 The mic: and was Vi dilutio Ianging bath eq‘ “01 th. tmpel’a‘. differeI F01 EXPERIMENTAL Apparatus Sedimentation-velocity and sedimentation-equilibrium studies were performed in a Spinco Model E analytical ultracentrifuge equipped with an RTIC temperature control unit and a phase plate as a schlieren~diaphragm. A capillary-type synthetic boundary cell was used for most of the velocity studies and also to determine the initial concentrations in terms of optical areas. Equilibrium experiments were performed in a double-sector cell. Both of these centerpieces were the filledéEpon type. For some of the velocity studies a single sector, analytical cell with a Kel-F plastic centerpiece was used. The 12 mm cells equipped with quartz windows were used in all determinations. Centrifugation was performed in an An-D duralumin rotor and a General Electric AH-6 mercury lamp served as the light source. The schlieren patterns were preserved on Kodak metallographic glass plates and measured with a Nikon.Model 6 Shadowgraph microcomparator. The micrometer stage traveled 25 mm on the y-axis, 50 mm on the Xaaxis and was read directly to 0.002 mm. Viscosities were determined with a Cannon-Ubbelohde semi-micro (tilution viscometer which could be operated with 8 Volume of solution ranging from 1 to 20 ml. A circulating, Precision Scientific water bath equipped with a Precision micro-set thermoregulator was used to con- tnaol the temperature for viscosity and density determinations. The temperature was controlled to j; 0.0l° C as indicated by a Philadelphia differential thermometer. For precise weighing, a Cahn Gram Electrobalance was used which has 65 a tota range. places titrat equili H1 and ratio. using were d 66 a total capacity of 1 g on the maximum range and 1 mg on the minimum range. The balance setting of the wheatstone bridge reads to four places on all ranges. A Starrett microsyringe-burette was used for sulfhydryl group. titrations and to fill the ultracentrifuge cell for short-column equilibrium experiments. The microsyringe had a total capacity of 250 pl and the micrometer could be read to 0.1 ul. Spectrophotometric measurements were made with a Beckman DK-2 ratio-recording spectrophotometer or a Beckman DU spectrophotometer using quartz cells with a 1 cm light path. The hydrogen ion concentrations of various buffers and-solutions were determined with a Beckman.Model G pH meter using a glass electrode. Materials were dried in temperature. controlled Cenco vacuum oven. Chemicals ‘pdfiercuribenzoate (PMB) was obtained from the Sigma Chemical Company. Glutathione (GSH) was purchased from'Matheson Company. Guanidine . HCl (GU) and Z-mercaptoethanol (ME) were Eastman Organic Chemical products. The guanidine - HCl was recrystallized from a 1 : 1 (v/v) mixture of absolute methanol and ethyl ether as described by Greenstein and Jenrette (1942). The urea, obtained from Mallinckrodt, 'was recrystallized from 60% ethanol and dried below 60° C in the vacuum oven. Sodium dodecyl sulfate (SDS) was obtained from Fisher Scientific and used without further purification. Fluorocarbon oil FC-43, purchased from Spinco, was used to form. a false bottom for ultracentrifugal studies._ N-acetylneuraminic acid (NANA), used to obtain a standard curve for NANA determination, was supplied by General Biochemicals. Unles acid were . time 1 from . the 13] Va £110: in 50C m1 of dIOps hour, 67 Unless otherwise specified, all chemicals were reagent grade quality. Chemical Methods Amino Acid Analysis1 The amino acid analyses were performed on 24, 48, and 72 hour acid hydrolysate, using a Beckman Acid Analyzer. The values obtained were extrapolated to zero time for acids whoée values decreased with time of hydrolysis. The quantity of protein analyzed was determined from a micro-Kjeldahl nitrogen analysis. Percentage of nitrogen in the protein was determined on samples dried at lOS°"C over P205 in the vacuum oven for 48 hours. Nitrogen The digestion mixture consisted of 5 g SeO2 and 5 g CuSO4-5H20 in 500 ml of concentrated H2804. Protein (l-Smg) was digested with 1 m1 of digestion mixture on an electric burner for 4 hours, then 5 drops of 30% H202 was added and digestion continued for another half hour, After cooling, 2 m1 of redistilled water was added. Immediately before attaching the digestion flask to the distillation Lmit, the sohltion was neutralized with 23 9 m1 of 40% KOH solution. The ammonia Was distilled by heating with a microburner and collected in 5 ml Of a 47. boric acid solution containing 3 drops of indicator. Standard 0' 1038 _I\_I_ HCl diluted 5 times was used to titrate the ammonia. The normality of the standard was determined by titration with Sigma 121: trig (hydroxymethyl) aminomethane. 1 Th Rie amino acid analysis was obtained through the courtesy of DIS. J. P. elltn J. C. Speck and H. A. Lillevik of the Biochemistry Department. 3 anon nitro sampl (Mote and F Weig} Ferrc acid 68 The recovery of nitrogen from standards was 100.2 :1; 2.9% using ammonium sulfate and 100.0 1: 3.3% using tryptophan. The per cent nitrogen in B-lactoglobulin determined in quadruplicate on 33 5 mg samples was 15.63 i .03% as compared to a reported values of 15.60% (McMeekin, 1954). Sulfur The total sulfur was determined by Spang Microanalytical Laboratory, Ann Arbor, Michigan. Prior to analysis, the protein was dried over P205 at 105° C for 48 hours in a vacuum oven. Phosphorus Phosphorus was determined colorimetrically in a wet digest (112804 and H202) of the protein~-- dried in the vacuum oven over P205 and weighed with the microbalance -- by the method of Sumner (1944). Ferrous sulfate was added in slight excess to reduce the phosphomolybdic acid to a blue eclor which was read at 610 mu. NeAcetylneuraminic Acid The method used was reported by Aminoff (1961). Similar to the Procedure given by Warren (1959), the method is also specific for the free acid. The protein was dried to constant weight in the vacuum oven OVer P O at 105° C and weighed with the microbalance. A Standard '25 curve was prepared using commerically available NANA. An amount of protein corresponding to 20 - 40 ug of NANA was dissolved in 0.5 m1 of 0. 1 3 H2504 and hydrolyzed for 1 hour at 80° C thus releasing the NANA. Per iod ate reagent was added and the sample incubated at 37° C for 30 min ’ after which the excess periodate was reduced with sodium aresnite. Thio ing extr mine ing a to pH remov 69 Thiobarbituric acid reagent was added and the color developed by heat- ing in a boiling water bath for 7.5 min. The color compound: was extracted in 5 m1 of acid-butanol and the absorbency at 549 mu deter- mined in a Beckman DU Spectrophotometer. Disulfide Bond Reduction The disulfide bonds of k-casein were reduced with mercaptoethanol (ME) at a level of 5:3 2 ul/mg of protein in 5 _M guanidine . HCl (GU) adjusted to pH 8.4 with methylamine (Anfinsen and Haber 1961). Follow- ing a reduction period of about 24 hours, the solution was adjusted to pH 3 with glacial acetic acid. The reducing agent and GU were removed by passing the solution over G-25 Sephadex equilibrated with 0. 1 g acetic acid. Complete elution of the protein was achieved approximately 40 to 50 m1 prior to the elution of ME. Reoxidation 0f the sulfhydryl groups should be negligible under the acidic con- ditions of this elution. Sulfhydryl Group Titration The sulfhydryl groups were titrated essentially according to the Procedure described by Boyer (1954) and reviewed by Benesch and Benesch (1962). However, to prevent reoxidation of the reduced disulfide bonds following the removal of ME, the following procedure was devised: a) a protein determination was made on an aliquot of the prOtein solution immediately following its elution in 0.1 Li acetic acid f rom the Sephadex c-25 columns, b) excess PMB was added directly to th e reduced protein in 0.1 M acetic acid, and c) the pH was adjusted to 7 with 0.1 g NaOH. the dete EDOU to t1 quant centr titra (Bene: solutj miCrob method tratio1 by Boy. fErred the 501 IEaCtio reacted 63H 801: Volumes a hm prOte 5“ and 70 The volume of protein solution was accurately measured after removing the aliquot and before adding the PMB solution. The concentration was determined on the aliquot by. measuring the absorbency at 280 mu. The amount of portein in the reaction mixture was usually 15 - 20 mg. A known quantity of PMB dissolved in redistilled water was added to the reduced k-casein and amounted to approximately ten times the quantity required to react with the newly formed SH groups. The con- centration of PMB in solution was determined just prior to addition by titration with a freshly prepared standard glutathione (GSH): solution (Benesch and Benesch, 1962). The concentration of the standard GSH solution was determined by weighing the desiccated crystals on the microbalance and dissolving with water in a volumetric flask. This method provided results which were in good agreement with the concen- tration calculated by using the extinction coefficient at 232 up given by Boyer (1954). Fifty milliliters of protein solution were quantitatively trans- ferred to a 100 ml volumetric flask. After standing for 1 hour at pH 7, the solution was made 7 M with respect to urea to insure complete reaction of the SH groups and the solution made to volume. The un- 1"matted 1MB in aliquots of this solution was titrated with the standard GSH 8Olution used previously to titrate the PMB solution. Thus, standard GSH Solution was added to both the sample and reference cell in equal volumes With a Starrett microsyringe and the end point determined by f0llowit-lg the change in absorption at 250 up. A blank consisting of a volume of 0.1 M acetic acid was treated in exactly the same manner as the Protein solution. The difference between the amount of PMB in the blan k and the sample represented the quantity of 343 bound to the protein. 71 One determination of the number of SH groups was made by a direct titration with standard PMB solution. In this experiment, however, a clear solution was not obtained unless the pH was adjusted to 9. Some re-oxidation could occur under these conditions. Nitroprusside The test was conducted in the presence of 5 M guanidine - HCl according to the procedure described by Hamilton (1960). One part of sodium nitroprusside to two parts of sodium carbonate were ground in a mortar until no crystals of the former could be distinguished. About 10 mg of this mixture was added to 1 ml of the protein solution after diaplacing the air with nitrogen. The test was performed on a reagent blank and a sample of B-lactoglobulin as a positive control concurrently with each experiment. Physical Methods Ultracentrifugation Determination of the protein concentiation. The freeze-dried Protein was weighed on a microbalance and dissolved in an appropriate 801vent. The weight was corrected for the moisture content as deter- mifled on a portion of the freeze-dried sample by drying to constant weight in the vacuum oven at 105° C over P205. Following dialysis against the SOlvent;, the solution volume was measured with a Hemilton Microliter sYT-‘inge. In some instances the concentration was rechecked by nitrogen. analysis. In several cases, the concentration was determined by measuring the area of the schlieren pattern for the initial boundary formed in 72 synthetic boundary cell. The amount of protein was determined from a standard plot of area and concentration constructed from data obtained in a similar manner. Sedimentation-equilibrium. The short-column equilibrium technique described by Van Holde and Baldwin (1958) was used. Centrifugations were performed in the double sector cell at 25.0° C using solution Column lengths of 1 to 2 mm. A period of 24 hours was allowed for attainment of equilibrium. This time was more than adequate as indi- cated by characteristics of the schlieren patterns and calculations made from an assumed,approximate diffusion coefficient. Schlieren patterns for the concentrated urea and GU solvents may also exhibit some curvature even at the low speed used. Consequently, the column which contained the protein solution was bracketed by the solvent column, ie., the false bottom (flourocarbon oil, FC-43) of the solvent column was slightly below and the air-liquid meniscus slightly above that for the solution column. To achieve this physical relationship the amount of solution required to produce the desired column length was calculated and precisely measured into the cell with a Starrett micro- syringe along with predetermined quantities of flourooarbon oil and solvent. Normally, the amounts added were a) 110 pl of oil and 70 pl of solution in the solution sector and b) 100 pl of oil and 90 ul of solvent in the solvent sector, resulting in a 10 ul differential on each side of the solution column. The protein solutions were dialyzed against their respective solvents for approximately one week at 20° 0 prior to sedimentation analyses. Optical areas obtained with the synthetic boundary cell were used as a measure of the concentration for molecular weight calculations. Details of the procedure are presented in Appendix II. 73 Diffusion coefficients were calculated from patterns obtained during the approach to equilibrium experiments according to the method of Sophianopoulos, Rhodes, Holcomb, and Van Holde (1962). Approach:§o-Eguilibrigm, This procedure was used principally to determine the molecular weight of the low-molecular weight component in a heterogeneous system. Experiments were performed at various rotor speeds and the data plotted as suggested by Trautman (1956) and adapted to heterogeneous systems by Erlander and Foster (1959). The details of this procedure are given in Appendix II. Since the concentrated GU, urea, and SDS solvents formed a gradient under these conditions, pre- caution was taken to assure that exactly the same volumes of solution and solvent were placed in the double sector cell. The patterns thus obtained showed only one liquid-air meniscus. In some trials, only one rotor speed was used in which case the weight-average molecular weight was calculated as described by Schachman (1957). .Sedimentationdvelocity. These experiments were performed at 20 or 25° C using a rotor speed of 59,780 RPM. The low sedimentation coefficients observed for the proteins in the presence of the dissoci- ating solvents precluded the use of the synthetic boundary cell in these studies. Accurate sedimentation coefficients were obtained with this cell for coefficients less than 1 S (Schachman, 1959) and the problem of restricted diffusion at the meniscus was eliminated. Furthermore, concentrated solutions of GU, urea, and SDS also form gradients at the meniscus making observations of the protein boundary difficult unless a synthetic boundary cell is used. Then, the protein boundary is formed at the start of the experiment and at a considerable distance from the 74 air-liquid meniscus. The cell was filled by placing 300 pl of protein solution in the lower compartment and 100 pl of solvent, which had been dialyzed against the solution, in the upper compartment. Sedimentation coefficients, determined by plotting the logarithm of the maximum ordinate against time, were corrected to values corres- ponding to water at 20° C (S The concentration corresponding to 20,w)° a particular $20 was the average concentration of the first and last w 9 frame used to obtain the $20 and was corrected for radial dilution ) according to Schachman (1959). Detailed calculations are presented in Appendix II. Densities and partial specific volume. The solvent densities were determined with 25 m1 pycnometers at 25.0 :;0.0l° C. Solution densities were calculated according to Fujita (1962) using the relation ,0 solution = ,0 solvent + ( 1 - ? psolvent) c where :1: is the partial specific volume of the protein and 3 its con- centration in g/ml. Freshly boiled, redistilled water was used to calibrate the pycnometers before each determination. Duplicate deter- minations agreed to;i 0.0001 or better. The partial specific volume was calculated from the amino acid analysis of the protein using the relation 3: W1 1 Vi Z“ i where E is the partial specific volume of the protein, Hi the specific volume of the iFh amino acid residue, and Ki the weight per cent of the 75 1th amino acid residue (Cohn and Edsall, 1943; McMeekin, Groves, and Hipp, 1949). The weight per cent of the amino acid residues is given by 18 M. 1 Wi = gi/IOOg prote1n where g, is the grams of the 1th amino acid and M1 the molecular_Weight of this amino acid. Intrinsic viscosity determinations. The relative Viscosities were determined at 25.0 i 0.0l° C. The outflow of time for water was 285.8 seconds. Four to six observations of the outflow time were obtained for each determination. All solutions were filtered through a sintered- glass filter. The relative viscosity was calculated according to the relation t p ”A. = "e: p": where Q, 1;, Bare the viscosity, outflow time, and solution density and 20,. 5,, "and 8, represent similar quantities .for the solvent. The solution density was calculated'as previously described. The intrinsic viscosity [ Q] was determined by plotting the reduced viscosity (Qsp/c) against the protein concentration in g/ml, where ’sta = - 1. O RESULTS Composition The amino acid composition of k-casein is shown in Table 4. These values were based on a nitrogen content of 15.3% as determined by micro-Kjeldahl analysis. The values for Thr, Ser and NH3 were obtained by extrapolation to zero hydrolysis time. The weight per cent of the amino acid residues (Wi) are presented together with a compari- son of the number of residues per 28,000 g of protein for a) purified Preparation No. 3A, b) the k-casein studied by Jollés g£_al, (1962), and c) a3-casein reported by Hipp £E.§l: (1961). The residues were calculated from their data which were reported as g/100 g protein. A summation of the residue weights which includes in addition to the amino acid residues, the values for NANA and phosphorus, as well as the values of Jollés §E_§l, (1962) for Cys,’Tr', galactose, and galactosamine amounts to 95 per cent. The partial specific volume was calculated from the amino acid residue weights and the concentration of NANA (1.4%) for the purified Preparation No. 3A. The weight percentages for Cys (1.30%),'Try' (0.96%), galactose (1.4%), and galactosamine (1.2%) were reported by Jolles 31331. (1962). By this method a E of 0.729 was obtained, _ 2: vi “1 v = = 68.925 = 0.729. W1 94.55 3.8. , Partial specific volumes of 0.62 for hexoses and hexosamines and 0.59 for sialic acid were used (Bezkorovainy and Doherty, 1962). The average of several determinations gave values of 0.22% phos- phorus and 1.4% NANA for purified Preparation No. 3A. The percentage 76 77 of P reported by JollEs g£.§1, (1962) and Thompson and Pepper (1962) was in good agreement with this value (0.217 and 0.22, respectively). The values reported for NANA have been subject to considerable variation depending upon the method of preparation (Marier, Tessier, and Rose, 1963). The purified kacasein preparation (No. 3A) contained 0.70;: 0.02% sulfur. The calculated molecular weights in terms of the per- centages of NANA, Try, P, and S are shown in Table 5. Physical Pgoperties of k-Casein Properties in Non-Dissociating Solvents The results of sedimentationdvelocity studies performed on differ- ent lots of purified No. 3A at temperatures of 3° and 25° C and ionic strengths from 0.1 to 0.5 are shown in Table 6. The S20 value for a McKenzieéWake preparation has also been included. Temperature and ionic strength had no significant effect on the sedimentation co- efficient. The data were fitted to the equation $20 = $20 (l-kc) ,giving the result 820 = 15.6 (1 - 0.0165C) 1:0.25, where 320 is in Svedbergs and p.1n mg/ml. The viscosity of this preparation was studied in 0.1 M,NaCl at 25.0° C. Fitting the data to the Huggins equation (Tanford, 1961) gave asp/c = 9.5 -+ (0.217) (90.25) c i 0.06, where the intrinsic viscosity was 9.5, the Huggins constant 0.217, and E_was in mg/ml. The data are plotted in curve C of Figure 12. Properties in Dissociating Solvents Sedimentation characteristics in SDS solutions. k-Casein 78 (purified No. 3A) was sedimented in buffers at pH 8.6 or 9.25 which contained from 2.5 to 40 mg/ml SDS. Usually the solutions were placed in the regular analytical cell without prior dialysis. Thus, a boundary due to SDS did not appear until the unbound SDS concentration exceeded the critical micelle concentration, i.e., 0.11% (Hofman and Harrison, 1963). An SDS boundary became apparent as the weight-ratio of SDS/protein approached 1. Apparently 0.5 to l g SDS was bound per gram of protein. Since the SDS and protein boundary were not com- pletely separated, the pattern was measured on the Trautman z-scale and X2 ZSY’plotted against X to correct for radial dilution. Gaussian curves were fitted to these plots to obtain the maximum ordinate for each peak. The calculated sedimentation coefficients are recorded in Table 7. A value of approximately 2.3 S was obtained for the sedimentation coefficient in SDS concentrations ranging from 2.5 to 20 mg/ml. Archibald molecular weights were obtained for k-casein solutions in veronal buffer containing 10 mg/ml SDS and 5 and 10 mg/ml of protein. The solvent, dialyzed against the protein solution, was used as a reference. The apparent molecular weights were corrected according to the equation M=MaPI/l+x1(i:;pp) , where X1 represents the grams of SDS bound by one gram of protein, 30 the/partial specific volume of SDS, E? the partial specific volume " of the protein, and £2 the solution density (Schachman, 1960). Even at a maximum x1, i.e. 1.0, molecular weights were calculated to be approximately 100,000. The sedimentation coefficient decreased as the 79 SDS concentration was increased to 30 or 40 mg/ml. This characteristic could be caused by unfolding or swelling as well as by molecular dissociation. Furthermore, the steep refractive index gradients due to free SDS, and the charge effect caused by bound SDS at these high concentrations make molecular weight calculations of questionable reliability. Consequently, further studies along those lines were not performed. Sedimentation and viscosity characteristics in 5.0 M GU solutions. The results of short-column equilibrium experiments performed on kncasein (purified No. 3A) in 5.0 §.G5 at pH 4.8 are recorded in Table 8 and Figure 13 (filled circles). A molecular weight of 126,000 was obtained by a leastnsquares extrapolation to zero concentration. A marked concentration dependence of the apparent weight-average molecular weight was noted. The ratio Mz/Mw was greater at a concen- tration of 3 mg/ml. Additionally, a Van Holde-Baldwin plot of the data showed considerable deviation from linearity (Figure 14B). The molecular weight of the small component present in a 3 mg/ ml solution of k-casein in 5.0‘M.GU was determined from a Trautman plot of approach-to-equilibrium data (see Figure 15) and yielded a value of 54,000. Sedimentation-velocity data were best represented by the linear relation SZO,w = 3.18 (190.0178 c), where S20,W is in Svedbergs and g.in mg/ml. A value of 3.1 Ficks “D20,w‘ was calculated from a short-column equilibrium experiment at a protein concentration of 10.3 mg/ml. An approximation of the 80 molecular weight was obtained by combining this value with the sedi- mentation coefficient determined at the same concentration. This method assumes that the sedimentation and diffusiun coefficients have the same concentration dependence. Nevertheless, an apparent molecular weight of 75,000 was obtained which was in agreement with the equilibrium data determined at the concentration. An intrinsic viscosity of 31 ml/g was determined from an evaluation of a reduced viscosity plot, see Figure 12B. The Scherage-Mandelkern constant, 9, calculated from these data was 2.00 x 106. The data are summarized in Table 9. Sedimentation and viscosity characteristics in concentrated acetic acid solutions adjusted to 0.15 M NaCl. The short-column equilibrium data obtained in 67% acetic acid are represented by the open circles in Figure 13, and compared with the values for reduced k-casein, Table 10. The apparent weight-average molecular weights, which were strongly concentration dependent, yielded a value of 124,000 at zero concentration by least-squares extrapolation. Similar values were obtained from an experiment performed on purified Fraction S. Ratios for theirelationship‘Mz/Mw were nearly one in all cases. Moreover, the Van Holde-Baldwin plots were linear for these'data, see Figure 14A. In.33% acetic acid, however, k-casein appeared to be polydispersed. ,AfTrautman plot of approach-to-equilibrium data for a 6.8 mg/ml solution.is shown in Figure 16. The molecular weight of the small component evaluated from the final slope was 58,000. Selected hydrodynamic properties were determined in 67% acetic acid and are summarized in Table 9. The sedimentation coefficients 81 were fitted to the equation 320,w = 2.7 (1 - 0.0302 c) where S20 w is in Svedbergs and £_in.mg/ml. A value of 2.4 Ficks was ) obtained for the diffusion coefficient at zero concentration by extrapolation of values calculated from the short-column equilibrium experiments. At a concentration of 7.5 mg/ml, the value was 1.5 Ficks. Substitution of these data into the Svedberg equation yielded a molecular weight of 101,000 which is in relatively good agreement with the value obtained by the short-column equilibrium technique. The intrinsic viscosity was found to be 35 m1/g as determined from a reduced viscosity plot (Figure 12A). A value for Q of 1.8 X 106 was calculated . Sedimentation characteristics in 7.0 M urea solutions at pH 8.5. k-Casein (purified No. 3A) was polydispersed in this solvent as indi- cated by the results obtained from a short-column equilibrium experi- ment. At a protein concentration of 5 mg/ml the Mw and M2 were 108,000 and 213,000, respectively, giving a ratio, Mz/"Mw of 1.97. Approach-to- equilibrium studies of this solution resulted in values of 57,000, 118,000, and 144,000 for the molecular weights of the small component, MW, and M2, respectively. A Trautman plot of the data is shown in Figure 17. Sedimentation and viscosity characteristics in pH 12,2 phosphate 2&2. Archibald molecular weights (MW), calculated from the meniscus patterns, were averaged to approximately 24,000 for a 10 mg/ml solu- tion of purified Preparation No. 3. A molecular weight of 23,400 was 0 O btalned from a Trautman plot of approach-to-equilibrium data for a 82 5.3 mg/ml solution of purified Preparation No. 3A. Unlike k-casein in 7.0 1.1 urea, 5.0 11 GU, and 33% acetic acid, the Trautman plot for these data fitted a straight line, see Figure 18. The diffusion coefficient for a protein concentration of 8 mg/ml was 5.8 Ficks, obtained in the Tiselius electrophoresis cell by the height-area method. The sedimentation coefficients fitted the equa- tion 320,w = 1.4 (1 - 0.0172 c) where 820,W is in Svedbergs and £_in mg/ml. A molecular weight of 22,300 was obtained by inserting these data into the Svedberg equation. The reduced viscosity was related to the concentration according to the equation ’Isp c = [II] + 0.305 [W2 c rt 0.21, where the intrinsic viscosity, [an , was 15.1. The calculated value for the Scheraga-Mandelkern constant was 2.04 X 106. These data are compared to those for k-casein in other solvents in Table 9. Physical Properties of Reduced k-Casein Sedimentation Characteristics in 5.0 fl;GU The experiments were performed with the PMB derivative of reduced k-casein to prevent re-oxidation of the disulfide bonds. Apparent molecular weights for the derivative remaining after removing aliquots for -8H group determination are shown as a function of the concentra- tion in Figure 19 (filled circles). These values were obtained from the slopes of Van Holde-Bladwin plots which were linear for these equilibrium measurements. The apparent molecular weights, compared to those of k-casein in Table 8, did not show marked concentration 83 dependence. A molecular weight of 28,700 was obtained by least-squares extrapolation to zero concentration. A weight-average molecular weight of 21,000 was obtained for a reduced, purified Fraction S in.5.0'§_GU at pH 8.4 and containing ME. The diffusion and sedimentation coefficients were 5.8 Ficks and 1.3 S at a protein concentration of 9 mg/ml. A molecular weight of 20,000 was obtained when these values were inserted into the Svedberg equation. The concentration dependence of a later preparation of the PMB-derivative gave the result SZO,w = 1.88 (1 - 0.0106 c). However, equilibrium molecular weight analysis showed Mw,= 34,000, Mz = 50,000, and a non-linear Van Holde-Baldwin plot, indicating either limited re-oxidation had occurred before the PMB was added or complete reduction was not achieved. This material was not used for -SH group analysis. Sedimentation Characteristics in 67% Acetic Acid Adjusted to 0.15 fl,NaCl In these studies, the protein was reduced in.5 !;GU at pH 8.4. The solution was adjusted to pH 3 with glacial acetic acid and dialyzed against the solvent. There should be no re-oxidation of -SH groups under these conditions. The protein concentration was determined by measuring the area under the curve formed in the synthetic boundary cell. Concentration dependence of the apparent molecular weights is shown in Figure 19 (open circles). As observed for the case of the PMB-derivative in.5 g GU, the concentration dependence was not large. These values represent both the weight- average molecular weight for the entire cell contents and that obtained 84 from the slopes of the Van Holde-Baldwin plots. Since these plots were linear, the values obtained agreed within experimental error. Also the ratio‘Mz/Mw was approximately one as would be expected from the appearance of the Van Holde-Baldwin plots. The molecular weight obtained by least-squares extrapolation to zero concentration was 27,300. These results are compared to those for k-casein in the same solvent in Table 10. The sedimentation-velocity data for reduced k-casein are represented by the relation 320,w = 1.26 ( 1 - 0.0146 c) where $20,w is in Svedbergs and g.in mg/ml. The diffusion coefficient, obtained at 6.4 mg/ml protein concentration, was 5.7 Ficks. Assuming the D20,w is not concentration dependent, a molecular weight CM3,D) of 20,000 was calculated. Determinatipn of Sulfhydryl Groups The nitroprusside test for free sulfhydryl groups performed in 5 ELGU gave negative results for purified Preparation No. 3A and Purified Fraction S. following treatment with.ME, the test was positive and the PMB titration experiments showed the presence of 2-3 -SH groups per 28,000 g of k-casein. Analysis of total sulfur and methionine showed that by difference, 3 cysteine/28,000 existed. k-Casein held at pH 12 for 24-48 hours at 20° C did not show any SH groups upon subsequent reduction with.ME and titration with.PMB: The results are summarized in Table ll° 85 TABLE 4 The amino acid composition of k-caseins Residues/28,000 g Amino acid g/100 ga Wib This studyc Jolw Gig-casein!— Asp 7.72 6.68 16 15 16 Thr 6.74f '5.72 16 16 10 Ser 5.03f 4.17 13 16 15 Glu 19.80 17.38 38 33 34 Pro 10.95 9.24 27 21 25 Gly 1.23 0.93 5 5 . 5 Ala 5.40 4.31 ' 17 17 17 Cys --- --- --- alv6 1.6 Val 6.30 5.33 15 12 12 ‘Met 1.68 1.48 3 2 2 Ileu 7.10 6.13 15 13 14 Leu 6.11 5.27 13 13 14 Tyr 7.61 6.86 12 11 15 Phe 3.86 3.44 7 7 7 Lys 6.51 5.71 12 11 13 His 2.36 2.09 4 3 3 Arg 3.96 3.55 6 6 7 Try --- --- --- 1 2 NH3 1.94f 1.82 32 --- 35 8 ; u1 = 95.12 86 TABLE 4 - Continued 3 Based on a nitrogen content of 15.3%. 9 Weight per cent of the ith amino acid residue. C Analysis provided through the courtesy of Drs. J. P. Riehm, J. C. Speck, and H. A. Lillevik of the Biochemistry Department. d Calculated from the g/100 g protein reported by JOIIES gt _1, (1962). ' e Calculated from the g/100 g protein reported by Hipp g£_§1, (1961) for a3-casein. f Extrapolated values. g This summation includes in addition to the amino acid residues, the values for NANA and phosphorus (based on the HZPO residue) as well as the values for Cys, Try, galactose, and ga actosamine reported by JolleS 3521. (1962). 87 TABLE 5 Molecular weights of k-casein from chemical analysis Minimum Number molecular of ‘Melecular Component Percentage weight residues weight Sialic.acid (NANA) 1.4 22,000 1 22,000 Phosphorus 0.22 14,090 2 28,180 Sulfur 0.70 4,570 6 27,420 Tryptophana 1. 05 19 , 500 1 19 , 500 a Reported by JollEs 25 El- (1962). 88 TABLE 6 Sedimentation coefficients of k-caseins at neutral pH Concentration (mg/m1) 820 X 1013 Temperature °C Solvent 6.03 14.5 3 phosphate buffer, 0.1 f72 6.4a 13.9 25 phosphate buffer, 0.2 [“/2 8.13 13.02 25 0.5 1_1_ NaCl 8.28 13.41 25 phosPhate buffer, 0.2 [72 10.0b 13.44 24 phosphate buffer, 0.1 [‘72 10.08 13.11 25 phosphate buffer, 0.2 [‘72 a Parified preparation No. 3A. Preparation obtained by the method of McKenzie and Wake. 89 TABLE 7 Sedimentation coefficients of purified Preparation No. 3A in buffers containing SDS SDS Protein SDS 13 Buffer (mg/ml) (mg/m1) (g/g protein) $20,w X 10 Veronal 2.5 8 0.31 2.288 pH 8.6 5.0 8 0.63 2.11a r72 = 0.1 10.0 5 2.00 2,28,11.14b 10.0 8 1.25 2.19c 10.0 10 . 1.00 2.35c 20.0 9 2.22 2.328,0.84b Tris 0 8.2 -- 8.29a pH 9.25 10 8.2 1.22 2.00c r72 = 0.1 20 8.3 2.41 2.26, 0.98b 30 8.3 3.61 1.10, 0.80b 30 8.1 3.70 1.28,“ 40 6.0 6.67 1.1e Not corrected to water. Value for boundary due to SDS micelles. Boundary due to SDS micelles was observed. -Free SDS removed by chilling to 0° C and centrifuging out the precipitated SDS. Performed in synthetic boundary cell by layering, on the solution, buffer containing 40 mg/ml SDS which was dialyzed against the Solution. 3 b c d (D 90 TABLE 8 Equilibrium molecular weight data for k- and reduced k-casein in 5.0 11 CI], pH 4.8 k-Casein Reduced k-Casei_n_ Concentration _4 Concentration .4 (mg/ml) MW X 10 Mz/Mw (mg/ml) Mw X 10 0 12.61 0 2.87 3.0 11.28a 1.948‘ 3 2.77d 5.5 9.44 1.18 6 2.52d 7.2 8.73a 1.183 9 2.49d 10.3 7.38a 1.17a 7b 21"” a Average of several determinations. b Fraction S (Waugh and von Hippel, 1956), purified by ultracentrifu- gation and elution from Sephadex G-75. c Solvent contained 7 ul/ml ME at pH 8.4. d 1MB derivative. 91 TABLE 9 Hydrodynamic properties of k-casein in 5.0'MpGU, 67% acetic acid - 0.15‘M,NaCl and pH 12.2 phosphate buffer Phosphate 5.0 M GU 677. Acetic 3. Buffet Property pH 4.8 Acid (pH 12.2, 0.19 THIZ) o 13 SZO,w X 10 3.18 2.7 1.4 k3 0.0178 0.0302 0.0172 020 w x 107 3.1 2.4 (c = 0) 5.8 ’ (10.3 mg/ml) (8 mg/ml) [’7 (ml/g) 31 35 , 15. 1 Huggins constant 0.82 0.50 0.305 b f/fo 2. 7 3. 2 2. 3 8 x 10‘6 2.00C 1.8c 2.04d Ms D 75,000 101,000 22,300 , (10.3 mg/ml) (c = 0) (c = 0) a From the equation $20,w = SEC”, (1 - kC) b Calculated from sedimentation-velocity and molecular weight data. c A molecular weight of 120,000 was used in the calculation. d A molecular weight of 24,000 was used in the calculation. 92 TABLE 10 Equilibrium molecular weight data for k- and reduced k-casein in 67% acetic acid - 0.15'M NaCl k-Casein Reduced k-Casein Concentration Concentration (mg/m1) MW x 10'4 112/11w (mg/ml) MW x 10"“ Mz/Mw 0 12.42 0 2.73 1.83 12.09 1.11 3.2 2.65 0.99 2.40 10.60a -- 6.3 2.55 0.91 3.67 10.74 1.17 9.6 2.48 1.13 6.50 9.14 1.01 6.50b 8.713:b 1.143:b 7.34 8.75 0.98 11.00 7.03 0.91 a Value for one calculation. b Fraction 8 (Whugh and you Hippel, 1956), purified by ultracentri- fugation and elution from G-75 Sephadex. 93 TABLE 11 Determination of the total sulfhydryl groups Protein Method of SH Group Analysis Reduced k-casein k-Casein k-Casein prepared by the method of Waugh and von Hippel (1956) k-Casein treated at pH 12 followed by reduction Titration of un- reacted PMBa with GSHb Trial 1 (18.9 mg)c Trail 2 (15.5 mg)C Direct determina- tion of Mercaptide at pH 9.5 Sulfur and methio- nine analysis Titration of un- reacted PMB with GSH Number SH/ 28,000 g Nitroprusside + 1.93 2.67 1.82 2.98 - 0.1 - a p-Mercuribenzoate b Glutathione c Total amount of protein reacted with excess PMB 94 40r- . l l 1 L 0 .005 .0l0 .0 IS .020 Concentration (g/ml) Figure 12. A plot of reduced viscosity (ml/g) for k-casein in A) 67% acetic acid - 0.15 M_ NaCl, B)_ 5.0 M guanidine-HCl, and C) 0.1 M NaCl. 95 M NO" l I I l J I l l l l l 0 5 l0 Concentration (mg/ml) Figure 13. Plot showing the concentration dependence of the apparent molecular weight of k-casein in 5.0 M guanidine.HC1 and 67°/. acetic acid - 0.15 M NaCl. Legend: 0, Mw in 67% acetic acid; 0, Mw in 5.0 l__4_ guanidine- HC1;A, Mw in 677. acetic acid for purified Fraction S (Waugh and von Hippel, 1956). 96 aa Ha\wa am.a Aa .Smoaaoaaoaw m o.m an EEE o.m Am .32 m 2.0 .. Boo oaoooo .fio .cwommolx How mumv Edwunwaflsoo cadaoonuwoem wo muon aw3pammuoufiom Cm> so... u sumac .oa osawam 97 (fig/412 xm - IO’ (c.- c...) ~|02 0. 0. o '0 N _: o .0 T l d 3 - o. I! _ O z 0 <1 F1 151511311115" Trautman plot of approach-to-equilibrium data for k-casein Rm-.'- guanidine-H01. Legend: 0, 42,040 RPM;‘, 33,450 RPM; 0, 20,410 ’ ’ 12,590 RPM. ¥ 98 75,42,212 xm - 70' V '0 I use 03:: .o ”Em oomaom .‘mzmm 30.3.0 68on .862 Z mH.o I cfiom ofiuoom Nmm aw swommoux now some ESwHewdeoououufiomowmmm mo uoHa coEuSmHH .oa oudmwm p: O “flaw—o A “v.0 nvv . N n ¢ n o h o 1 d d d fl q q _I- 1 F 0 T. O o m 99 179’ x.-'°° X ( .5 802 .0 :5 23.8 .0 :5 823 3.35 08.3 .o "Eamon .0 6mm £6 mg «no»: .m o.n a“ swomoolx now mum—o gwwnfiwsoououusooougo mo ”.01“ £95535. .2 0.3me No. .3 ...ov _. OOOND.» 2 r 3 80.63... N: J n. O a. 6 . coed... 3: .Em 08.2 .0 “Em 08.3 .0 ”Em 08.3 .0 maoaooaoaooaoo Hon; or.“ .a uuaowwa .o w .Hommse oumfimmosm N.NH mm aw awommonx you name Ebwhnflawsootoulzom0Hamm mo uoam do quHH ma on: I. O NO—.A 0‘ 0v _ N n ... m o d a a a J 03.3 .2 mm 101 Figure 19. Plot showing the concentration dependence of the apparent molecular weights in dissociating solvents for reduced k-casein and k-caseinexposedto pH 12. Legend: 0, MW for'reduced k-casein in 677. acetic acid - 0.15 M NaCl; . , MW for PMB-k-casein in 5.0 M guanidine-RC1; A , MW for purified Fraction S (Waugh and von Hippel, 1956) in 5.0 1.1 guanidine, pH 8.4, containing 7 ul/ml Zmercaptoethanol; OM from Trautman plot of approach-to-equilibrium data for k-casein in pH 12.2 phosphate buffer, ['72 = 0.19:0 , Mw for k-casein treated at pH 12. 2, then-dialyzed against 5.0 M guanidine-H01. 102 on oaawam . _E\ OE v 5:228:00 QN Cd .-Owa DISCUSSION Composition of k-Casein A comparison of the amino acid composition of purified Prepara- tion No. 3A with that of JollEs gt_§g? (1962) for a'McKenzie-Wake preparation showed good agreement. The analyses, including the carbohydrate and phosphorus content, accounted for 95% of the total protein. JollBs g£_gl, (1962) accounted for only 85% of the residues in their analysis of k-casein. Perhaps a more thorough analysis of the carbohydrate moiety would account for the remaining portion. The total number of cationic and anionic groups per 28,000 g was esti- mated to be 22 and 58, respectively, assuming that a) the phospho- amino acid side chains were dibasic anionic groups and b) ignoring the amide nitrogen. When the 32 amide nitrogens are considered, then, there exists but four excess anionic groups. The large amount of proline is particularly noteworthy. The 1.4% of NANA found for purified Preparation No. 3A lies in the range reported for other k-casein preparations. iMarier g£__l, (1963) reported values of 0.65% and 1.25% for a McKenzie-Wake pre- paration, whereas, Thompson and Pepper (1962) and Jo11es at. 31. (1962) reported values of 2.5% and 2.4%, respectively, for preparations by the same method. A modification of the‘McKenzieAWake preparation yielded k-casein containing 1.8% NANA (Nitgzhmzhh\and_§ggby, 1963). Also, Marier, gt; 11. (1963) gave a value of 2.14% for a preparation obtained by the Swaisgood-Brunner method as previously published but not purified as described herein. Urea-starch gel electrophoresis showed more of the leading bands in their preparation. The extent of 103 104 variation due to analytical procedures and that due to differences in the preparations can only be speculated. Possibly some of the con- taminating proteins found in k-casein preparations contain NANA. This postulate is supported by the observation of Malpress (1961) that only 68% of the NANA in whole casein was released by rennin whereas Marier ggugl. (1963) found approximately 90% of the NANA was released from a Swaisgood-Brunner preparation of k-casein. To some extent the phosphorus content appears to be related to the purity of k-casein preparations. Waugh's Fraction S contained about 0.5% P. The crude k-casein prepared by Long (1958) contained 0.46% P, but following five centrifugations it contained 0.33% P. Purer preparations had a phosphorus content of approximately 0.22% (Jones _e_t_ _a_1_., 1962; Thompson and Pepper, 1962; Waugh, 1958). Preparations obtained during this study varied from approximately 0.35% P for crude k-caseins to 0.22% P for the purified material. The composition of ag-casein is similar to that of k-casein. This protein fraction is undoubtedly rich in k-casein as evidenced by its amino acid composition, phOSphorus and NANA contents, reaction with rennin, and ability to stabilizetIS-casein. Nevertheless, there are some noteworthy differences between these two proteins: a) the phosphorus content of ag-casein (0.35%) is similar to that of crude k-casein although electrophoresis and sedimentationdvelocity experi- ments showed only one boundary, b) its sedimentation coefficient (23 S) is much larger than that for k-casein, c) it is mughulg§§_soluble at pH 6.9 (0.26%), and d) its threonine content is significantly lower. Interestingly, this protein was prepared by the same procedure employed by Long (1958) with the following exceptions; a) only one centrifugation 105 at low speed was used to remove the as-casein and b) the final ultracentrifugations were performed in the absence of salt. Possibly the interaction observed by Long (1958) in the presence of salt participates in the formation of a stable complex in the absence of salt. The fact that repeated ultracentrifugation in salt solutions does not concentrate k-casein preparations beyond a certain degree of purity was observed by Hipp 33.21, (1961) and, also, during the course of this study. The formation of stable complexes between k-casein and one or more of the l-casein constituents would explain the discrepancies listed above. For example, the greatest difference between the amino acid composition of the l-casein fraction and k-casein as reported in this thesis was the lower content of threonine in X-casein. Furthermore, Hipp g£_§é, (1961) observed that the exposure of as-casein to pH 11.8 resulted in the formation of a number of electrophoretic peaks at pH 10.1 where previously only one peak was observed. In view of the above argument, this observation would indicate that the associated complex of casein's was dissociated in the high pH environment (Waugh and von Hippel, 1956). Molecular Size and Interactions of k-Casein Polymer Size of k-Casein in Neutral Salt Solutions k-Casein forms polymers in neutral buffers or NaCl solutions which are relatively independent of temperature or ionic strength. These polymers are not entirely uniform with respect to size as evidenced by the skewness of the sedimenting boundary. Nevertheless, the inter- action is unusually specific and an estimate of the aggregate size is noteworthy. 106 Although a direct determination of the molecular weight was not performed, an approximation can be obtained from the ScheragadMandelkern equation (Appendix 11) since 6 is relatively insensitive to particle shape. Thus, using the equation given by Schachman (1959), a molecular weight of approximately 650,000 was calculated from the sedimentation coefficient and intrinsic viscosity. Therefore, the polymer seems to be composed of 10 to 15 basic units (M.= 56,000). One must not con- clude that this aggregate is indigenous to the native micelle, however, this ability to form aggregates certainly demonstrates the proteins unusual capability for specific interaction. Dissociation of the k-Casein Polymers and the Properties of the Units Obtained Sedimentation, diffusionLgand viscosity characteristics of k-casein in dissociating solvents. The polymer size of k-casein was significantly reduced when dispersed in solvents containing SDS, 7.01; urea, 5.0'MLGU, concentrated acetic acid, or high concentrations of hydroxyl ion. Thus, the equilibrium molecular weights extrapolated to zero concentration in 67% acetic acid and 5.0 M;GU were approximately 125,000. Although there was some indication of an increase in the apparent molecular weights at low concentrations (Figure 13), the protein was essentially monodispersed in 67% acetic acid. At low concentrations in.5.0‘M_GU and in.7.0'M,urea or 33% acetic acid the protein exhibited properties characteristic of polydisperSity. In 5.0 M_ GU and 7.0 M urea, the Mw was similar to that obtained in 67% acetic acid but the M.z was nearly doubled. Determinations of the molecular weight of the light component in three solvents, i.e., 33% acetic acid, 5.0‘M_GU, and 7.0‘M,urea, resulted in an average value 107 of 56,000. These data may be interpreted to suggest that k-casein existed in these solvents in the form of both lower and higher polymer species. For example, lowering the concentration of acetic acid or lowering the concentration of protein in 5.0 M_GU may favor a monomer- polymer equilibrium rather than the dimer form observed in 67% acetic acid. The basic unit of k-casein appears to have a molecular weight of approximately 56,000. The molecular weight of k-casein in solutions containing 10 mg/ml SDS was roughly 100,000. The polymer size was probably similar to that in acetic acid, 00, and urea solutions since this calculation assumed one gram of SDS bound per gram of protein; a figure most likely over- estimated. However, the molecular weight at pH 12 was approximately 24,000 or about one-half the value for the basic unit. This result compares well with the value reported by McKenzie and Wake (1959b). Molecular weight values were not corrected for charge effects or specific protein-solvent interaction-~except for an estimate in the case of SDS-~because of a lack of satisfactory correction values. However, the charge effect should not be large since the solutions contained relatively high concentrations of salt (e.g., 0.15 rH/Z) (Svedberg and Petersen, 1940; Schachman, 1959; Tanford, 1961). Pre- ferential interactions with SDS and concentrated acetic acid should not effect the molecular weight greatly since their partial specific volumes are nearly the reciprocal of the density of the solution (Schachman, 1960). Kielley and Harrington (1960) found that <35% GU was proteinebound for two dissimilar proteins (i.e., ribonuclease and myosin) dispersed in 5.0 M,GU. Further, these investigators obtained a molecular weight of 13,000 1:500 for ribonuclease which agrees with 108 the known value.. The fact that similar molecular weights were obtained in all of the solvents employed in this study suggests that large charge effects or preferential interactions were not operative. These studies demonstrated that the polymers were not completely dissociated in any of the solvents employed except the pH 12 buffer. Although a smaller unit was present, a tendency towards larger polymer size was more apparent in 7.0 M urea, 33% acetic acid, and 5.0 M GU at low protein concentrations than in 67% acetic acid. Conceivably an unusually strong interaction occurs between the basic units of k-casein since most proteins would be completely dissociated under these con- ditions. The sedimentation coefficients, especially in 67% acetic acid, exhibited a considerable concentration dependence. Presumably this characteristic can be attributed to the non-ideality of the system as demonstrated by the large dependence of the molecular weights on concentration. However, the diffusion coefficient in 67% acetic acid showed only a small increase with decreasing concentration. The intrinsic Viscosities in 67% acetic acid and 5.0 M;GU were greatly increased over that in neutral salt solutions or pH 12 phos- phate buffer. This observation was similar to that of Harrup and Woods (1961) who found that the intrinsic Viscosities of bovine serum albumin, egg albumin, B-lactoglobulin, lysozyme, and insulin were much greater in anhydrous formic acid than in aqueous solutions. The calculated frictional ratios as well as the large intrinsic Viscosities suggest that the molecules were extended. However, a combination of thermodynamic and hydrodynamic data in the form of the Scheraga-Manderkern conStant indicates that the molecule underwent 109 an isotropic swelling rather than an extension in the polypeptide chains. 6, 2.001: 106, and 2.04 x 106 The calculated values for B were 1.8 X 10 in 67% acetic acid, 5.0 M;GU, and pH 12 phosphate buffer, respectively. The theoretical minimum value is 2.12 X 106, however, the values reported here are within the experimental error of this minimum. Scheraga and Mandelkern (1953) discuss the case of horse serum albumin in urea solutions where the frictional ratio indicated an axial ratio of approximately 20 : 1, whereas the calculated B's ranged from 1.98 to 2.05 X 106. They concluded that the protein molecules swelled in the presence of urea. Values lower than the theoretical minimum have been reported for other proteins (Schachman, 1959; Yang, 1961). Harrup and Woods (1961), on the basis of light scattering data, suggested that isotropic swelling occurred in bovine serum albumin rather than a linear extension of the chains in anhydrous formic acid. The dissociation of k-casein in 67% acetic acid and 5.0'M,GU was reversible with respect to the sedimentation-velocity characteristics. k-Casein which was held for two weeks in 67% acetic acid at room temperature exhibited the same NANA content and reaction with rennin following dialysis against water as it did prior to the-exposure to the dissociating system. Holding the protein in 5.0 M GU or 7.0M urea followed by dialysis against water yielded a k-casein solution which reacted normally with rennin. Some difficulty was experienced in completely removing the bound SDS from the protein as indicated by a lower sedimentation coefficient (e.g., 83 108). Sedimentatign and diffusion characteristics of reduced k-casein in dissociating solvents. Following cleavage of the disulfide bonds with ME, the molecular weight of k-casein was approximately 28,000 in 110 both 5.0 5:63 and 67% acetic acid. The protein appeared monodispersed as evidenced by linear Van Holde-Baldwin plots and ratios onMZIMw approaching one. As in the case of k-casein, the molecular weights were not corrected for charge effects or preferential interactions. Concentration dependence was not marked for either the molecular weights or the sedimentation coefficients. Diffusion coefficients were determined at only one concentration in both solvents giving values of 5.8 Ficks at 9 mg/ml in 5.0 M GU and 5.7 Ficks at 6.4 mg/ml in 67% acetic acid. Comparison of k-casein and reduced k-casein. Reduction of the disulfide bonds clearly caused a decrease in the observed molecular weight of k-casein. This fact was obvious even from a qualitative observation of the sedimentation-equilibrium patterns, see Figure 20. Furthermore, a comparison of the sedimentation and diffusion coeffi- cients showed that cleavage of the disulfide bonds caused a reduction in the $20,w and an increase in the D20,w' The concentration de- pendency of the sedimentation coefficients for reduced k-casein were approximately half that for k-casein. This was as expected since the apparent molecular weights for reduced k-casein were much less con- centration dependent. These data are summarized in Table 12. The basic unit (M.= 56,000) of k-casein observed in various dissociating solvents appears to be composed of two sub-units having a molecular weight of approximately 28,000. The proposed sub-unit weight is in accord with the chemical analyses which indicated an average minimum molecular weight of 24,300. Furthermore, it is pro- posed that the two sub-units are joined by disulfide bonds. This postulate is supported by the presence of three -SH groups per 28,000 g 111 as determined by PMB titrations, analyses for total sulfur and methionine, 1. (1962) and and the cysteic acid values reported by Jolles gt Hipp g£_§1, (1961). Since k-casein does not contain free «SH groups, an odd number of -SH groups per sub-unit weight precludes at least one inter-molecular disulfide bond. The results obtained were not a con- sequence of the particular method of preparation employed in this study since the same observations were made on a freshly prepared, purified Fraction S preparation. A comparison of the properties of k-casein in pH 12 phosphate buffer to those of reduced kacasein indicated that the disulfide bonds were destroyed in this buffer. Also, k-casein dissolved in the phos- phate buffer and then dialyzed against 5.0 M_GU exhibited an equilibrium molecular weight of 24,500 at a protein concentration of 9 mg/ml, see Figure 19. Chemical evidence showing that alkaline- treated protein subsequently reduced with ME did not give the nitro- prusside reaction or react with PMB substantiated the conclusions derived from the physical studies. These results are not surprising considering the lability of disulfide bonds in proteins to alkali (Cecil and McPhee, 1959). Brown, Delaney, Levine and Van Vunakis (1959) observed an odor of hydrogen sulfide upon neutralization of aqueous solutions of ribonuclease which had been exposed to pH l2;7 for 30 min at room temperature. iMore recently, Young and Potté (1963) measured a 70% loss of half-cystine \ in ribonuclease after twd‘hours at pH 11 in.5 M;guanidine. Further evidence in favor of a basic unit weight for k-casein of approximately 56,000 comes from the work of Garnier 25.3}, (1962) and Beeby (1963). These researchers proposed that k-casein had a molecular 112 weight of 55,000 and 50,000, respectively. They based this conclusion on data pertaining to the release of protons and GMP resulting from the action of rennin on k-casein. If, as proposed by these workers, only one GMP is released per 56,000, then the two sub-units must be differ- ent but of approximately the same weight since the equilibrium patterns of reduced k-casein have shown it to be quite homogeneous. Further, a peptide other than GMP must be released by rennin since studies described earlier in this thesis showed that peptides of approximately 8,000 molecular weight were released from a 28,000 molecular weight unit. Reduced k-casein did not show the same tendency to participate in.a monomer-polymer equilibrium as did the non-reduced protein in the dissociating solvents employed. However, reduced k-casein in neutral phosphate buffer possessed the same sedimentation-velocity characteristics (820 = 13 S) as the non-reduced. This result was expected since a typical sedimentation pattern was restored upon returning the protein to pH 7.0 from pH 12 (Waugh and von Hippel, 1956). Thus, the disulfide bonds may have some influence on the interactions of k-casein but certainly, the effect is not great. Some other possible structures of k-casein. Recently nEEEZEEEEED\ land Beeby (1963) and Beeby (1963) proposed that k-casein was a com-_ plex and that the initial action of rennin was to break this complex. Seemingly, this proposal was based on the following observations. a) Fifteen per cent of their k-casein preparation was soluble at pH 4.7 and the precipitate became more insoluble at pH 7 with each precipitation. b) When a 6g urea solution of the protein was dialyzed 113 against pH 4.7 acetate buffer, a precipitate formed leaving 20% of the protein in the supernatant. The precipitate was partially insoluble at pH 7. c) The insoluble material at pH 7 appeared similar to para-k- casein and the soluble portion from a) and b) similar to GMP. d) And finally, very low rennin concentrations, the nitrogen and NANA initially appeared more rapidly in the pH 4.7 soluble portion than in the 12% TCA soluble material. Despite these evidences of a k-casein complex, one must consider that neither the k-casein preparation nor the various fractions were well characterized. Furthermore, it is possible that l-casein fraction was present in their preparations. Fraction S, prepared in this laboratory by a similar method, contained large quantities of the l-casein fraction. A.McKenzie4Wake preparation of k-casein also contained l-casein. The percentage of nitrogen reported for one of - their preparations (Beeby, 1963) was 14.6%, whereas, the purified k- casein studied here contained 15.3%. Moreover, purified Preparation No. 3A did not precipitate when solutions in 5.0 !.GU at pH 4.8, 7.0 §_urea, SDS, or acetic acid were dialyzed against water. The physical characteristics and reaction with rennin were normal for k-casein which had been treated in GU, urea, or acetic acid. Furthermore, a molecular weight of something less than 50,000 was not observed for k-casein in.7.0 E_urea as would be expected if a complex existed and was broken as was suggested. Several alternative interpretations of the data of Nitschmann and Beeby are considered. 1. The material which was soluble at pH 4.7 as described above in a) and b) may be enriched with the 114 lwcasein fraction. Possibly the l-casein fraction con- tained the NANA not found in the GMP (see previous dis- cussion). Also, some of the constituents of this frac- tion must have a relatively low molecular weight as evidenced by anMw = 20,000 and M2 = 90,000 in.5.0 y;GU. These characteristics could cause the k-casein fraction, or some component thereof, to appear similar to GMP. The fraction of the precipitate obtained in a) and b) above which was insoluble at pH 7 (similar to para-k- casein) may have been an irreversible aggregate of k- casein and l-casein fraction similar to aé-casein. Interactions of the l-casein fraction with k-casein would also explain the more rapid increase in the amount of nitrogen and NANA soluble at pH 4.7 when compared to that in 12% TCA following the action of rennin. Observa- tions in this laboratory indicated that some k-casein may be soluble at pH 4.7, e.g., k-casein preparations containing 35-50Z - or possibly less - of the l-casein fraction does not cloud when treated with rennin in di- lute salt solutions (< 0.1'fl)g. The amount of NPN liberated definitely indicated that GMP was being re- leased (Note: Nitschmann and Beeby stated that k-casein was not in the pH 4.7 supernatant since the solution did not cloud with rennin). Further, the supernatant ob- tained at pH 4.4 during the urea-TCA preparative method contained in addition to large proportions of B-casein a fraction similar to crude k-casein as evidenced by free- 115 boundary electrophoresis. Thus, it can be argued that the NANA-containing material which is soluble in 12% TCA prior to the action of rennin may be one or more of the l-casein constituents. This fraction may not be entirely soluble in 12% TCA under all conditions which would explain the conclusion arrived at by Wake (1959), namely that all of the GMP was not soluble. The initial stage of the action of rennin on k-casein would cleave the GMP from the protein, thus, increasing the ratio of k-casein to unaltered k- casein. At pH 4.7, then, more of the k-casein in addi- tion to l-casein would remain soluble. In fact, Beeby (1963) found that the NANA/N ratio increased with time and suggested that "the intact sialic acidecontaining component carries with it part of the insoluble compon- ents of the k-casein complex". However, only the initial proportion of the l-casein fraction and the released GMP would be soluble in 12% TCA. After the complete reaction has occurred, the only NANA-containing pro- teins would be GMP and the l-casein fraction. If any of the latter was not previously soluble in TCA, inter- action with GMP could lead to complete solubility, thus, all of the NANA would be determined as soluble NANA. 2. The faster increase of pH 4.7 - soluble nitrogen and - NANA compared to that which was soluble in 12% TCA can be explained in another manner. The complete rennin action may be a result of two cleavages. Thus, the initial 116 cleavage would yield a large peptide of 12,000-16,000 molecular weight which is soluble at pH 4.7 but not in 12% TCA. An extended reaction would cleave this peptide approximately in half giving the GMP and another peptide. This would account for one proton, as suggested by Garnier ggmgl, (1962) and a single GMP per 56,000 molecular weight unit. An assay of the total soluble peptides re- leased indicated that units of 6,000-8,000 molecular weight were released per 28,000 molecular weight. ” Nevertheless, if the complex exists as proposed by Nitschmannlapd_ Eeeby (1963), then another interpretation of the physical data pre- sented here is tenable, namely; that the complex should consist of two proteins of approximately the same molecular weight (28,000) since equilibrium studies of the reduced material indicated homogeneity. Secondly, the disulfide bonds probably would not be inter-molecular, but rather intra-molecularily located and possibly in only one of the proteins. And finally, the disulfide bonds must facilitate the complex formation since cleavage of these bonds re- sulted in reduction of the molecular weight to 28,000 units. .aomz.m.ma.ou whom owumom Nmm was cowumcHaumuov mfinu How can: uco>aom u .uon GmausmHH m Bonn pmawaumuwv mm pamcomsoo u:wfiH may mo unwam3 Hmanomaoz a .Ha\wa m mo sowumuuamucoo awououm Q How uLwH03VHmaaooHoa mwmum>muunwwm3 m yp 1 ........................ I 1“! ‘I,I ' I A III 1‘ --- --- so;m --- o.~ m.H --- e.oH x m m.H o.~ m.~ --- N.N N.m --- .m\w --.. -..- H .2 ...... 8 mm m .a $35 E m.m a.m w.m --- H.m m.~ --- “an x 3.0Nn mm aoHo.o oaao.o Nano.o --- wsao.o mono.o mono.o x mm.a 0N.H e.H --- mH.m A.N o.ma mHoH x 3.0m sooo.am aooo.sm o.pooo.mm oo~.m~ oom.s~ ooo.q~ mooo.maa oooromfi ooo.e~H oooromo 2 mu m o.m I 82 2 ma 3.5 m o; 8 .m o.m I 82 cg E 333$ a mH.o-eaoa gunman z mH.o-uau< gunman oauuo< aka mumaamoam oauooa ans «unnamoem .mmwmnoux consumm awumuulx awommoax pounce“ paw aflommoux mo wowuuonoun Hmugmhzn onu mo humEESm NH mamr rm - r 1 b2} these become Cb - cm =13? $9. dr rIn r c° = 1' 1 P 2> rm Since m1 = tan 0 AY (21) and r 31 . a 5 HC equation (18) becomes and Using equation (16) (19) and (20) FET‘ (22), 139 Kb - - 1 AX tan 6 Z 2 A — r - r 1 X tanwO IIX b m Y s . bounda REP a'b' Mc M. p A yn ry x1]“ (23) If the same schlieren diaphragm angle is used for both photographs, this equation simplifies to [XX [KY equil MaPP = 2 A . M“) m (24) rb ' r x X m Afi— ZP AY syn. boundary whererb=_%b_ and rm=_§m_ . o M5 Weight-average molecular weight at r (Van Holde - Baldwin plot). Equation (14) is used directly as 'Mapp c . Although g.cannot be evaluated r A y obtained relative to the concentration at the l_dc d 1 r directly, it is readi meniscus, gm. Thus, by adding and subtracting Em from g. Mapp [(9 - 9m) + cm] . , (25) .1.‘_.9.= rdr A Applying equations (16), (21), and (22) as before, the following expressions are derived; 1 . tan a A Y _ Mapp. 14x tan 9 xAr ""‘ '1r-‘—r-r-- ""*-' x/M° R,P a b ‘Mc A a'b' Md'Mo + constant or ZSY =‘MaPP. Z)__ i Y + constant. (26) 140 Therefore Al was plotted against A; AY and Mapp was obtained X M. from the slope since Mapp =- A Mo Slope.- (27) Zquerage molecular weight for the entire cell contents. This method utilizes information derived from the intersections of the plot described in the above paragraph with the meniscus and the cell bottom. From equation (14), the following expression is developed: MaPP rb fie): H‘ )m= . (28) Applying equations (l6), (19), (21), and (22) the above equation becomes: 1 tan 9 . tan 9 rb R3Pa 1) MC WAYb- rm Rspa'b'Mc AYm = MaPP _Ax t -9 f A an‘ , RSP a'b' MC Mo A Y equil Xm or Mapp= Aml§¥blr - (SYN/rm x . M :3 AY equil . (29) xm ' Example Calculation The microcomparator readings and the calculations for an equili- brium pattern are presented in this section. The symbol Rn refers to the distance from the inner reference line to the designated position as measured by the microcomparator on the photographic plate. For the 141 'Model E used, r = 5.70 cm at low speeds and Mo = 2.103. inner Appendix Table 2 Data and example calculation from a short-column equilibrium pattern Protein: Reduced k-Casein Speed: 14,290 RPM Conc.: 3.2 mg/ml Temperature: 25.0° C Buffer: 677. acetic acid - 0.15 2; NaCl 8: 60° n Rn Xn AYn Z Yn AYn/Xn X103A_}_(_ZAYnX103 Rn + 5.70 Mo . Mo 0 2.025 0.110* 1 2.030 0.112* 0.112 2 2.040 0.114* 0.226 3 2.050 0.117* 0.343 4 2.060 0.120* 0.463 5 2.070 0.123* 0.586 6 2.080 0.126* 0.712 7 2.090 0.128* 0.840 8 2.100 14.087 0.1312 0.9712 9.31 4.62 9 2.110 14.097 0.1322 1.1034 9.38 5.25 10 2.120 14.107 0.1356 1.2390 9.61 5.89 11 2.130 14.117 0.1426 1.3816 10.10 6.57 12 2.140 14.127 0.1434 1.5250 10.15 7.25 13 2.150 14.137 0.1464 1.6714 10.36 17.95 14 2.160 14.147 0.1484 1.8208 10.56 8.66 15 2.170 14.157 0.1526 1.9734 10.78 9.38 16 2.180 14.167 0.1534 2.1268 10.83 10.11 17 2.190 14.177 0.1560 2.2828 11.00 10.85 18 2.200 14.187 0.1584 2.4412 11.17 11.61 19 2.210 14.197 0.1622 2.6034 11.42 12.38 20 2.220 14.207 0.1650 2.7684 11.61 13.16 21 2.230 14.217 0.1658 2.9342 11.66 13.95 22 2.240 14.227 .0.1694 3.1036 11.91 14.76 23 2.240 14.237 0.1746 3.2782 12.26 15.59 24 2.260 14.247 0.1762 3. 4544 12.37 16.43 25 2.270 14.257 0.1794 3.6338 12.58 17.28 26 2.280 14.267 0.1818 3.8156 12.74 18.14 27 2. 290 14. 277 0. 1834 3. 9990 12.85 19.02 28 2.300 14.287 0.1868 4.1858 13.07 19.90 29 2.310 14.297 0.1900 4.3758 13.29 20.81 30 2.320 14.307 0.1940 4.5698 13.56 21.73 31 2.330 14.317 0.1972 4.7670 13.77 22.67 32 2. 340 14. 327 0. 2030 4. 9700 l4. 17 23.63 33 2.350 14.337 0.2068 5.1768 14.42 24.61 34 2.360 14.347 0.2104. 5.3872 14.67 25.62 35 2. 370 14. 357 0. 2132 5.6004 14.85 26.63 (continuation of Table 2) 142 36 2.380 14.367 0.2178 5.8182 15.16 27.67 37 2.390 14.377 0.2210 6.0392 15.37 28.72 38 2.400 14.387 0.2240 6.2632 15.57 29.78 39 2.410 0.228* 6.4912 40 2.420 0.232* 6.7232 41 2.430 0.236* 6.9592 42 2.440 0.239* '7.l982 43 2.4465 0.241* Rb = 2.448 rb = xb = 6.864 AYm = 0.110 Rm = 2.025 M. AYb = 0.242 rIn = Xm = 6.663 MO * Values obtained by extrapolation. 42 Cb " Cm "-' % ZAYII + Rb :- (R42 ‘1" g )AY 1 R. +~l1X Mo [42 7+3)» 1122+ 0 01 0 003 c c- = —A-—-— . 2 + -.9——— . = . . b cm 2.103 (7 198 ) 2.103 (0 241) 0 03457 Note that the first value for ZSY used in the summation was obtained at Xm + QR and similarly, theAY was “obtained at the midpoint of the small interval remaining at the bottom. Essentially, this method-represents trapezoidal integration. g. is obtained by similar measurements of a synthetic boundary pattern obtained §£.the same schlieren diaphragm angle, ie. ”a; mfAY xm syn. boundary for this case. = 0.04904 Other quantities necessary for the calculations are a) psolution _ » R T (8.314;: 107) (298) 1.0688 b = A = ‘* 4 = ’ ) (1»- '61))LJ‘I (1 - 0.73 x 1.0688) (2.23936 x 10“’ 1 50,290, and c) 2 2 = 0.368. The different average molecular r - r b m Weights are (obtained of:.follows: 143 a) weight-average molecular weight for the total cell contents; from equation (24) MSPP = (2) (50,290) (0,368) 9323221. u: 26,100 0.04904 b) z-average molecular weight for the total cell contents; from equation (29) 0.242/6,864 - 0.11946.663 8: 27,260 0.03457 m??? = 50,290 2 c) weight-average molecular weight from the Van Holde-Baldwin plot; from equation (26). The slope is obtained from a plot onXYn/X X.103 ' 3 3PP = = - against £>§_ [SYh X 10 , then.M MQA Slope (2.103) (50,290)(O.242) — 11 25,600. Calculation of the Diffusion Coefficient The diffusion coefficient can be obtained by combining measure- ments of photographs taken after the plateau region disappears from the sedimentation-equilibrium pattern. The simplest method for such calculations is that proposed by Sophianopoulos g£_§1, (1962) utilizing the following equation; 2 c , he D _ = - A t 30 In (%) eq (5r t constant ‘gjrb _ rm’AI II , ( ) ' mid point ._ where 3 1‘b Eh 2 AI: 1+Tr—Y rb+rm and 2 2 _- _ AII=1+<_rb_,£e> 11./J” VD) M , 2 RI 2- 144 By removing the optical constants, equation (30) becomes . 2 1 Y - Y = constant - —I[——]-)— t . 31 n ( [8 eq ls t)mid point r% _ r% AIAII ( ) Thus, log (lsfléq’-15Yf) measured at the mid point of the solution column was plotted against the time (min), letting the first photo- graph be zero time since only the time interval is important. The slope of this plot is given by ...ij AIAII slope — 2 . 2.303 (rb - rm) -1 Changing the diffusion coefficient from cm2 min to cm2 sec-1, then, gives D = #2:.43‘03 (rb-rm)2 (slope) 2 -1 (32) 712 AI AH 60 Approach-to-Equilibrium Theoretical The flow equation for the ultracentrifuge has been derived from the laws of irreversible thermodynamics and for incompressible Systems is q t t 1 Ji=-Z Lik %§a (i: 1, .60., <1)- (33) k=1 t Differentiating equation (3) with respect t°.£ at constant time gives Ba 1101231 t=(311k )t-w 145 For an isothermal system the chemical potential is a function of the pressure and concentrations of all solutes, therefore =2)_§___111 152 where l is a shape factor. Note that [[2] is in dl/g ( [[2] in ml/g 2= 100 [II] in dl/g). Since hydrodynamic properties depend on both the particle size and shape, these authors pointed out that two hydrodynamic parameters must be obtained to define the size and shape. The frictional coefficient for a sphere having a volume equal to ’yg_is f0 = 6 fl" Il'ao . (57) Substituting this equation into equation (54) gives 0 M (1 - $20,)? DZOJE) V (58) S = 20 w ' 0 ° , NAV f/fo 67Tii20,w a 3 V 1/3 But ao = Zjfi; and substituting equation (56) for Va and rearranging results in the following expression: 1/3 300 M ”TV NAv M (1 -' R7 ) .. o 20& pZOLw = f/fo 6 "”2 S 20,W NAV 09W 1/3 1/3 . f /f NA = NAV SEOLW [’1] 42013: (59) 0 16200 2/3 - 5 B’ - Tr M (1 " V20,w p20,w) and [In -- and a thermo- or Thus, two hydrodynamic properties - SEO,w dynamically determined molecular weight were used in these studies to calculate gt However, §_is not very sensitive to particle shape and therefore the effective dimensions of the molecules were not calculated. Since 6 is not very sensitive to particle shape, an approximate molecular weight can be calculated from the intrinsic viscosity and sedimentation coefficient. Assuming an average value for Q (2.16 X 106) and substituting the value for the viscosity of water at 20° C in equation (59) gives 153 3/2 1/2 4690 (sgo’w) [q] (1 " V20,w p20,w) M ___ <60) APPENDIX III Appendix Table 3 A comparison of the amino acid analysis for k-casein and the l-casein fraction Amino k-Casein al-Casein Fraction Acid g/100 g g/100 g ASp 7.72 8.18 Thr 6. 743 1.65"=1 Ser 5.03a 3. 17a Glu 19.80 24.11 Pro 10.95 7.16 Gly 1.23 ' 2.63 Ala 5.40 2.86 Val 6.30 5.89 Met 1.68 1.99 Ilen 7.10 5.18 Leu 6.11 8.23 Tyr 7.61 5.673 Phe 3.86 4.55 Lys 6.51 9.04 His 2.36 3.83 Arg 3.96 4.17 m3 1.94‘al 1.90 a Extrapolated to zero hydrolysis time. 154 cHEmsmv LIBRARY "7th171114111111fliflilllifllflflflyfljflflm“