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Modification of ASI-Casein by 2-phenyl-1,4-dibromoacetoin Beveridge, Herbert James Thomas 1970

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MODIFICATION OF asi--CASEIN BY 2-PHENYL-l,U-DIBROMOACETOIN by HERBERT JAMES THOMAS BEVERIDGE B.S.A., University of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Food Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1970 In presenting th i s thes i s in pa r t i a l f u l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree tha permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i cat ion of th i s thes i s f o r f i nanc ia l gain sha l l not be allowed without my wr i t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada i i ABSTRACT The histidine specific reagent 2-phenyl-l,U-dibromoacetoin (PDA) has been applied to OSl-casein B. Reaction of (XSI-casein with PDA for 26 hours resulted in the loss of about two residues each of histidine and methionine per (ZSI-casein monomer (molecular weight = 27,000). The modified protein was 90% soluble in 8 mM calcium chloride and precipitated quantita-tively at 13 mM calcium chloride. The control was precipitated quantitatively at 8 mM. The calcium binding capacity of the modified asi-casein was reduced to about U . £ calcium ions per PDA <ZSi_c-asein monomer from 12.k calcium ions per GtSl-casein monomer. Reaction of asi-casein with PDA resulted in the . production of aggregated material which remained at the origin on poly-acrylamide gel electrophoresis in the presence of urea and 2-mercaptoethanol and which was eluted at the void volume of a Sephadex G-200 column. While following the time course of the reaction of PDA with CZSI-casein, i t was found by N-bromosuccinimide (NBS) spectrophotometry titration (6 M urea-acetate-formate buffer, pH U.O) that two residues of tryptophan per monomer asi-casein could be detected at zero reaction time but 3.1 residues could be detected after 30 hours. Comparison of the results obtained with asi-casein, PDA OSl-casein, ^3-lactoglobulin and a-chymotrypsinogen using both the NBS titration procedure and the p-dimethylaminobenzaldehyde method of Spies and Chambers (U6) suggest the presence of a "buried" tryptophan residue in asi-casein. PDA has a negligible effect on the NBS titration procedure so the residue "exposed" cannot be an artifact generated by PDA bound to the protein. Increasing the urea concentration to 10 M did not expose the third tryptophan residue to NBS. i i i TABLE OF CONTENTS Page INTRODUCTION 1 SURVEY OF THE LITERATURE 3 Variations in the asi-Caseins 3 Molecular Weight ii Properties of Gtsi-Casein 5 Chemical Modification of Caseins 13 METHODS AND MATERIALS 18 Preparation of Glsi-Casein 18 2-Phenyl-l,U-dibromoacetoin 18 Solubility of as i -Casein in Calcium Chloride 19 Stabilization of a si-Casein by K-Casein 20 Nitrogen 21 Amino Acid Analysis 21 Tryptophan 22 Electrophoresis * 23 Phosphorous 2h Calcium Binding 2$ Gel Filtration 26 RESULTS AND DISCUSSION 27 Genetic Variant of asi -Casein 27 Reaction Time of asi-Casein with PDA 29 Production of PDA as i -Casein 33 iv Amino Acid Analysis 35 General Observations on PDA asi-Casein 37 Calcium Sensitivity of asi-Casein and PDA asi-Casein U-0 Stabilization by K-Casein h2 Gel F i l t r a t i o n U5 Tryptophan in asi-Casein U8 General Discussion $2 BIBLIOGRAPHY 55 APPENDIX Computer Program 60 LIST OF TABLES Table Page I pH Adjustment of asi-casein reacted with PDA for 31 various times. II Amino acid analysis of asi-casein and PDA asi-casein. 36 III Binding of calcium to asi-casein and PDA ast-casein. U3 IV Comparison of tryptophan values obtained by the NBS k9 t i t r a t i o n procedure and by the method of Spies and Chambers LIST OF FIGURES Figure Page 1 Polyacrylamide gel electropherogram of the milk from 28 four individual cows and of the asi-casein used in this study. 2 Time course of the reaction between PDA and asi-casein 32 3 Electrophoretic patterns of the 0, 8 , 12, 2h and 30 3k hour PDA asi-casein reaction products. U Ultraviolet absorbance of asi-casein and PDA asi-casein. 39 5 Solubility of asi-casein and PDA asi-casein as a hi function of calcium chloride concentration. 6 Stabilization of asi-casein and PDA asi-casein by hh K-casein. 7 Sephadex G-200 gel f i l t r a t i o n of asi-casein and PDA U6 asi-casein. 8 Consumption of NBS by protein and by PDA. 51 ACKNOWLEDGEMENTS I wish to express ray gratitude to Dr. S. Nakai for supervision and guidance throughout the course of this work, to Miss Lynne Robinson for helpful guidance during the writing of the computer program, and to Miss Arlene Gelder for assistance with the amino acid analysis. I am indebted to the National Research Council for financial support in the form of two scholarships held during the course of this work. INTRODUCTION The caseins of bovine milk interact with themselves, with each other and with calcium ions to form a stable colloidal system consisting of micelles that appear spherical under the electronmicroscope and range in size from U O to 300 nm in diameter ( 2 3 ) . The stability of the milk system during pro-cessing procedures such as pasteurization and canning is directly related to the stability of the micelle and to the reactions and interactions which allow the micelle to retain its integrity. The study of the micelle and of the caseins which make up the micelle then, has practical value as well as being of interest as an unexplained natural phenomenon. In 1956, Waugh and Von Hippel (56) demonstrated the presence of a casein fraction of milk acting as a "protective colloid" and capable of protecting other casein fractions—notably the (X S -caseins—against precipitation by calcium ions. Since that time the K - T as-casein interaction has been directly demonstrated ( 3 0 ) as has been the protective effect of je-casein towards d'?S*^ casein (65). However, the nature of the interaction between these two proteins has not been fully elucidated. Attempts to explain the nature of the K - , a S -casein interaction have led to an appreciation of the role played by hydrophobic interactions in the formation of casein micelles (lit), but the mechanism of interaction has remained unclear. One approach to explaining the underlying mechanism of interaction has been to chemically modify one or more amino acid residues in the caseins, observing whether or not these modifications induce changes in an interacting system. Interest in this type of study has centered mainly upon the K-casein molecule. Ehotooxidation (69) and reaction with N-bromosuccinimide (25), 2-phenyl-l,ii-dibromoacetoin (PDA) and diazo-l'H-tetrazole (26) have 2 indicated that histidine residues in K -casein play an important role in allowing the interaction between K- and as-caseins to take place since, in a l l cases, modification of histidine was followed by the inability of K-casein to exert a protective effect towards as-casein. In comparison to the data available for K* -casein, data on the effect of residue modification in a Si-casein is limited (66,6U). During their work on the effect of histidine modification on *c-casein, Nakai and Perrin (27) found that PDA modified only one histidine residue and that, except for a small decrease in lysine, the reagent was apparently specific for histidine. With the availability of a histidine specific reagent i t was decided to investigate the role of histidine in aSl-casein. The object of this work, then, is to prepare the PDA derivative of OSl-casein and to determine the effect of this modification upon the calcium sensitivity of the protein and upon the ability of K-casein to protect the modified as i-casein from precipitation by calcium ions. 3 SURVEY OF THE LITERATURE Variations in the a S -Caseins Current practice has been to classify the caseins in terms of their mobility in starch or polyacrylamide gels at alkaline pH and in the presence of 2-mercaptoethanol. Use of this technique combined with column chromato-graphy has shown that a s -casein is not a single protein but a "family" of proteins which migrate in the same general region of a gel and have mobilities which l i e between 0.88 and 1.18 (1). If the relative mobility of the a $4 band is taken as 1.00 (12), then the relative mobilities of the remaining components on starch gels are C t S O(l.l8), GtSl ( l . l l i ) , CtS2(l.ll), as3 (1.06) and as5 (0.88). Of these fractions, OSl-casein is the most impor-tant since i t constitutes by far the major component of the CIS-casein complex. aSI-Casein has been shown by Thompson et al (51,5>3,2l) to consist of three genetically controlled variants. These have been named Ct Si-casein A, B and C in order of their decreasing electrophoretic mobility. Study of selected cows has indicated that the variance is controlled by three allelic autosomal genes with no dominance—each allele being responsible for the production of one of the three forms of asi-casein. From this, the six possible types of aSl-casein and their corresponding genotypes are A(A/A), B(B/B), C(C/C), AB(A/B), AC(A/C) and BC(B/C). All six types have been demon-strated in the milk of individual cows. Studies on the occurrence of the various OSi-caseins in different breeds of cattle (21) have shown that the most common type is a s I-casein B with asi-casein C being relatively rare and a Si-casein A a very rare type of casein. Further, i t is interest-ing to note that in 98 Ayrshire cattle examined, only the B/B genotype was found. Recently, a fourth genetic variant of 01 S I-casein has been reported (9) having an electrophoretic mobility intermediate between that of asi-casein A and asi-casein B. This casein has been designated asi-casein D and has been found i n the milk of the Flemish breed and i s very rare indeed. The differences between asi-casein B, C and D are minor and probably result from single amino acid substitutions. Thus, asi-casein B contains one more glutamic acid than the C variant while the C variant contains one more glycine than the <XSI-casein B. S i m i l a r l y , comparing asi-casein D with the B variant, asi-casein D contains one more residue of proline and one less residue of serine. dSl-Casein A, however, contains one more lysine but one less each of arginine, aspartic acid, alanine and valine, two less phenylalanines and three less leucines or, a net deletion of eight amino acid residues when compared to the B variant. Fingerprint studies on chymo-t r y p t i c digests of the A variant (5h) suggest that these differences are due to a mutational event involving a lysine/asparagine substitution and to the deletion of eight amino acid residues which are l i k e l y to be sequential. End-group analysis (19,58) has indicated that a l l three asi-casein variants have an N-terminal arginine residue and the C-terminal sequence -leu-leu-tryp-COOH. Molecular Weight Swaisgood and Timasheff (U9) have obtained a value of 26,900 ± 2,700 daltons by l i g h t - s c a t t e r i n g experiments carried out under structure breaking conditions (2-chloroethanol, anhydrous formic acid and water-methanol mixtures). In 6.5 M urea (pH Iu5), Waugh et a l (58) found by osmotic pressure a molecular weight of 27,500 for asl,2-casein, which i s probably a mixture of the B and C genetic variants. Noelken (31) obtained a value of 2U,600 daltons for 5 as i-casein B from equilibrium sedimentation experiments i n 3 M guanidine. Light-scattering measurements at pH 12 on asl,2-casein yielded a value of 27,500 (11) and a molecular weight of 27,500 has been deduced from end-group analysis ( 5 8 ) . In his excellent review of milk proteins, McKenzie (23) adopted a value of 27,000 for the molecular weight of OSl-casein monomer and this seems a reasonable value to assume i n the study to be reported. Properties of aSI-Casein Waugh et a l ( 5 8 ) have defined a S-casein as the casein component which interacts with K* -casein at appropriate stoichiometric weight ratios and which forms complexes with K*-casein i n the absence of divalent cations and micelles clottable with rennin i n the presence of divalent cations. While this definition i s not really satisfactory, /S -casein also interacts with K-casein and divalent cations, i t serves to emphasize two of the most important properties of <jS I-casein: 1. the sensitivity of the protein to precipitation by calcium ions; 2. the property of interaction with K -casein to form stable micelles i n the presence of calcium. aS-Casein, classically, i s the casein fraction exhibiting marked sensitivity towards calcium ions being quantitatively precipitated at a calcium chloride concentration of 8 mM ( 6 6 ) . The property i s somewhat variable, however, depending upon temperature and genetic variant. The genetic variants B, C and D would be expected to behave quite similarly since the differences between them apparently involve only single amino acid sub-stitutions. Work by Noble and Waugh (30) on a casein which i s probably a mixture of aSl-caseins B and C tends to support this, but, i t has not been ex p l i c i t l y demonstrated. However, OSl-casein A has been shown by Thompson et a l ( 5 5 ) to differ considerably from a s t - c a s e i n B in i t s solubility 6 properties. At 1 C, a s 1-5 i s quantitatively precipitated by 50 mM calcium chloride but, as the calcium chloride concentration exceeds 150 mM, a gradual "salting i n " effect occurs. Temperature has no effect upon the solubility and the protein remains insoluble with increased temperature. At 37 C, aSl-B begins to precipitate abruptly at 5 mM calcium chloride and precipita-tion i s complete at 8 mM. This phenomenon has also been observed by Noble and Waugh (30). This behavior offers an alternative method to electrophoresis for distinguishing a s i-casein from /9-casein since $-casein i s soluble i n calcium chloride solutions at 1 C and remains soluble un t i l the temperature reaches 18 C whereupon abrupt precipitation takes place. The reaction i s reversible and j3-casein w i l l redissolve i f the temperature i s lowered to 15 to 16 C. The behavior of a SI-casein A in calcium chloride solutions i s in contrast to that of the other OSI-casein fractions. Like OSI-B, at 1 C OSI-A i s quantitatively precipitated at about 10 mM calcium chloride but, unlike aS l-B, this s o l u b i l i t y i s reversed at about 100 mM calcium chloride, the protein becoming completely soluble. This behavior i s dependent upon the •i salt concentration. Increasing the potassium chloride concentration decreases the depth of the dip in the calcium chloride solubility profile until at O.lii M potassium chloride the dip i s barely discernable. At 37 C, a s i - A pre-cipitates at 5 mM calcium chloride but precipitation i s not complete at 10 mM calcium chloride in contrast to a s i-B. Temperature also affects the solubility of OSI-casein A. The protein i s soluble at 1 C in 0.U0 M calcium chloride but upon increasing the temperature to 33 C abrupt precip-itation takes place. Precipitation is reversible by cooling the solution to about 28 C. Thus, a s i-casein A exhibits properties intermediate between those of a s i-casein B and C and )S-casein. It has been suggested ( 5 U ) 7 i that these changes in the properties of CtSl-casein A may be due to the sequential deletion of eight apolar amino acids in the peptide chain sequence. Creamer and Waugh (7) have shown that about ten atoms of calcium are bound per as-casein monomer when the protein is 2,0% soluble and, apparently, about thirteen sites of similar calcium binding power at pH 6.6 exist (55) for aSI-caseins B and C. When calcium binding exceeds this critical level, preci-pitation occurs. Noble and Waugh (30) have estimated that eleven calcium ions are bound per mole of asl,2-casein precipitated by addition of calcium chloride. Thompson et al (55) have a calcium binding value of about seventeen calcium ions per 27,000 molecular weight for aSl-Casein A or about four more than a s i -casein B. The difference in number of available binding sites cannot be attri-buted to a difference in the number of phosphate groups since each genetic vari-ant contains about 1.0$ phosphate or about ten phosphates per mole of protein. If micelles are harvested from skimmilk by high speed centrifugation and the calcium removed from these micelles by chelating agents such as citrate, a clear protein solution will be obtained which has been termed by Waugh et al (30,57) first cycle casein. In the ultracentrifuge at low temperature this material reveals two peaks at 1.3 S and 7.5 S which have been identified as representing )9-casein and complexes of a S l - and AC-casein respectively. At 25 C this material gives rise mainly to a symetrical peak at 7.8 S (1$ protein, pH 7.0) which shows l i t t l e boundary spreading suggesting that the product of the interaction is a monodisperse complex (57). This evidence for the association of a s i - , <- and -casein was obtained by Waugh and co-workers prior to l°6l. Also i t was shown that the only essential components for micelle formation were a Si-casein, /<-casein and a divalent cation, usually calcium. In a subsequent paper (30), Noble and Waugh demonstrated 8 the presence i n an ul t r a c e n t r i f u g a l pattern of the asi-casein i n t e r -action product obtained by mixing together the p u r i f i e d components. During t h e i r studies on the formation of micelles, Noble and Waugh (30,59) found that by adding calcium chloride to a mixture of O S I - and K-casein a characteristic s o l u b i l i t y curve was obtained. The f i r s t portion of the curve p a r a l l e l e d the asi-casein s o l u b i l i t y curve consisting of an i n i t i a l plateau followed by a sharp drop (the dip) i n supernatant protein at about 5 mM calcium chloride. This was followed by a dramatic rise i n super-natant protein to a plateau at about 10 mM calcium chloride. I t was considered by Noble and Waugh that the descending leg of the dip consisted of p r e c i p i -t a t i n g calcium asl-caseinate and the ascending leg of the dip consisted of calcium as l-caseinate coated with /c-casein. The largest weight r a t i o (OS I /K ) at which complete s t a b i l i z a t i o n could occur was found to be 10 (0.07 M potassium chloride, 0.01 M imidazole, pH 7.1). Decreasing the weight r a t i o caused a progressive lessening of the dip u n t i l i t could not be observed at a r a t i o of 1. However, under the experimental conditions used, complete micelle s t a b i -l i z a t i o n could be obtained i n 10 to 20 mM calcium chloride at an asl/Vc weight r a t i o of 10. This data i s i n opposition t o the e a r l i e r idea that /c-casein and asi-casein interacted stiochioraetrically i n the weight r a t i o k ( O S I / K ) (57) which i s approximately the r a t i o i n which the two components occur i n milk. I t should also be noted that while K- and asi-casein appar-ently interact to form a complex, i t requires a calcium concentration i n excess of that required to precipitate asi-casein to cause the complex to form stable micelles. This would suggest some essential role f o r calcium ions i n the formation of stable micelles. One drawback to the Noble and Waugh technique for determining micelle 9 stabilization is that relatively large quantities of material are required. Zittle (65) has introduced a test which overcomes this difficulty. By incu-bating a S l - and K-casein together at different known weight ratios in the presence of a constant amount of calcium, the protein remaining in the super-natant after low speed centrifugation may be determined spectrophotometryally. Plotting a SI / K weight ratio against supernatant protein yields character-istic S-shaped curves to which the stability of a test system may be related. asi-Casein, like the other caseins, has a strong tendency to associate (Ii2,U8), this tendency apparently being different among the genetic variants. The forces driving these association reactions are considered to be those involved during the formation of hydrophobic interactions or hydrophobic bonds (35). Formation of hydrophobic bonds in proteins is an endothermic process accompanied by an increase in both the enthalpy and the entropy of the system. The large possibility for hydrophobic interactions in the caseins is suggested by the high content of apolar amino acids present in these proteins (23,1*0). Payens and Schmidt (33) have made a study of the association of OS I-caseins B and C in the ultracentrifuge. They found that at pH 6.h each of these caseins undergo a rapid endothermic polymerization and that the particle weight of the polymerizing unit is 87,500 for aSl-casein B and 113,000 for asi-casein C. They considered that there were consecutive polymers present up to the pentamer of the polymerizing unit and, using a modification of the Steiner (hi) procedure, association constants were calculated for stepwise polymerization at 2 C, 9 C and lU C. For a s i -casein C, AH and AS were found to be positive and the free enthalpy was decreased by a constant amount of -3.2 kcal/raole at each association step up to the pentamer. Payens (35) and Payens and Schmidt (33) have suggested that a s i - c a s e i n associates through the formation of hydrophobic bonds between polymerizing units and that, because of the equality of the enthalpy change at each step, the same bond i s formed between the hydrophobic s i t e s on the subunits. This i s considered to imply that these interacting s i t e s are s u f f i c i e n t l y f a r enough apart to avoid more than one contact between subunits thus l i m i t i n g the number of possible configurations for the polymerized species. In a study of the polymerization of a s i - c a s e i n B by osmotic pressure measurements at neutral pH, Ho and Chen (16) found that the degree of poly-merization depended on ionic strength. In 0.01 M potassium chloride at h C and 20 C molecular weights of 31,000 and 29,UOO respectively were found and i n 0.1 M potassium chloride at h C and 20 C molecular weights of 122,300 and 91,700. These l a t t e r molecular weights would be expected to correspond to the "tetramer" and "trimer" of the O S J-casein B monomer. From these results i t was suggested that e l e c t r o s t a t i c interactions play an important role i n the polymerization of a s i-casein. To determine the shape of C J S I -casein B i n the monomeric and polymeric states, these authors measured the v i s c o s i t y of CISI-B solutions at the two i o n i c strengths. In 0.01 M potassium chloride the i n t r i n s i c v i s c o s i t i e s of monomer casein vary from 11.8 ml/g to 10.2 ml/g as the temperature i s increased from U.9 C to 37 C. In the presence of 0.1 M potassium chloride at pH 7, the i n t r i n s i c v i s c o s i t i e s of the trimer or tetramer vary from 9.3 ml/g to 7.7 ml/g from i ; C to 20 C. Also the d i f f u s i o n constant (0.1 M potassium chloride, pH 7.08) I>20jW i s 3.7 x 10"? cm2/sec. Since i t i s known that the i n t r i n s i c v i s c o s i t y of common globular proteins vary from 3.3 to luO ml/g and the d i f f u s i o n coefficients are less by a factor of about two, i t was concluded that GtSl-casein B i s not a spherical molecule. Furthermore, studies of i n t r i n s i c v i s c o s i t y i n 6 M guanidine was about twice the value obtained in the absence of guanidine hydrochloride. Consequently, Ho and Chen conclude that asi-casein B does not exist as a random coil characterizing the structure of a denatured protein but that the asi-casein molecule is relatively compact as compared to a > denatured protein. Swaisgood and Timasheff (h9) have examined the association of CtSl-casein C by light-scattering, ultracentrifugal and viscometric techniques. These authors have confirmed the observation of Ho and Chen (l6) that the association is sensitive to ionic strength—higher polymers being found at higher ionic strength. Further, these authors found that between pH 8 and 9 the most stable unit was the dimer and at pH values above 9 (ionic strength 0.3'!) the monomer appeared. From their viscometric data, Swaisgood and Timasheff were unable to classify ClSi-casein C as a solid ellipsoid of revolution or as a random coil since their data was consistent with either model. However the dimer could be described well in terms of a rigid globular compact structure. A tetramer of molecular weight 110,000 could be described as a prolate ellipsoid of revolution with an axial ratio of 20-20 or as a rigid rod of length 5>60 A and cross-sectional area of 12.0 A. From the data noted above i t is clear that the conformation of the OS I-caseins is not known. However, two general conclusions about the shape of the molecule may be inferred from this data: 1. The asl-casein monomers appear unlikely to be random coils in solution and are more likely to be present in a relatively compact, folded non-spherical structure. 2. asi-Casein monomers polymerize readily to form dimers, trimers, tetramers etc. the exact species present depending upon the pH and ionic strength. At t h i s point i t i s of interest to return to the work of Payens and Schmidt and note that t h e i r work was carried out at 9 C and an ionic strength of 0 . 2 , pH 6 . 6 . From the data of Ho and Chen ( 1 6 ) , i t would be expected that the predominating species under these conditions would be the tetramer. From t h i s i t i s i n t e r e s t i n g to speculate that polymerization of a s i-casein involves an i n i t i a l series of polymerization steps from the monomer to the tetramer involving e l e c t r o s t a t i c interactions to y i e l d stable aggregates which can, i n turn, undergo weaker reversible polymerization involving hydrophobic bonds. Optical rotatory dispersion (ORD) measurements (13) and l i g h t - s c a t t e r i n g data ( 2 2 ) indicate that a s i-casein exists i n solution at neutral pH as a random c o i l almost completely devoid of a - h e l i c a l organization and, i n t h i s respect, resembles denatured proteins. Herskovits (13) has reported the percent a - h e l i x i n a s I-casein as being h-1%% depending upon the procedure used i n the calculation. I t has been recognized for some time that proline residues tend to disrupt a - h e l i c a l structure i n proteins. Szent-Gyorgyi and Cohen ( 5 0 ) observed that about 8 percent proline s t a t i s t i c a l l y distributed along a peptide chain w i l l deform the backbone of the polypeptide into a random c o i l . Further, Blout and co-workers ( 5 , 2 0 ) have postulated that amino acids such as valine and isoleucine which have branched side chains tend to r e s i s t h e l i x formation. a s i-Casein i s r e l a t i v e l y r i c h i n valine and isoleucine and contains 8 . 8 mole % proline (13) . Also structure breaking solvents such as urea or formic acid have only a small effect upon the o p t i c a l rotatory properties of a s i - c a s e i n , the Moffitt-Yang parameter b Q going from -30 i n potassium chloride buffer of neutral pH to 0 i n 8 M urea (13) . These findings are a l l consistent with the assignment to a S I-casein of a disordered, non-helical, random c o i l configuration. 13 These ideas on the conformation of asi-casein may be contrasted with suggestions of Ho and Chen (16) and Swaisgood and Timasheff (h9) which in-dicate that asi-casein does not exist as a random coil but that the a s i -casein molecule has a relatively compact, folded, non-spherical structure. Chemical Modification of Caseins In recent years a common method of investigating the biological properties of proteins has involved chemical modification of one or more reactive groups in the protein molecule. The changes induced in the biological properties often yield information concerning the importance of the modified group in maintaining the functional integrity of the protein and may yield information on the mechanism by which the protein performs its function. Of the dozens of reagents and procedures available for the modification of proteins (13>), only a l i t t l e over a half dozen of these have been employed in casein chemis-try. One of the earliest attempts to modify the caseins involved the enzyme phosphoprotein phosphatase prepared from beef spleen (36). Dephosphorylation of ic-casein did not significantly affect its stabilizing ability towards OS-casein. However, dephosphorylation of as-casein markedly decreased its ability to interact with K-casein to form stable micelles in the pre-sence of calcium. Examination of the dephosphorylated proteins in the ultracentrifuge indicated that the modification did not affect the ability of either protein to form complexes. From this data i t was concluded that the formation of i c - , as-casein complexes is not influenced by the presence of esterified phosphate groups and the growth of stable micelles does not depend upon the formation of calcium phosphate interaction products within the as-casein complex. However, i t would be inferred that the growth Ik of stable micelles from the /c-, a S -casein complexes i n the presence of calcium i s dependent upon the interaction of calcium and esterified phosphate groups i n the as-casein part of the complex. Zit t l e et a l (66,68,69) have investigated the effect of photooxidation in the presence of methylene blue on the properties of the Q S - , and K-caseins. Generally, i n a l l three proteins, aggregation sufficient to impede the movement of the proteins into a polyacrylamide gel during electro-phoresis was observed. The major amino acids oxidized were histidine, tryptophan and some tyrosine i n decreasing extent of oxidation, and the optical density i n the trough region around 200 ran i n the ultraviolet spectra of the proteins showed marked increases as the oxidation proceeded. Photooxidized K -casein was incapable of protecting a S \ -casein from precipitation with calcium and was not susceptible to rennin attack. Photooxidation of (3 -casein resulted i n a marked decrease in the sensitivity of this protein towards precipitation by calcium. Oxidation to the extent of 10 moles of 0 2 per 30,000 g /S-casein resulted i n a protein, normally precipitated by 10 mM calcium chloride at 30 C, that was precipitated only to the extent of about 20$ in 1*0 mM calcium chloride. Concurrent with this change in calcium sensitivity, the a b i l i t y of j8 -casein to bind calcium ions dropped from 21 to 27 moles of calcium per 10^ g fB -casein to 10 moles per 10-* g at the upper extent of oxidation. No explanation of this phenomenon was apparent. When as»-casein was modified by photooxidation changes similar to those reported for /8 -casein were observed. asi-Casein (pH 7.0) i s quantitatively precipitated in 8 mM calcium chloride whereas after photo-oxidation none i s precipitated by 8 mM calcium chloride and only 2$% i s precipitated with twice as much calcium. Calcium binding i n this oxidized 15 protein was not reported. From the results reported by Z i t t l e for oxidized caseins, i t appeared that h i s t i d i n e and tryptophan were the most l i k e l y candidates to be of impor-tance i n the formation of * c - , a S t-casein complexes. Nakai et a l (25»26) have investigated the effect of modifying tryptophan and h i s t i d i n e i n * c -casein on the K-, a SI-casein interaction. Careful oxidation of the four residues of tryptophan i n K-casein (molecular weight = 5k,000) by N-bromo-succinimide resulted i n no loss of s t a b i l i z i n g a b i l i t y as determined by the calcium chloride-centrifuge method. However, modification of h i s t i d i n e with 2-phenyl-l,U-dibromoacetoin and diazo-l«H-tetrazole resulted i n drastic loss i n the a b i l i t y of K -casein to protect a S i-casein. Carboxymethylation, however, did not affect the s t a b i l i z i n g a b i l i t y . Woychik and Wondolowski (6I4.) have investigated the effect of n i t r a t i o n of t r y s o y l residues i n and a s i-caseins with the reagent t e t r a n i t r o -methane. I t was found that n i t r a t i o n of up to four of the eight tyrosine residues i n K -casein affected i t s a b i l i t y to s t a b i l i z e a s i - c a s e i n only s l i g h t l y . However, on n i t r a t i o n of the f i f t h tyrosine a sharp drop i n s t a b i l i z i n g ability,was observed. On n i t r a t i o n of a S I-casein, i t was found that after n i t r a t i o n of 38$ of the tyrosine, the a s i - c a s e i n was unable to enter into the formation of a protective complex with K-casein. Further, a S i-casein was found to lose some of i t s calcium s e n s i t i v i t y with increased levels of n i t r a t i o n but p r e c i p i t a t i o n was e s s e n t i a l l y complete at 20 mM calcium chloride. In the case of both proteins, the f u l l y nitrated caseins were unable to penetrate the pores of an 8% acrylamide gel and impacted i n the st a r t i n g s l o t despite the presence of 7 M urea and 2-mercaptoethanol. The reagent S-ethyl-trifluorothioacetate has been utilized by Woychik (63) to study the role of free amino groups in /c-casein. These are mainly the € -amino groups of lysine. Blockage of the amino groups inhibited the formation of stabilizing complexes with a s I-casein but did not affect the ability of rennin to cleave /c-casein into para K -casein and glycomacro-peptide. The para /c-casein formed from the trifluoroacetylated K-casein (TFA- /c-casein) did not however form an insoluble coagulum. Removal of the blocking trifluoroacetyl groups restored the stabilizing ability of the /c-casein. Furthermore, TFA- /c-casein sedimented in the ultracentrifuge as a hypersharp boundary travelling about one half as fast as native K -casein. It was suggested that the changed TFA para- K -casein solubility properties and the modified ultracentrifugal behavior were probably due to the increased net negative charge of the derivatized protein resulting in a lower degree of aggregation. Hoagland (17,18) has studied the effect of acylation on the properties of J3 -casein. This author observed that succinylated jS-casein was 100% soluble in 33 mM calcium chloride and that acetylated )8 -casein was hh% soluble in 2E> mM calcium chloride at neutral pH. Addition of /c-casein to the acetylated /3 -casein increased the solubility of the derivatized fi -casein indicating that acetylated /3 -casein was capable of complexing with /c-casein to form a stable system. Recently, Nakai and Perrin ( 27 ) have observed that when /c-casein A2 is modified by 2-phenyl-l,U-dibromoacetoin (PDA), only one histidine residue per 20,000 g /c -casein is modified. A very slight loss in lysine was also noted. When the single histidine residue was modified, i t was found that x-casein aggregated and would not penetrate the pores of an acrylamide' gel during electrophoresis. Further, PDA modified K-casein lost completely its ability to stabilize QSi-casein against precipitation by calcium ions. 18 METHODS AND MATERIALS Unless otherwise stated in the procedure, a l l chemicals used in the following methods were of reagent grade and were used without further p u r i f i -cation. The water used was tap water purified by passage through a cartridge of mixed bed ion exchange resin (Barnstead No. 0 8 0 8 ) . Preparation of q sI-Casein as I-Casein was prepared from the milk of a single cow (Ayrshire) by the procedure of Zittle and Custer ( 6 7 ) . The purified a Si-casein was readily soluble i n water and gave water clear solutions. 2-Phenyl-l,U-dibromoacetoin (PDA) This reagent was prepared by S. Nakai^ as outlined by Schramn ( U 3 ) . The product was recrystallized three times from benzene petroleum-ether and dried in a desiccator before use. The reagent was protected from light when not in use. In use, a 1 0 x molar excess of PDA (based on the histidine content of the protein) was dissolved i n sufficient methanol so that the f i n i a l concen-tration of methanol after addition to the aqueous protein solution would be 16-17$. The protein ( asi-casein) was dissolved in 0 . 1 M phosphate buffer pH 6 . 7 and the methanolic PDA solution was added. The reaction was allowed to proceed at room temperature, i n the dark, for the required time. The xDepartment of Food Science. University of British Columbia, Vancouver 8 , B. C. Canada. reaction was terminated by placing the reaction mixture i n small diameter ( l cm) d i a l y s i s tubing and dialysing against a large volume of deionized water pre-cooled to U C. D i a l y s i s was carried out f o r 72 hours with the water being changed twice d a i l y . I t i s important that the narrow d i a l y s i s tubing be used as large diameter tubing does not allow f o r s u f f i c i e n t l y rapid removal of excess reagent. S o l u b i l i t y of asi-Casein i n Calcium Chloride Reagentst 0.001 M Imidazole buffer pH 7.5 80 mM Calcium chloride 1 N Sodium c i t r a t e pH 11.5 Procedure: To 3.0 ml of imidazole buffer was added 1.0 ml of 0.5$ aqueous solution of O S I-casein. S u f f i c i e n t water was added so that the f i n i a l volume after addition of the calcium solution would be 5.0 ml. Calcium chloride solution was added i n increments of 0.1 ml from 0 ml to 1.0 ml. The tubes were allowed to stand at room temperature f o r 15 minutes and then were centrifuged at 3000 g for 10 minutes. 3.0 ml of supernatant from each tube was transferred to a second tube and 2.0 ml 1 N sodium c i t r a t e was added. A blank containing water i n place of the protein was run i n p a r a l l e l . The o p t i c a l density at 280 nm was determined with a Beckman D.B. spectrophotometer using the blank as a zero o p t i c a l density reading. 20 S t a b i l i z a t i o n of dS l-Casein by K-Casein The procedure used i n t h i s study was based upon the published procedures of Z i t t l e ( 6 9 , 6 0 ) . The /c-casein had been prepared according to the method Z i t t l e and Custer ( 67 ) and gave s l i g h t l y opalescent water solutions. To c l a r i f y the solution, the pH was adjusted-to 11 with 0.1 N sodium hydroxide and immediately returned to pH 7 .0 with 0.1 N hydrochloric acid. This procedure yielded a clear solution of /c-casein which was used the same day i t was prepared. Reagents: 1 mg/ml K-Casein 0.01 M Imidazole buffer pH 7.0 00 mM Calcium chloride 0.2 N Sodium hydroxide Procedure: K-Casein (1.0 ml) and 2.0 ml imidazole buffer were mixed with s u f f i c i e n t water so that the f i n i a l volume after subsequent additions would be 0.0 ml. OSr-Casein (20 mg/ml) was added to the tubes i n increments of 0.00 ml, 0.1 ml, 0.2 ml up to 1.0 ml to give a range of a s I //c weight ratios from one to twenty. 00 mM calcium chloride (1.0 ml) was added to each tube and the tubes were incubated at 30 C for 10 minutes. In addition, a tube was set up containing everything except that water replaced the calcium chloride. This tube represented 100$ OS l-casein i n solution. The tubes were centrifuges at 3000 g for 0 minutes and dilutions of each tube super-natant were made i n 0.2 N sodium hydroxide so as to bring the o p t i c a l density at 280 nm within the range of a Beckman D.B. spectrophotometer. When suitable d i l u t i o n s had been found f o r each tube, d i l u t i o n s of the K-casein solution were made so as to determine the contribution to the o p t i c a l density of the K -casein. After suitable correction f o r d i l u t i o n , the results were expressed according to the formula: $ asl-casein i n solution - O.D.Qa - O.D.^  °'D'NoCa " °*D'/c where O.D.Ca = o p t i c a l density of the s t a b i l i t y t e s t , O.D.K = contribution made by K-casein to the t o t a l o p t i c a l density and O.D. Q^ a " op t i c a l density of the 100$ s o l u b i l i t y control tube. Nitrogen Determination Nitrogen was determined according to the micro-kjeldahl procedure (2). The nitrogen content of as i-casein was 15.25$ and t h i s figure was used i n the calculation of protein concentration. Amino Acid Analysis Amino acid analysis was carried out on a Phoenix Model M6800 (Phoenix Precision Instrument Co.) amino acid analyser using the Moore-Stein two column system. The samples were analysed as follows. Approximately 50 mg protein sample were dissolved i n water and made to 10.0 ml with water. Two aliquots of 1 ml each were taken from t h i s solution f o r Kjeldahl nitrogen determination and 1 ml of the solution was pipetted into each of three hydrolysis tubes and freeze-dried. Glass d i s t i l l e d hydrochloric acid (6 N, 10 ml) was pipetted into the hydrolysis tubes and the resulting solu-'tion was frozen i n a dry ice-methanol mixture and evacuated to 50-100 microns Hg pressure. During evacuation the frozen solution was allowed to thaw. When thawing was complete, the vacuum was turned off and the tube was flushed with nitrogen and again evacuated. This flushing procedure (without freezing) was repeated three times and on the finial evacuation the tube was sealed. Hydrolysis was carried out in a circulating hot air oven at 107 C for 2k, k& and 72 hours. The hydrolysis tubes were removed from the oven and transferred quantitatively to a round bottom flask and reduced to dryness on a rotary evaporator at hO C. A small quantity of water was introduced into the flask and re-evaporated to remove any remaining traces of hydrochloric acid. The residue was taken up in citrate buffer pH 2.2 (38), transferred quantitatively to a 10 ml volumetric flask and made to 10.0 ml with citrate buffer. This solution was applied to the ion exchange column of the analyser. The protein content of the sample was based upon a Kjeldahl nitrogen value and calcula-tion of the results was carried out on an I.B.M. 360 computer (see appendix) available through the University of British Columbia computing center. Analysis of each hydrolysis was carried out in triplicate, the values for serine and threonine were regressed to zero time of hydrolysis and the standard error calculated. The remainder of the amino acid values were averaged and standard deviations calculated. The results were expressed as a mean and a 95% confidence limit. Tryptophan Tryptophan was determined by both the method of Spies and Chambers (U6) and by the N-bromosuccinimide (NBS) spectrophotometry titration method as described by Spande and Witkop (hh). p-Dimethylaminobenzaldehyde (practical) was obtained from Eastman Organic Chemicals and was recrystallized twice from ethanol by adding water to a warm ethanolic solution until crystallization was just initiated and then cooling the solution. A suspension of slightly greenish-white crystals was obtained. These crystals were harvested by suction filtration, dried in air and kept in a desiccator away from light until used. N-bromosuccinimide (NBS) was purchased from Fisher Scientific Company and was recrystaliized from warm water. About 5 g NBS were dissolved in 2^ 0 ml water by stirring and warming. The aqueous NBS solution was placed in an ice-water bath and crystallization allowed to proceed. The white, crystalline material obtained was collected by suction filtration, washed with ice cold water and dried overnight in a vacuum desiccator. The recovery was about 6k%, Extensive heating of the NBS solution is to be avoided since NBS apparently decomposes on heating, the solution turning yellow-orange and smelling strongly of bromine. Urea-acetate-formate buffer was made by dissolving 3.U g sodium acetate* 3H20 and 180 g urea in about 250 ml of water. The pH was adjusted to U.O with formic acid and the solution was diluted to 500 ml with water. This buffer was kept at h C when not in use. The NBS solution used for the titration of tryptophan was made by dissolving approximately 22 mg NBS in urea-acetate-formate buffer immediately before use. The remainder of the procedure was as described by Spande and Witkop (hh). Electrophoresis Polyacrylamide gel electrophoresis was carried out in tris-EDTA-borate buffer at pH 9.1 as described by Thompson et al (52). The buffer was made by dissolving 30.25 g Tris (tris (hydroxymethyl) aminomethane), 3.90 g disodium EDTA and 2.30 g boric acid in about 700 ml water. The pH was adjusted to 9.1 and the s o l u t i o n made to 1 l i t r e . This stock so lu t i on was d i l u t e d 1 to 3 before use. Gels f o r e lec t rophores i s were made by d i s s o l v i n g 10 g acrylamide and 0.0 g N,N'-methylenebisacrylamide i n about 70 ml d i l u t e d b u f f e r . U0 g urea were added and the so l u t i on d i l u t e d to 100 ml with d i l u t e d bu f fe r . The gels were set by add i t i on of 1.0 ml 2-mercaptoethanol, 1.0 ml 10$ ammonium per su l f a te and 1.0 ml TMED (30% N,N,N',N'-tetramethylenediamine (V/V) i n 90$ ethanol) and pour ing i n to a mold. Samples were app l ied to the ge l by soaking 3 MM f i l t e r paper s t r i p s ( 2 x 0 mm) i n the sample so lu t ion and i n s e r t -ing these s t r i p s i n the ge l with the a id of a razor b lade. The solvent used fo r samples cons i s ted of 2 ml d i l u t e d b u f f e r , 0.6 g urea and 0.1 ml 2-mercaptoethanol. For mi lk , 0.2 ml of t h i s s o l u t i o n was added to 0.1 ml mi lk and the f i l t e r paper was soaked i n t h i s s o l u t i o n . For d r i ed samples, the sample was weighed i n to a tube and d i s so lved i n 0.2 ml of the so lvent . E lec t rophores i s was c a r r i e d out i n a ho r i zon ta l apparatus f o r 22-2U hours i n a co l d room at h C. The voltage across the ge l was maintained at 300 V. The separated bands were v i s u a l i z e d by s t a in ing i n 1% amido b lack 10B conta in ing 10$ a ce t i c ac id f o r 3 minutes and destained by washing the ge l i n 10$ a ce t i c ac id u n t i l the des i red degree of background c l a r i t y was obtained. Phosphorous Phosphorous was determined according to the method of Morrison (2k). Calcium Binding This method was designed to measure the calcium remaining i n solution after p r e c i p i t a t i o n of as i-casein by a standard calcium chloride solution. The EDTA t i t r a t i o n procedure of Ntailianas and Whitney (32) was u t i l i z e d for calcium determinations. Reagents: 1. Calcium chloride 80 mM 1.176 g CaCl2'2H20 Was dissolved i n 100 ml water. A standard calcium chloride solution was made by d i l u t i n g the 80 mM solution one i n ten. 2. EDTA k g disodium EDTA and 2 g sodium hydroxide p e l l e t s were dissolved i n water and made to one l i t r e . This solution was t i t r a t e d against the standard calcium chloride to obtain the normality. 3. Calcein indicator 0.2 g calcein (Fisher Scientic Company) was dissolved i n 25 ml of 0.1 N sodium hydroxide and dilute d to 100 ml. t h i s solution was stored i n the dark when not i n use. ii. Imidazole buffer—-0.01 M imidazole, 0.07 M potassium chloride, pH 7.0. Procedure: The following solutions were pipetted into centrifuge tubes (10-20 ml): 1. 2.0 ml protein solution (0.0$) 2. 0.0 ml (6.0, U.0) imidazole buffer 3. 3.0 ml (2.0, 3.0) 80 mM calcium chloride In addition a series of blanks were set up in which the protein was replaced by water. These tubes served as zero binding controls. The tubes were mixed and allowed to stand at room temperature for 30 minutes and then centrifuged at 30,000 g for 30 minutes at 20 C. 3.0 ml of the supernatant was withdrawn to a 00 ml flask, 10.0 ml of EDTA and 3 drops of calcein indicator were added and the excess EDTA was back titrated with standard calcium chloride solution. From this, the calcium bound to the precipitated protein may be calculated i f the protein concentration is known from a Kjeldahl nitrogen determination. Gel Filtration Gel filtration was carried out in Sephadex G-200 in a column fitted with flow adapters and operated in an upward flow manner. The eluting buffer was 0.00 M phosphate buffer pH 7.0 and the sample was applied in a semi-automatic manner by means of a three-way valve (37). 27 RESULTS AND DISCUSSION Genetic Variant of a SI-Casein The QSi-casein used in this study had been isolated from a single Ayshire cow from the University herd. This cow had not been typed with respect to casein genetic variance so an attempt was made to determine the OSI-casein genetic type of this cow. Since no authentic samples of the various OSl-caseins were available i t became necessary to run polyacrylamide gel electropherograms on cows in the University herd in the hope of discover-ing a milk which contained identifyable variants. Of fourteen cows examined, no recognizable variation was found in the OSI-casein fraction and the a s i -band of a l l of the milks examined coincided with the band produced by the purified OSI-casein used in this study. A typical electropherogram is shown in Figure 1. At first glance i t would appear that milk 607 contains an OSi-band moving more slowly than the other OSl-bands but measurement of the relative mobility of this band does not support this, and close examination of the point of application will show that the origin is set slightly behind the other starting points. This illustrates the major drawback to the f i l t e r papers-razor blade technique used in this study. It is extremely difficult to make the starting points for a l l five samples on a gel line up exactly. Kiddy et al (21) examined the milk of 1,378 individual cows and found that 97.0$ of the cows tested had OSI-casein B, 21.5$ OSI-casein C and 6.5$ OSI-casein A. This data does not necessarily reflect the frequencies that would be found in random samples since efforts were made to sample large numbers of daughters of specific sires especially in two breeds 601 403 asi 301 607 Fig. 1. Polyacrylamide gel electropherogram of the milk from four i n d i v i d u a l Ayrshire cows and of the Otsi-Casein used i n t h i s study. Numbers 601, U03, 301 and 607 are patterns obtained from the milk of the indiv i d u a l cows. Ctsi= a s i-Casein used i n t h i s study. Conditions: 7% acrylamide gel, lw5> M urea, pH 9.1, 0.66 ml 2-mercapto-ethanol per 100 ml gel. 29 (Holsteins and Guernseys). However, the relative abundance of the B variant is quite apparent. Furthermore, a l l of the 98 Ayrshire cows examined were homozygous for the B variant. Aschaffenburg (3) on the basis of the study mentioned above and upon a study of 52 cows in Great Britain has assigned a value of 1.00 to the frequency of OSi-casein B in the Ayrshire breed. From this data i t is apparent that the failure to find genetic variants in a sample of fourteen cows is not unusual especially since some of the cows examined were Ayrshires. Since the B variant i s so common, especially in the Ayrshire breed, and since the CISl-casein used in this study came from an Ayrshire cow and coincided with a l l of the GtSi-bands in the milks examined, i t seems very probable that the (z SI-casein used in this study was asi-casein B. Reaction Time of asi-Casein with PDA Nakai (28) found that reaction of K-casein with PDA for 2h hours resulted in the modification of one histidine residue per 20,000 g <-casein so i t was expected that asi-casein would have approximately the same reaction time. To determine the required reaction time and to follow the course of the reaction, 20lu6 mg asi-casein were dissolved in 50 ml 0.1 M phosphate buffer pH 6.7 and at time zero 10 ml of a methanol solution containing 8.3 mg/ml PDA was added to the protein solution. Immediately after mixing, a 10 ml aliquot was removed to a small diameter dialysis bag, the bag was labelled as zero time of reaction and dialysis was begun immed-iately. Samples (10 ml) were withdrawn at U, 8, 12, 2h and 30 hours and dialysed as described previously. After dialysis the samples were placed in small flasks, the pH and appearance noted, and the pH adjusted with 0.1 N sodium hydroxide u n t i l a clear solution was obtained. This data may be seen i n Table I. The samples were freeze-dried and stored i n a desiccator. The freeze-dried samples were dissolved i n 0.0 ml of water. An aliquot of each sample was hydrolysed for 2h hours as described under Methods and Materials and analysed for basic amino acids using the short column of the amino acid analyser. Each hydrolysate was analysed three times. From these runs, h i s t i d i n e was evaluated. Further aliquots were taken for nitrogen, tryptophan (NBS method) and electrophoretic analysis. The results of the h i s t i d i n e and tryptophan analysis are shown i n Figure 2. This figure shows a decrease i n h i s t i d i n e from 0.7 moles/mole CtSI-casein to luO moles/mole. During t h i s reaction period no si g n i f i c a n t changes were observed i n any other basic amino acid. I t i s apparent that PDA modifies about 2 moles of h i s t i d i n e per mole of GtSl-casein (molecular weight = 27,000) i n contrast to the results observed for K-casein ( 2 8 ) . The l i n e was very nearly f l a t between 2h and 30 hours reaction time and 26 hours was chosen as the reaction time for the main portion of the exper-iment . Tryptophan determination by the NBS oxidation procedure yielded two rather unexpected results (Fig. 2). F i r s t , the value of 2 moles tryptophan per mole of as i-casein was obtained at zero reaction time. A value approach-ing 3 moles/mole (23) had been expected. Secondly, the value for tryptophan increased from 2.0 moles/mole to 3.1 moles/mole over the reaction period of t h i r t y hours. I t has been reported (hh) that some proteins possess trypto-phan residues refractory to NBS oxidation which react either very slowly or not at a l l . This behavior may r e f l e c t tryptophan residues buried i n Table I. pH Adjustment of OSl-casein reacted with PDA for various times. Time of Initial Finial Initial Sample (hr.) pH pH Appearance 0 0.63 7.30 Milky k 0.08 7.20 Milky 8 0.60 7.30 Milky 12 0.00 7.20 Opalescent 2h 0.60 7.27 Very slightly opalescent 30 0.76 7.30 Very slightly opalescent 2. Time course of the reaction between PDA and a si-Casein. Histidine was determined by amino acid analyser short column, tryptophan by the NBS titration method. The vertical lines on the histidine line represent the 95>$ confidence limit at each point. (Molecular weight of asi-casein = 27,000) hydrophobic regions of the protein but such residues are usually exposed by conducting the t i t r a t i o n i n concentrated urea. Since the tryptophan deter-minations reported here were conducted i n 6 M urea, i t i s d i f f i c u l t to imagine tryptophan buried i n a hydrophobic region. However, the results do suggest the presence of a buried or unreactive tryptophan residue i n CtSI-casein. This observation w i l l be examined i n more d e t a i l i n a l a t e r portion of the t h e s i s . Polyacrylamide gel electrophoretic patterns of the 0, 8, 12, 2k and 30 hour samples are shown i n Figure 3. As the reaction proceeds, two intermediate products are formed followed by the production of material - which remains at the o r i g i n and cannot penetrate the gel pores even i n the presence of urea and mercaptoethanol. Product production i s accompanied by progressive depletion of the a s i-band. The production of material remaining at the o r i g i n of electropherograms i s strongly reminiscent of the patterns obtained by Z i t t l e (68,69) for caseins photooxidized i n the presence of methy-lene blue. The phenomenon has been attributed by Z i t t l e to aggregation of the protein. In the present case i t i s apparent that a l l of the a S i-casein has not undergone reaction but, i n the l a t t e r stages, the material remaining at the o r i g i n i s the major product. Further, i t i s apparent that the product of the reaction i s heterogeneous and that some of the CIS l-casein remains indistinguishable from i t s "native" form. Production of PDA asi-Casein asi-Casein was reacted with PDA as described previously f o r 26 hours. On the f i r s t attempt to carry out t h i s reaction, the reaction mixture (about 150 ml) was placed i n a large diameter (lu5> cm\ d i a l y s i s bag and dialysed 8 12 24 3 0 H O U R S Fig. 3. Electrophoretic patterns of the 0, 8, 12, 2h and 30 hour PDA ast-casein reaction products. Conditions: 7% acry-lamide gel, U.0 M urea, pH 9.1, 0.66 ml 2-mercaptoehanol per 100 ml gel. overnight against deionized water. The next day a gelatinous precipitate was found i n the bag which would not dissolve at pH 10 and which on c e n t r i -fugation at 3000 g for 10 minutes yielded a clear supernatant over a f a i n t l y yellow gelatinous p e l l e t . Urea (100 g) was dissolved into a suspension of the precipitate (200 ml) to obtain a cle a r , f a i n t l y yellow solution of the PDA derivative of OS l-casein. D i a l y s i s of t h i s solution against 20 l i t e r s of 0.$% sodium chloride, pH 7.1, f o r 2h hours resulted i n the reappearance of the j e l l y - l i k e p r e c i p i t a t e . This behavior i s apparently a manifestation of the aggregation of O S i -casein induced by reaction with PDA beyond 30 hours since no p r e c i p i t a t i o n had been observed i n the experiment performed previously. The major d i f f e r -ence between the two experiments involved the use of large diameter d i a l y s i s tubing i n t h i s case and i t was considered that the large d i a l y s i s tubing did not allow s u f f i c i e n t l y rapid removal of excess PDA. When the experiment was repeated using small diameter d i a l y s i s tubing no precipitate was encountered. Amino Acid Analysis Amino acid analysis of OS l-casein and of the PDA derivative of O S l -casein were performed as described under Materials and Methods. Table I I shows the results of t h i s analysis and of the phosphorous analysis. This data i s i n reasonable agreement with analyses reported i n the l i t e r a t u r e (23,1|0) and the values of 10.2 moles of glycine and hh to hS moles of glutamic acid per 27,000 g of OS l-casein are consistent with the assignment of the B genetic variant to the OS l-casein used i n t h i s study. The f a i l u r e to find cysteine i n any of the analyses provides supporting evidence that the preparation i s not contaminated with either K-casein or with minor Table I I , Amino acid analysis of asi-casein and PDA O S I - c a s e i n . (Residues per monomer molecular weight = 27,000) 95$ 95% Amino Acid OSI-Casein Confidence PDA OSI-Casein Confidence Limit Limit Lysine 16.1 0.35 16.1 0.10 Histidine 6.0 0.21 ii.3 0.17 Arginine 7.2 0.25 7.0 0.15 Tryptophan^ 2.0 3.2 Aspartic Acid 17. k o .55 17.3 0.17 Threonine^ 6.2 0.21 5.9 0.16 Serine^ 1U.9 0.3U 1U. 7 0.2li Glutamic Acid UU .6 1.60 U5.6 0.38 Proline 19.7 0.83 18.9 1.00 Glysine 10.2 0.26 10. h 0.11 Alanine 10.7 0.10 10. k 0.10 Cystine \ 0.0 0.0 0.ii3 Valine 12.3 0.U6 12.ii Methionine 5.8 0.19 i i . i i 0.28 Isoleucine 12.6 1.10 12.3 0.22 Leucine 19.7 l . U o 19.2 0.81 Tyrosine 11.5 0.1+9 11.3 0.18 Phenyl alanine 8.9 0.09 9.0 0.27 Ammonia 32.0 1.30 3U.2 2.90 Phosphorous-^ 1.0$ 1.0$ Tryptophan was determined by the NBS t i t r a t i o n procedure and i s the average of duplicate analyses. ^Extrapolated to zero time of hydrolysis. 3Average of duplicate analyses. CtSl-casein fractions which have been reported by Annan and Manson (18) to contain sulfur either i n the form of d i s u l f i d e bonds or of sulfhydryl group-ings. The phosphorous analysis i s i n good agreement with reported values (23,UO) and was not affected by the reaction of PDA with CtSl-casein. The decrease i n h i s t i d i n e of about 2 residues per 27,000 g asi-casein confirms the e a r l i e r observation of the extent and s p e c i f i c i t y of PDA towards aS l-casein. That the difference i s r e a l i s evidenced by the fact that the difference between the means i s greater than the apparent experimental error as indicated by the confidence l i m i t s . The tryptophan values lend supporting evidence to the e a r l i e r observation that tryptophan, as determined by the NBS t i t r a t i o n method, increases about one residue per 27,000 g during reaction of O S l-casein with PDA. Further, the analysis reveals the unexpected result that methionine decreases one to two residues per 27,000 g over the reaction period. Methionine modification was not observed i n K-casein where a very s l i g h t decrease i n lysine was observed (28). General Observations on PDA asi-Casein PDA C tS l-casein freeze-dried at pH 7.0 i s a f a i n t l y yellow s o l i d that i s indistinguishable from freeze-dried asi-casein except f o r the yellowish color. The derivative i s soluble i n water at lower concentrations but attempts to dissolve the dry protein at concentrations greater than U-5 mg/ml resulted i n the formation of a viscous gel which could not be dispersed by agitation. Freezing of solutions of PDA asi-casein at concentrations of h-S mg/ml resulted i n gel formation but at lower concentrations no gel formed. D i l u t i o n of the gelled protein resulted i n fragmentation but no noticeable dissolution. Adjustment of the pH of a gel solution to pH 11 resulted i n rapid clearing of the solution and was accompanied by a v i s u a l l y noticeable drop i n the visco-s i t y . On adjustment of the pH back to 7.0 the cleared solution remained clear and the protein showed no further tendency to precipitate or gel unless the solution was frozen. The pH adjustment procedure was adopted whenever i t was necessary to obtain solutions of concentration greater than U-5> mg/ml as was required to determine s t a b i l i z a t i o n by K-casein. Wherever possible the pH adjustment procedure was avoided by the use of lower protein con-centrations . The u l t r a v i o l e t absorption spectrum of G lS l-casein and PDA a s i - c a s e i n i s shown i n Figure k as determined on a Unicam SP800 B recording spectro-photometer. Calculation of the absorbance at 280 nm per ml of PDA O S l -casein and OS I-casein w i l l y i e l d values of 2.16 and 1.09 respectively. From t h i s i t i s apparent that the absorbance of OS I-casein i s about doubled upon reaction with PDA. I t i s known that PDA alone absorbs i n the region around 280 nm so t h i s increase may indicate incorporation of the phenyl group of PDA i n t o O S I-casein. The increase i n absorption around 2$0 nm may also be due to the phenyl group of PDA but curves of s i m i l a r shape to t h i s have been observed when proteins are photooxidized (68,69). This has been a t t r i -buted to oxidation of aromatic amino acids ( 6 0 ) , especially h i s t i d i n e , and t h i s intrepretation would be consistent with the amino acid analysis. PDA asi-CASEIN asi-CASE!N 250 275 300 325 W A V E L E N G T H (nm.) F i g . h. U l t r a v i o l e t absorbance of a s i - c a s e i n and PDA C t s i - c a s e i n . Conditions: 0.1 M Na?HP0j, b u f f e r , pH 7.0 i n 1 cm c e l l s . asi-Casein = l.hh mg/ml, PDA a s i-casein - 0 .60 mg/ml. ho Calcium S e n s i t i v i t y of CtSI- and PDA CtSl-Casein The s o l u b i l i t y of a S i - and PDA CISI-casein i n calcium chloride i s shown i n Figure 0. The CtSl-casein used i n t h i s study shows the t y p i c a l calcium s o l u b i l i t y curve associated with Ct S i-casein B being quantitatively precipitated by 8 mM calcium chloride. At t h i s calcium concentration the PDA derivative i s s t i l l about 90$ soluble and i s not completely insoluble u n t i l the calcium concentration has reached about 13 mM. This difference cannot be ascribed to changes i n the amount of phosphate organic ester i n the protein since both a s i-casein and i t s PDA derivative contain 1.0$ phosporous. In some respects t h i s result was not unexpected since the caseins have been reported to lose t h e i r s e n s i t i v i t y to calcium on photooxidation (66,68), n i t r a t i o n (6U), and acylation (18). However, no explaination of t h i s pheno-menon has yet been forthcoming. Creamer and Waugh (7) have shown that bind-ing of calcium to CtSl-casein i s accompanied i n i t i a l l y by increasing monomer aggregation and eventually by precipitate formation. That the phenomenon i s related to binding i s shown by the fact that the exact concentration of calcium which i n i t i a t e s p r e c i p i t a t i o n i s dependent upon protein concentration (30). I f p r e c i p i t a t i o n were not a binding phenomenon, i t would be independent of concentration. Thus the p r e c i p i t a t i o n of CtSl-casein appears to involve binding of calcium ions with concurrent monomer aggregation u n t i l precipi^, t a t i o n i s i n i t i a t e d and s i x to eight calcium ions are bound. This process continues u n t i l f u l l p r e c i p i t a t i o n occurs and eleven to thirteen calcium ions are bound per 27,000 g CtSl-casein (30,00). Calcium binding, then, i s a c r i t i c a l factor governing CtSl-casein s o l u b i l i t y and studying the changes i n t h i s property may shed some l i g h t upon the reason f o r the lack of calcium CALCIUM CHLORIDE (mM) F i g . 0. S o l u b i l i t y of asi-casein and PDA asi-casein as a function of calcium chloride concentration at room temperature. h2 s e n s i t i v i t y i n modified CIS I-caseins. Calcium binding studies were performed as described i n Methods and Materials and the r e s u l t s are shown i n Table I I I . The value of 12.h moles of calcium bound per 27,000 g CtSI-casein i s i n reasonable agreement with values reported i n the l i t e r a t u r e ( 3 0,55). PDA CiSI-casein shows a marked decrease i n the a b i l i t y to bind calcium and binds only It.5 to 1+.8 moles calcium per mole a S I-casein. I t has been suggested by Chien and Waugh (6) using infrared spectroscopy that organic phosphate groups are the primary, but not the sole s i t e s for calcium binding i n CtSI-casein. There are nine organic phosporous atoms per molecule of asi-casein (30) and, since only about 5 moles of calcium are bound per mole PDA CtSI-casein, i t i s evident the phosphate groups no longer bind calcium as avidly as they once did. Since no change was observed i n the phosphorous content of CtSI-casein on modifica-t i o n with PDA and since the amino acid analysis shows no apparent decrease i n groups which might be expected to bind calcium (ie carboxyl groups), the reason f o r the reduced calcium binding of PDA CtSI-casein remains obscure. S t a b i l i z a t i o n by K-Casein One of the most important properties of CtSI-casein i s i t s a b i l i t y to interact with K-casein to form a complex that i s resistant to p r e c i p i t a t i o n by calcium ions. This property of PDA CtS I-casein was tested as described i n Methods and Materials and the results are shown i n Figure 6. This test was the only one i n which i t was necessary to carry out the pH adjustment procedure on PDA asi-casein. At f i r s t glance i t appears from these curves that the a b i l i t y of <z S i-casein t o interact with *c-casein i s impaired Table I I I . Binding of calcium to CtSl-casein and PDA CtSl-casein as determined by a centrif u g a l method. F i n i a l Protein Calcium Concentration Calcium Bound (mM) (moles/mole^) CtS I-Casein 16 12. U 2k 12.h 28 12.U PDA a Sl-Casein 16 h.h9 2h h.h9 28 h.Bh •Finial protein concentration was 0.1 mg/ml. •Molecular weight asi-casein = 27,000. hh 0 5 10 15 20 as\/« F i g 6 . S t a b i l i z a t i o n of asi-casein and PDA as»-casein by K-casein. Calcium concentration i n the t e s t i n both cases i s 10 mM. only to the extent of about 20%. However, when i t i s considered that PDA GtSl-casein i s about 27% soluble i n 10 mM calcium chloride a different picture emerges. I f the s o l u b i l i t y of PDA Ct SI-casein under these conditions i s subtracted from the s t a b i l i t y curve, then the maximum amount of protection obtained from K-casein i s reduced to about %0%. Recalling the gel electro-pherograms of PDA asi-casein, i t i s noted that some CtSI-casein remains apparently unreacted and t h i s w i l l probably account for some of the i n t e r -action products observed i n Figure 6. Further, there exists an intermediate reaction product or products which may be capable of interaction with K-casein to form a system stable towards calcium ions. When these two possi-b i l i t i e s are taken into consideration i t seems apparent that when CtSI-casein i s modified by PDA to the extent that i t i s no longer capable of entering the pores of an electrophoretic g e l , i t i n a l l p r o b a b i l i t y can no longer interact with K-casein to form a system stable towards calcium ions. Gel F i l t r a t i o n The results of Sephadex G-200 gel f i l t r a t i o n are shown i n Figure 7. The same column was used i n a l l three cases. In t h i s figure the elution p r o f i l e of asi-casein i s compared to the elution p r o f i l e of PDA CtSI-casein subjected to two treatments. The f i r s t ( s o l i d l i n e ) involved direct d i s -solution of freeze-dried PDA a SI-casein into the elu t i n g buffer before application to the column. The second (dot-dash l i n e ) involved dissolution of the protein i n buffer and adjustment of the pH to 11. The solution was held at t h i s pH f o r $ minutes, the pH was returned to 7 and the sample was immediately applied to the column. The dashed v e r t i c a l l i n e s at the beginning and end of the elution p r o f i l e s mark the elution positions of blue U6a Fig. 7. Sephadex G-200 gel f i l t r a t i o n of asi-casein and PDA asi-casein. Conditions: gel bed size 2.0 x 33 cm., flow rate 30 ml/hr, eluting buffer 0.00 M Na^HPO^, pH 7.0, column operated i n an upward flow manner. dextran and potassium dichromate respectively, measured at the position of maximum peak height. The pattern obtained for Ct S i-casein i s reasonably t y p i c a l for t h i s protein. Reaction of C t S l-casein with PDA causes two major changes to occur i n the elution p r o f i l e . F i r s t , a portion of the material was eluted at the void volume suggesting the presence of large aggregates or polymers of PDA O S l-casein. This i s consistent with the electrophoretic patterns which showed material present that remained at the o r i g i n . Secondly, the preparation appears to contain a heterogeneous range of polymerized forms as indicated by the r e l a t i v e flatness of the p r o f i l e s between 60 ml and 100 ml elution volume. The heterogeneity i s further suggested by the presence of numerous small peaks i n the PDA O S l-casein p r o f i l e . Treatment of the a S l - c a s e i n derivative with base for a short time did not a l t e r the p r o f i l e markedly, although a peak at about 107 ml that may re-present unaltered CtS l-casein has disappeared and been replaced by a peak at about 80 ml i n the base treated sample. I t has been reported (68) that photooxidized /8-casein can interact with "native" /3-casein to form interaction products which are not precipitated by calcium. I f the 107 ml peak i s "native" C t S l-casein, i t may be that the base treatment has opened the PDA C tS l-casein structure s u f f i c i e n t l y to allow interaction to occur. Recalling the calcium s o l u b i l i t y p r o f i l e of PDA C t S l-casein, i t i s noted that t h i s derivative precipitated abruptly at about 8 mM calcium chloride. I f t h i s preparation contains "native" C tS l-casein and i f no interaction takes place between C tS l-casein and PDA O S l-casein, then a step would be expected to occur i n the s o l u b i l i t y p r o f i l e as the O S l-casein began to "precipitate at about 2 mM calcium chloride. The fact that the curve remained h8 f l a t u n t i l about 8 mM calcium chloride suggests that PDA a SI-casein exerts a protective effect upon OSI-casein. I f such an interaction does occur, i t could explain both the disappearance of the 107 peak and the appearance of a peak i n the base treated PDA asi-casein p r o f i l e at a smaller elution volume. Tryptophan i n aSI-Casein I t was suggested e a r l i e r that asi-casein contained a tryptophan residue refractory to NBS oxidation i n spite of the fact that the spectrophotometric t i t r a t i o n had been conducted i n the presence of 6 M urea. This observation was investigated more close l y by comparing the results obtained with <XSI-and PDA asi-caseins with those obtained with two better known proteins, P-lactoglobulin and a-chymotrypsinogen. The l a t t e r proteins were obtained from N u t r i t i o n a l Biochemical Corporation, a-chymotrypsinogen as s a l t free crystals and /9-lactoglobulin as cr y s t a l s three times r e c r y s t a l l i z e d . Both proteins were used without further p u r i f i c a t i o n . The results obtained with the four protein preparations by NBS t i t r a t i o n were further compared by obtaining tryptophan values by the method of Spies and Chambers (U6). The results of these analyses are shown i n Table IV i n which each value represents the average of duplicate analyses. Good correlation was observed between the values obtained by the NBS t i t r a t i o n method and the method of Spies and Chambers for a-chymotrypsinogen and )S-lactoglobulin. In asi-casein, the difference between the two methods approaches one residue thus offering supporting evidence for the presence of a buried residue i n OSI-casein. The value obtained f o r PDA OSI-casein by the Spies and Chambers method i s perhaps a l i t t l e low although i t f a l l s within the reported range of values. Table IV. Comparison of tryptophan values obtained by the NBS t i t r a t i o n procedure and by the method of Spies and Chambers. Tryptophan (moles/mole) NBS Spies and Protein T i t r a t i o n Chambers Literature <XSI-Casein2 2.2 3.3 2.0-3.3 PDA aS I-Casein 3.2 2.7 /3 -Lactoglobulin3 2.0 2.1 1.8-2.0 a-Chymotrypsinogen^ 6.7 6.8 7.0 1 aSI-Casein and )S-lactoglobulin values taken from (23). Ct -Chymotrypsinogen values from (8). ^Molecular weight CtSl-casein = 27,000. ^Molecular weight /8-lactoglobulin = 18,000. ^Molecular weight a-chymotrypsinogen = 23,000. 50 The reason for the low value i s not known. The consumption of N-bromosuccinimide by the various proteins and by PDA i t s e l f i s shown i n Figure 8. Clearly, the effect of NBS on the absorbance of PDA at 280 nm i s very small. Furthermore, the tendency i s to increase absorbance on addition of NBS which i s exactly the opposite to the effect on protein. Since the calculation of tryptophan from NBS t i t r a t i o n data i s based upon the decreased i n absorbance at 280 nm with NBS consumption, an increase i n absorbance due to PDA reaction with NBS would tend to decrease the difference between the i n i t i a l and f i n i a l absorbance values obtained from t i t r a t i o n data. This would result i n a decreased tryptophan value rather than an increased value such as has been obtained f o r PDA CiSI-casein. Furthermore, the amount of PDA treated with NBS i n t h i s test (l . l i l mg) i s f a r i n excess of the amount which would be expected to be bound to 1 to 2 mg of protein i f only h i s t i d i n e and methionine were involved, and the increase i n absorbance i s only 0.02 absorbance units with l . U l mg PDA. From t h i s i t appears that PDA has a negligible effect upon the NBS t i t r a t i o n of tryptophan and that the tryptophan residue exposed by reaction of asi-casein with PDA i s not an a r t i f a c t generated by PDA bound to the protein. Determination of tryptophan i n GtSl-casein by NBS t i t r a t i o n i n urea buffers from 3.6 to 10 M urea did not reveal any changes i n the tryptophan content with increasing urea concentration. I t was not possible to deter-mine tryptophan i n the absence of urea since CtSI-casein i s insoluble at the acid pH values required f o r the determination. 51 E c O 00 CM o LU O < CQ rr o oo m < a-CHYSVIOTRYPSINOGEN /3-LACT0G10BULIN asi-CASEIN 0.80 P D A 0.60 0.40 020 \ 20 40 60 80 100 /AM NBS CONSUMED (XI0" 2) F i g . 8. Consumption of NBS by protein and by PDA i n 6 M urea-formate -acetate buffer, pH lj.0. Weights of components analysed are: PDA l.Ul mg, Ctsi-casein l.Ul mg, /3-l a c t o g l o b u l i n 1.36 mg, a-chymotrypsinogen O.U°6 mg. 52 General Discussion I t i s evident from some of the results obtained i n t h i s study, that s i g n i f i c a n t changes occur i n the conformation of CtSl-casein on reaction with PDA. Evidence which suggests t h i s i s : 1. material remaining at the o r i g i n on gel electrophoresis; 2. production of material which i s larger than the exclusion l i m i t of Sephadex G-200; 3. exposure of a "buried" tryptophan residue and U. the unusual s o l u b i l i t y properties of PDA CtSl-casein. Changes such as observed above may be brought about by at least three possible changes i n the CtSl-casein. F i r s t , any folded structure present i n the caseins as suggested by Ho and Chen (16) may have been disrupted causing the formation of long threads of disordered CtSl-casein. Secondly, the protein or i t s disorganized derivative may aggregate through non-covalent bonds to form large polymers. Thirdly, since the reagent (2-phenyl-l,li-dibromoacetoin) i s b i f u n c t i o n a l , the p o s s i b i l i t y of intermolecular or i n t r a -molecular crosslinking cannot be ignored. One s t r i k i n g feature of the results obtained i n t h i s study i s the s i m i l a r i t y between them and the results obtained by Z i t t l e for the photo-oxidized caseins (66,68,69). Photooxidation i s considered to take place through a c y c l i c free r a d i c a l mechanism whereby the ligh t - e x c i t e d dye i s reduced by the substrate while the l a t t e r i s being oxidized (62). During the operation of t h i s cycle an amino acid (protein) free r a d i c a l or an amino acid hydroperoxide (39) i s formed. Protein polymerization has been reported to be induced by free r a d i c a l l i p i d peroxidation (1*1) and by peroxydisulfate (29). Both of these l a t t e r reactions are considered to take place through free r a d i c a l polymerization and covalent crosslinking of proteins, and the f a i l u r e of the photooxidized caseins to enter a 53 polyacrylaraide gel even i n the presence of urea and 2-mercaptoethanol may be a manifestation of these reactions. Furthermore, Woychik and Wondolowski (6U) report that complete n i t r a t i o n of OSl-casein with tetranitromethane caused the protein to become incapable of entering the pores of an acrylamide gel made up i n urea and 2-mercaptoethanol. Crosslinking of proteins with t e t r a -nitromethane (10) has been reported. From these reports, crosslinking of aS I-casein by PDA seems to be a d e f i n i t e p o s s i b i l i t y considering the nature of the properties observed for PDA asi-casein. Gel f i l t r a t i o n of Sephadex G-200 d e f i n i t e l y suggests an increase i n molecular size and heterogeneity of molecular size such as might be expected i f random crosslinking reactions had occurred. The reason for the decreased calcium binding a b i l i t y of PDA CtSl-casein i s unknown. The conformation of CtSl-casein may affect i t s calcium binding a b i l i t y since C S l-casein A binds 17 moles calcium per 27,000 g protein (55), and i t has been suggested that t h i s and other changes i n the properties of asi-casein A may be due to the conformational changes induced by the deletion of hydrophobic amino acids. I t may be that unfolding of the OSI-casein molecule has allowed charged groups which would otherwise have bound calcium to interact with groups of opposite charge on the same or neighbour-ing molecules thus reducing the number of calcium binding s i t e s on the mole-cule and increasing the tendency of the molecules to aggregate. PDA a s i-casein shows reduced a b i l i t y to be s t a b i l i z e d by K -casein. I f the complications of aggregation or crosslinking are set aside, i t appears that h i s t i d i n e plays an important role i n the interaction between a s i-casein and /c-casein. Methionine appears u n l i k e l y to have a role since photooxida-t i o n of K- and /3 -casein i n which methionine was altered only s l i g h t l y showed results somewhat s i m i l a r to those obtained i n t h i s study. However, the p o s s i b i l i t y of r a d i c a l conformational changes induced by polymerization or aggregation being responsible for the observed behavior cannot be ruled out and may i n fact be the dominant factor governing the interaction, or lack of int e r a c t i o n , between PDA OSI-casein and K-casein. To the best of t h i s author's knowledge, the observation of a "buried" tryptophan i n OSI-casein i s the f i r s t report of such a residue. The finding of "buried" residues suggests the presence of folded structure i n OSI-casein and thus lends support to the report of Ho and Chen (16). Furthermore, the f a i l u r e of urea to expose the tryptophan residue to NBS suggests that the peptide backbone has a strong influence on the conformation of O S I -casein i n the region of t h i s tryptophan residue. The secondary forces which contribute to protein structure, and which are disrupted by urea, are appar-ently of reduced importance i n t h i s region of the molecule. BIBLIOGRAPHY Annan, ¥. D. and W. Manson. 1 969 . A Fractionation of the as -Casein Complex of Bovine Milk. J. Dairy Res., 3 6 : 209 . A. 0. A. C. Methods of Analysis. I960. Association of O f f i c i a l A g r i c u l t u r a l Chemists. P. 0 . Box 0UO, Benjamin Franklin Station. Washington, D. C. 200ltLu 10th e d i t i o n . Aschaffenburg, R. 1968 . Reviews of the Progress of Dairy Science. Section G. Genetics. 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N i t r a t i o n of Tyrosyl Residues i n K - and Cis i-Caseins. J. Dairy S c i . , 52:1669. Z i t t l e , C. A. 1961. S t a b i l i z a t i o n of Calcium-Sensitive ( a s ) Casein by Kappa-Casein: Effect of Chymotrypsin and Heat on Kappa-Casein. J. Dairy S c i . , UU:2101. Z i t t l e , C. A. 1963. Photooxidation of as-casein. J. Dairy S c i . , ii6:607. Z i t t l e , C. A. and J. H. Custer. 1963. P u r i f i c a t i o n and Some Properties of a s-Casein and K-Casein. J. Dairy S c i . , U6:ll83. Z i t t l e , C. A., E. B. Kalan, M. Walter and T. M. King. 196U. Photo-oxidation of /9 -Casein. J . Dairy S c i . , V7:1052. Z i t t l e , C. A. 1965. Some Properties of Photooxidized K-Casein. J. Dairy S c i . , W3:llU9. APPENDIX Computer Prog I M <w S < <T . . G 2 ! C u -— o _) 1— t — —* *> o m ~~ o i — *—« •» u> CO <a P J oo — — o t - t — * •« Z , — i C' II •—< z —- c >—' t — '• «—1 C; o z: — o c <S. o UJ CT .—. L P <. UJ •—< cr: z: 1- <r * c or. t - H *—* U J <t * CJ <I 2 * LP . >—< .—! v^-II UJ -> LP. — t_i <j < 0 U J C or a: cr -4- O I P . rvi — C M f\i - C\i I—J II - > II • 3 — O -—> • —-« - v O - 5 h - U- — 00 O 3 s ^ s ; £3 O in < l -< — ' ~ 2 — <f -< — O r - O II I P - I P C M — I P , I— I P . L P t — — <1 — ~ < o su o a x: a. <i <. ac c. a. UJ CC a •dUJ C cc cc a. a n o 11 UJ c * E » C l I - (J — 2:: a. 0 < r c - l -o < u> o o cr 11 o -J c U J _ J 2: — • O — 21 CJ — .—• *>;-r—I O w c — ^ 0 O C ><• •• O 2: — >-( 3: u_ ou X -><• O 0 O ro I P X JS X tl_i t »• O _ J a O 1 3 I P . h-^ r • LP. UJ 0 _ J O C .—< 0 • u; U- c X C U- X 0 cr _ J • u> .—1 u. 0 ip. X • (XI • X a; 11 •> IT-. h - II L P • w <f • ZD • — <: C: 5r L U 5:. X « 3 ex — - _ J < I L X — UJ U_ !^r LL : C- u.. -: U- O: U. c Lf^  »—« •—1 a 3 a a CJ o o o m o t/l a 101 CONTINUE I F ( J . E 0 . N ) G D TO IC C PRINTING T I T L E (CNF CARD ALLOWED FOR T I T L E > 3C WR I TE ( 6 , 57 ) T ITt.E 5 2 FORMAT('1•»//?QA4) C PRINTING HEADINGS  300 WPITF<6,42) 42 FORMAT ( »o ' , 33X, '24 HP. F Y DPOL YS I S • , 16 X, • 4 8 HR . . H YDR OL Y-S I S « * 16 X , • 72 1 HR. HYnsQLYSIS*/39X»17(IH.) , 1 6 X , 1 7 ( I H . ) , 1 6 X , 1 7 ( I H . )/ 1GX, * AMINO AC 2 IH » , 14X , ' RUN l ' ^ X ^ R U N 2',6X, ,RUN 3',6X,'RUN 1«,6X,«RUN 2 ,»6X, ,RU 3 N 3 ' ,6X ,'RUN 1 • ,6X,« RUN 2'» 6X,•RUN 3' ) WRITE(6,43)  43 FORMAT( 1 OX, 10( 1F. ), 14X,5( 1H.),6X,51 IH.),6X,5( IH. I ,6X,5(1H.) ,6X ,5(1 1 H . ) , 6 X , 5 ( 1 H . ) , 6 X , 5 ( 1 H . ) , 6 X , 5 ( 1 H . ) , 6 X , 5 ( 1 H . ) ) C INT IMG RESULTS DG 2 J = 1,N WRITE(6 ,44) (AC I 0 ( J , I) , I = 1 , 4 ) , i A A ( J , K ) ,K = 1,9 ) 4 4 FORMAT( «0't 4X,4A4,7X,9( 5X ,F6.2) )  2 CONTINUE C COMPUTING MOLECULAR WEIGHTS WPITE(6,43) DO 4 K= 1, N MW(K ) =0 nn 4 J=I»N MW( K. )=MW(K)+AA(J,K)*AAMW(J ?-A A ( J , K ) * 1 B  4 CONTINUE WR I T E ( 6 , 4 6 ) ( M W I K ) ,K = 1,9) 46 FORMAT( «0',4X, 'MOLECULAR WE IGHT',7X,9[5X,I 6 ) ) I F ! ITROL.GT.OGG TO 32 C CORECTING AMINO AGIO LEVELS IF(CGND(7).EG.O )GO TO 32  DO 200 K-=l,9 FACT 1=CONDI 7)/A A( 1, K) FACT2=CCNC<8)/A A(4,K) F A G T= ( F AC T 1 + F AC T2 ) / 2 DO 200 J = ] , IF> A A ( J , K ) ~A A ( J , K ) »FACT  200 CONTINUE DO 201 K = l , 9 F A C T l = C 0 N n ( 9 ) / A M ( I E + 6 ) , K > F A C T 2 = C O N D ( 1 0 ) / A A ( ( 1 8 + 7 ) , K ) FACT=(FACTL+FACT2)/2 ID=1B+1  DO 201 J=IO,N A A ( J , K.) - A A ( J » K ) * F ACT 201 CONTINUE ITRGLMO WRITE (6 ,47) 47 F0fiHAT( '1',//IPX,'CORRECTED AMINO ACID LEVELS' ) GO TO 300 C PUNCHING CARDS 3 2 I F ( I C . E O . 0 ) G O TO 31 DO 7 K = l , 9 WRITE(7 ,48) ( A A ( J , K ) ,J=1,N) 48. FORMAT ( 6X» 1 2F 6. 2/6X , 1 2F 6. 2)  .7 CONTINUE 31 STOP END 

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