Modification of histidine in K-casein by 2-phenyl-1, 4-dibromoacetoin

The main object of this thesis was to specifically modify histidine residues in κ-casein and KA1 and KA2 with the reagent 2-phenyl-1 ,4-dibromoacetoin (PDA) for the overall purpose of testing the hypothesis that histidine plays an important role in the stabilizing ability of κ-casein. Experiments designed to test the hypothesis that PDA causes κ-casein to aggregate by cross-linking were also carried out and these included the preparation of 2-phenyl-4-bromcacetoin (PMA) and reacting this with κ-casein and KA1 and KA2, as well as reacting PDA with histidine. Preliminary objectives of this thesis were to purify large amounts of κ-casein by scaling up the method of Zittle (66), to improve the electrophoresis technique described by Perrin (4l) and Peveridge (2) so that κ-caseir; subfractions could be seen, and to purify the sub-fractions by DEAE cellulose chromatography using the method of Mercier (28). The lowest yield of κ-casein during four attempts of scaling up Zittle's method was 6.0% and the highest yield was 7.5%. Two preparations of κ-casein resembled those published by McKenzie (23) both in amino acid composition and electrophoretic heterogeneity. Modifications in the electrophoresis technique which gave promising results included steps to increase the voltage through the gel equilibrating the gels by carrying out preliminary runs without samples, lowering the ionic strength of the system, changing the bridges, and increasing the concentration of urea in the gel. Suggestions by Mercier (28) which proved to be important in the separation of the subfractions of κ-casein by DEAE cellulose chromatography included sifting the cellulose before use, recrystallizing the urea, and using concentrations of NaOH and HCl below 1.0 molar during regeneration of the cellulose. Reactions of PDA and PMA with κ-casein, KA1 and KA2 did not support the hypothesis that histidine plays an important role in the stabilizing ability of κ-casein because l) PDA κ-casein was prepared, in low ionic strength buffer, which dissolved, normally and had the same stabilizing ability as untreated κ-casein, even though 0.6 histidine residue was modified; and 2) PMA KA1 and PMA KA2 were prepared which, although having 1.05 histidine and 1.60 lysine residues less than untreated KA1 and KA2, nevertheless had the same stabilizing ability as the untreated protein. Rather, these results support the contentions that l) the low stabilizing ability of PDA-κ-casein results from aggregation caused, by cross-linking of histidine residues on separate κ-casein molecules; and 2) the cross-linking may be dependent upon the ionic strength of the reaction. Removing one bromine from PDA to make PMA seemed to result in a change of selectivity from histidine to lysine. PMA did not react on κ-casein in the same way as it did on KA1 and KA2 and this supports the statements that future work on the modification of amino acids in κ-casein and studies on the interaction of this protein with κ-casein should be carried out on the subfraction rather than on κ-casein. Reactions involving PDA and histidine did not lead to an explanation of the mechanism of action of PDA because the yields of the products were too low to allow chemical analysis. Products having Rf ‘s of 0.62, 0.58, and 0.40 were produced and purified by cellex and paper chromatography in Jones' solvent (33) for the thin layer chromatographic separation of amino acids. These products reacted positively to Pauley's reagent, indicating that they were histidine addition products.Land and Food Systems, Faculty ofGraduat