Whole casein 4 Individual casein components 5 Micellar casein 12 Proposed Models for the Structure of Casein Micelles 16 Core-coat type of structure 16 Dispersed type of structure 20 Experimental Approaches 22 Chemical modification 22 Enzymatic hydrolysis 25 EXPERIMENTAL 27 Materials 27 Water 27 Milk 27 Reagents 27 Apparatus 29 Dialysis tubing 29 Water bath pH meter Centrifuges Freeze-drying apparatus Fraction collector Conductivity apparatus Chromatographic columns Spectrophotometers Starch-gel electrophoresis apparatus Densitometer iii Methods 34 Horizontal starch-iirea-2-mercaptoethanolgel electrophoresis 34 Preparation of skim milk 39 Preparation of isoelectric casein 40 Preparation of Og-, and K-caseins 40 Estimation of protein 43 Determination of the extinction coefficient of 2-hydroxy-5-nitrobenzyl bromide 44 Fractionation of casein by DEAE-cellulose chromatography 44 Modification of tryptophan residues in casein with 2-hydroxy-5-nitrobenzyl bromide Casein samples from the skim milk reacted with 2-hydroxy-5-nitrobenzyl bromide (HNB) Estimation of modified tryptophan residues in each casein component Tryptic hydrolysis 57 Ratio of ttg-/ p-and K-caseins in the trypsin treated micellar and isoelectric casein 61 RESULTS AND DISCUSSION 62 Effects of Urea on pHs of Buffers Imidazole-citric acid-urea buffer for alkylation Tris-citric acid-urea buffers DEAE-Cellulose Chromatography Purification of urea Sample size of alkylated whole casein ' Identification of casein fractions 72 Regeneration of cellulose columns 75 Milk Samples 77 Modification of Tryptophan Residues in Casein Micelles 2-Hydroxy-5-nitrobenzyl bromide (HNB) Reaction of HNB with skim milk and casein solution Reaction of alkylated casein samples with HNB casein component iv Tryptic Hydrolysis Substrate to enzyme ratio Tryptic hydrolysis of micellar and solubilized isoelectric caseins Casein component^ in trypsin-treated micelles and isoelectric casein SUMMARY AND CONCLUSIONS 101 BIBLIOGRAPHY 105 ACKNOWLEDGMENTS 115
