Comparative study of methods for the isolation and purification of bovine kappa-casein and its hydrolysis by chymosin.
kappa-Casein was purified from a single batch of whole acid casein (kappa-A variant) using different methods in order to compare their merits in producing a purified material with a carbohydrate and phosphate heterogeneity representative of the whole kappa-casein complement in milk. Ion-exchange methods of purification gave products of higher purity than precipitation techniques involving final purification by ethanol fractionation, but all methods resulted in kappa-caseins of apparently similar heterogeneity and chemical composition. The purified kappa-caseins were hydrolysed with chymosin and the derived macropeptides isolated. These were all virtually identical as determined by reversed-phase chromatography and gel electrophoresis. Some observations on chymosin hydrolysis of kappa-casein were made. In addition to formation of the major para-kappa-casein (Glu1-Phe105) and macropeptide (Met106-Val169), chymosin hydrolysis at pH 6.6 also resulted in two minor para-kappa-caseins with N-termini corresponding to Phe18 and Ser33 of kappa-casein. At pH 5.5 and 4.5 para-kappa-casein was rapidly hydrolysed into at least six fragments, one of which had an N-terminus corresponding to Trp76 of kappa-casein. At pH 6.6, 5.5 and 4.5 the kappa-casein macropeptide was stable to chymosin, but at pH 2.3 it was hydrolysed by chymosin into fragments with N-termini corresponding to Met106, Ile125, Ala138, Val139, Thr145 and Glu147 of kappa-casein.
Coolbear
K P
KP
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Elgar
D F
DF
Coolbear
T
T
Ayers
J S
JS
D000818:Animals; D002364:Caseins / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D012085:Chymosin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism
D002364:Caseins; D010446:Peptide Fragments; D012085:Chymosin