David Francis Elgar

Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene–divinylbenzene

A precise, sensitive and reliable RP-HPLC method was developed to enable not only unequivocal determination of alpha-lactalbumin and beta-lactoglobulin in bovine whey samples, but also simultaneous measurement of proteose peptone, caseinomacropeptide, bovine serum albumin and immunoglobulin G. The optimised method on the Resource RPC column allowed separation of the proteins in 30 min and could be applied to the analysis of soluble proteins in a variety of commercial and laboratory whey products. Furthermore, some qualitative information on protein heterogeneity and quality could be derived from the RP-HPLC analyses with additional data available from on-line electrospray mass spectrometry. Within- and between-day repeatability over a wide range of concentrations was excellent (RSD< or =5%) for all proteins except immunoglobulin G and bovine serum albumin where RSD was 7-10%. Analysis of grouped data from whey protein concentrate and whey protein isolate samples gave a limit of detection of < or =0.3% powder mass and a limit of quantitation of < or =1.0% powder mass for all proteins except immunoglobulin G. Limits of detection and quantitation were 0.6% and 2.0%, respectively, for this protein. Quantitative data obtained by the RP-HPLC method compared very favourably with data obtained by alternative methods of whey protein analysis.

Detection and quantitation of lactoferrin in bovine whey samples by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene

An existing RP-HPLC method for the measurement of the major bovine whey proteins in bovine whey samples and powders has been extended to include the analysis of the minor bovine whey protein lactoferrin. Lactoferrin could be detected and quantitated at levels down to 0.2 microg and linear calibration was observed between 0.2 and 30 microg. Reliable quantitation of lactoferrin in whey samples could be achieved provided the bovine serum albumin to lactoferrin ratio did not exceed 10:1. Quantitative data obtained by the RP-HPLC method compared favourably with data obtained by Mono S analytical chromatography.

Profiling of genetic variants of bovine κ-casein macropeptide by electrophoretic and chromatographic techniques

Abstract Bovine κ-caseins of single variant phenotype (AA and BB) and mixed phenotype (AA, BB and AB) were purified from milk and the corresponding κ-casein macropeptides prepared by chymosin hydrolysis. The macropeptides were characterized by PAGE, Mono Q- and reverse-phase HPLC. Chromatographic profiles showed marked differences between the monovariant macropeptides, attributable to differences in glycosylation. The B variant macropeptide was found to be more highly glycosylated than the A variant with an apparently greater number of oligo-saccharide chains per peptide unit and a higher level of sialylation. The analytical profiles for the mixed variant sample were a composite of those for the individual variants, all components being accounted for by one or other variant. It was concluded that while the overall extent of glycosylation may vary, there are consistent patterns of glycosylation for each variant.

Comparative study of methods for the isolation and purification of bovine κ-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.