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.

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.

Ion exchange process for removing glucosyltransferase from crude glucoamylase.

The advant of a new range of high-protein capacity cellulosic ion exchangers suitable for use on an industrial scale made it worthwhile investigating the conditions necessary to remove the contaminating enzyme glucosyltransferase from a commercial preparation of crude glucoamylase. The potential of the SP derivative, SP Indion((R)), for achieving this separation is shown. At pH 2.5 the glucosyltransferase was selectively adsorbed by the ion exchanger, and 99% of the glucoamylase was recovered in the eluate from the column. Purification of an Aspergillus culture filtrate by this method will require careful control of the ionic strength of the culture medium if it is to be used without the additional step of cation exchange to lower than pH to 2.5.

Application of 1,1'-carbonyldiimidazole-activated agarose for the purification of proteins. II. The use of an activated matrix devoid of additional charged groups for the purification of thyroid proteins.

The use of 1,1'-carbonyldiimidazole-activated agarose for biospecific affinity chromatography is described. Activation of agarose with this carbonylating reagent gives a matrix devoid of additional charged groups. Conditions for the coupling of a range of ligands and leashes have been evaluated. The efficient purification of bovine trypsin, human thyroglobulin and sheep thyroid membrane glycoproteins demonstrates the suitability of the new activated matrix for affinity chromatography.

A novel method of activation of cross-linked agaroses with 1,1'-carbonyldiimidazole which gives a matrix for affinity chromatography devoid of additional charged groups.

1,1'-Carbonyldiimidazole, a carbonylating reagent, has been shown to be suitable for the activation of cross-linked agaroses for affinity chromatography. The activated matrix (an imidazolyl carbamate) is relatively stable to hydrolysis but smoothly reacts with N-nucleophiles such as those present in either affinity chromatography ligands or leashes, e.g. ethylenediamine or 6-aminohexanoic acid. If butylamine was attached via the 1,1'-carbonyldiimidazole method, the resulting product was devoid of charged groups and thus had the same titration curve as agarose. The suitability of this new matrix for affinity chromatography was demonstrated by the successful purification of trypsin by several different systems.