John Stephen Ayers

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.

Application of 1,1′-Carbonyldiimidazone-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

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

Investigation of the activation of various insoluble polysaccharides with 1,1′-carbonyldiimidazole and of the properties of the activated matrices

Abstract This report further characterises a new procedure for the preparation of affinity chromatographic supports, namely the activation of hydroxylic solid-phase supports with 1,1′-carbonyldiimidazole (CDI). Matrices with a controlled degree of substitution can be synthesised by the use of CDI, and if required, a high level of activation can readily be achieved e.g. up to 5 mmol/dry gram with cross-linked agarose. The CDI-activated agarose was found to have a half-life of greater than fourteen weeks when stored in dioxane. The conditions for coupling simple amines of differing pKa values to these active matrices were evaluated and the coupling yields analysed. Based on these results, conditions suitable for the coupling of proteins were established. The linkage of the amino group of the ligand to the support (an N-alkylcarbamate) was shown to possess good stability over a wide pH range. This stability was much greater than that of the isourea linkage obtained with the cyanogen bromide activation method. It is expected that this new activation procedure should prove to be particularly useful for a variety of affinity chromatographic experiments, including those which cannot tolerate the hydrolytic release of small amounts of the insolubilised ligand. The CDI method has been extended to other polysaccharide matrices e.g. cellulose and dextran derivatives, and to glycophase-coated glass beads. The activated glass bead derivative provides a suitable support for attachment of ligands for high-performance affinity 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.

Investigation of the activation of cross-linked agarose with carbonylating reagents and the preparation of matrices for affinity chromatography purifications

Abstract The activation reaction of cross-linked agarose with 1,1′-carbonyldiimidazole (CDI) has been extended to other carbonylating reagents, and has confirmed that CDI allows the facile preparation of activated matrices suitable for affinity chromatographic supports. These studies showed that 1,1′-carbonyldi-1,2,4-triazole (CDT) gave a more reactive activated matrix, while 1,1′-carbonyldi-1,2,3-benzotriazole reacted only slowly and inefficiently. Phosgene, in addition to the disadvantage of toxicity, does not give a high level of activation. The introduction of imidazolyl carbamate groups onto cross-linked agarose by generating CDI in situ from phosgene and imidazole gave one-third of the level of activation of that obtained with pure CDI. All of the activated matrices had sufficient stability to aqueous conditions to allow unhurried isolation of the washed, activated product. All carbonylated matrices when subsequently coupled with monoalkylamines were found to be devoid of any additional charged groups due to the activation process. The studies have demonstrated that CDI is the most effective and convenient of the carbonylating reagents studied for the preparation of activated matrices to be used in affinity chromatographic experiments. However, the CDT-activated matrix is much more reactive than the CDI matrix and may be useful for the coupling of unstable protein ligands where short coupling times are essential.

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.

Cross-linked hydroxypropylated cellulose gel for chromatography

Abstract A series of insoluble cross-linked hydroxypropylated celluloses (HP-Regcell) were prepared by reacting ground regenerated cellulose with epichlorohydrin and propylene oxide in the presence of strong aqueous sodium hydroxide at a temperature of 40°C or higher. The products had gel-like properties with pore sizes up to about 110 A and were useful as column packings for gel chromatography. They were also used to prepare ion-exchange derivatives with excellent capacities for protein adsorption.