Frequency of occurrence of novel milk protein variants in a New Zealand dairy cattle study population : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry, Massey University
Since the discovery of genetic polymorphism within milk protein genes, a considerable volume of research has been published relating milk protein genetic variants and milk production properties. Polymorphism of milk proteins can result in two effects: (a) changes in the biological and physico-chemical properties of systems containing the variant protein, (b) changes in the synthesis level of variant proteins. As a result several studies of milk protein variants have identified phenotypes which may be commercially advantageous for specific products. Currently employed methods to determine milk protein phenotypes are generally limited to electrophoretic techniques. The gel electrophoretic techniques commonly used are able to detect most milk protein variants that differ by their net electrical charge. However single amino acid substitutions that result in a change in net charge account for only 25% of the possible substitutions that could occur. The remaining 75% of potential variants are the result of a neutral residue substituted by another neutral residue - a 'silent' variant. Thus it is likely that some substitutions, and hence genetic variants have gone undetected in the past. The purpose of this study was to develop new methods for determining the phenotype of milk proteins, and to determine the frequency of occurrence of silent or other novel variants in a New Zealand dairy cattle study population. Polyacrylamide gel electrophoresis (PAGE), free zone capillary electrophoresis (CE), peptide mapping by reverse-phase HPLC and electrospray mass spectrometry (ESI-MS) were used in the characterisation of milk proteins purified from 109 individual dairy cows. Three different PAGE systems were used. Alkaline-urea PAGE enabled the detection of α si-casin
variants B and C, β-casein variants group A (variants A1, A2 and A3) and B, and K-casein variants A and B in the study population. Beta-casein variants A1, A2 and A3 were subsequently resolved in an acid-urea PAGE system. The whey proteins were very poorly resolved in PAGE systems containing urea. Alpha-lactalbumin A, and β-lactoglobulin (β-LG) variants A and B
were resolved in a non-denaturing 'native' PAGE system. The frequencies of the various milk protein variants corresponded closely to figures previously published. A free zone CE method that is able to resolve β-LG variants A, B and C was used to check the phenotype of purified β-LG samples. Three samples previously typed as β-LG BB were subsequently determined to be β-LG CC; one sample typed as β-LG BB was re-assigned as β-LG BC. This highlighted the limitations of PAGE systems for the detection of known variants. Tryptic hydrolysis of purified casein proteins and β-LG, followed by reverse-phase HPLC separation of the resultant peptides was used to create peptide 'maps' of the hydrolysis products. Differences in peptide maps were noted between protein variants. The differences corresponded to peptides containing a substitution site. All samples analysed in this way contained more peptide peaks than expected. Analysis revealed that some were the result of incomplete digestion; others the result of chymotryptic-like cleavages. No aberrant peptide maps, indicative of a silent mutation, were detected. Purified casein proteins and β-LG were subjected to ESI-MS for mass analysis. The mass of each protein species was determined as follows: Protein Average mass Std. dev.
as1-CN B-8P 23614.9 Da 1.2 Da
as2-CN A-11P 25228.9 Da 1.5 Da
β-CN A1-5P 24023.9 Da 3.1 Da
β-CN A2-5P 23983.5 Da 1.8 Da
β-CN B-5P 24092.6 Da n.d.
k-CN A-1P 19038.8 Da 1.5 Da
k-CN B-1P 19003.8 Da n.d.
β-LG A 18362.6 Da 1.0 Da
β-LG B 18277.0 Da 0.9 Da
β-LG C 18287.2 Da 0.6 Da
In all cases the experimentally determined mass corresponded to the mass calculated from published primary sequences of milk protein variants. In addition to the expected β-LG variant in each mass spectrum, additional species were detected differing from the mass of the β-LG species by increments of approximately 324 Da. Although less pronounced, the +324 Da molecular weight species were also detected in a sample of β-LG purchased from the Sigma Chemical Company. The additional species were also detected in whey prepared by ultra-centrifugation, although at a much lower level. The 324 Da molecular weight adducts observed in ESI-MS spectra of purified β-LG are consistent with an addition of a lactosyl residue to the protein. The observation that these species remain after heat denaturation, reduction and RP-HPLC treatment suggest that the linkage is covalent. Lactulosyl-lysine is known to form in milk products during some processing conditions, particularly during heating. The observation of these glycated species in gently treated, unheated milk suggests that glycation may occur to some extent in the udder of the cow. The association of the 324 Da molecule with β-LG does not alter the charge, molecular weight or hydrophobicity sufficiently to be detected by PAGE. CE or RP-HPLC.