Determination of the phosphorylation level and deamidation susceptibility of equine β‐casein
Determination of the phosphorylation level and deamidation susceptibility of equine β‐casein
复制标题
马β-酪蛋白磷酸化水平和脱酰胺敏感性的测定
作者：
J. Girardet;L. Miclo;S. Florent;D. Mollé;J. Gaillard
用反相高效液相色谱法从哈夫林格马奶中分离得到β-酪蛋白，尿素凝胶和双向凝胶电泳法显示出微观上的不均一性，这可能是由于不同程度的磷酸化所致。为了研究β-酪蛋白的磷酸化程度，通过胰酶水解法和多肽释放的MS以及碱性磷酸酶处理的蛋白质的MS测定了其一级结构。发现的哈夫林格马β-酪蛋白脱辅型的分子质量(25 514±3 Da)接近报道的序列(GenBank AAG43954)的理论质量，该序列通过插入由有时外显子(25 511.40 Da)编码的区域(残基 27-34)而修改。因此，从哈夫林格马奶中分离出的β-酪蛋白对应于226个 氨基酸残基的变体。后者由具有3~7个磷酸基团的高度多磷酸化的异构体组成，经2-DE测定，等电点为4.74~5.30。此外，马的β-酪蛋白能够在潜在的脱酰胺基序135Asn-Gly136中的Asn水平上自发脱酰胺。在生理条件下孵育96 h后，约有80%的蛋白质被脱胺。
β‐Casein was isolated from Haflinger mare's milk by RP‐HPLC, and displayed microheterogeneity by urea‐electrophoresis and 2‐DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine β‐casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo‐form of Haflinger mare's β‐casein (25 514 ± 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27–34) encoded by an exon sometimes out‐spliced (25 511.40 Da). Hence, the β‐casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi‐phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2‐DE, ranging from 4.74 to 5.30. Moreover, the equine β‐casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif 135Asn‐Gly136. Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.