Structures determination of biological peptides generated by hydrolyzation of modified milk casein

与葡萄糖反应后的酪蛋白经水透析、冷冻干燥后作为改性酪蛋白。赖氨酸残基的修饰度约为50%。将修饰后的酪蛋白用胃酶-胰酶水解法分离得到3,000、3,000-10,000和10,000以上三个组分。3种多肽的得率分别为75%、5%和19%。与天然酪蛋白相比，改性酪蛋白中300000Da以下的多肽减少了约20%。高效液相色谱检测结果表明，天然酪蛋白和改性酪蛋白的多肽图谱存在明显差异。此外，还从改性酪蛋白中获得了三种特殊的多肽。修饰后的酪蛋白中抑制血管紧张素转换酶的多肽的数量也减少了。此外，还合成了酪蛋白β结构的RDMPI多肽。将该多肽与葡萄糖在110℃下反应2 h，结果表明：(1)天然多肽和修饰多肽的高效液相色谱图谱明显不同，1、2、3、4、5、6号多肽似乎是环状的。(2)经MS分析，5号肽已糖化，峰中还含有RDMPI+1G和RDMPI+2G。(3)通过蛋白质测序仪分析，观察到RDMPI的N端精氨酸残基发生糖基化。

Casein after reaction with glucose was dialyzed against water, freeze dried and used as modified casein. The modified degree of lysine residue was about 50%. The modified casein was then hydrolyzed by pepsin-pancreatin, and isolated into three fractions of under 3,000Da, 3,000-10,000Da and over 10,000Da. The yield of 3 peptides fractions were 75%, 5% and 19% respectively. In comparison with native casein, the peptides under 3,0000Da from modified casein was deceased about 20%. The results by HPLC showed that there is clear difference between the peptides pattern of native casein and modified casein. Moreover, three special peptides were generated from modified casein. The quantity of peptides inhibit Angiotensin converting enzyme, from modified casein was also decreased.Besides, RDMPI peptide from beta-Casein structure was also synthesized. This peptide was reacted with glucose, at 110℃ for 2 h. The following was made clear: (1) HPLC pattern of native peptide and modified peptides was clearly different, it seemed that peptide No 1,2,3,4,5,6 were gycated. (2) By the MS analysis, peptide No 5 was glycated, and, peak was also contained RDMPI+1G and RDMPI+2G. (3) By the analysis of Protein Sequencer, the glycation of Nterminal Arginine residue of RDMPI was observed.