牡蛎源类蛋白反应修饰肽的分离纯化及肽锌螯合物的结构表征

Separation and Identification of Oyster Peptide Modified by Plastein Reaction and Characterization of Peptide-zinc Complexes

以牡蛎为原料制备了类蛋白反应修饰肽,利用Sephadex G-15凝胶层析柱和反向高效液相色谱(RP-HPLC)等分离技术得到1条锌离子螯合活性为161 mg/g的多肽(M_w=835),多肽序列为EVPPEEH.以测得的肽序列为模板合成多肽,将纯肽与锌离子进行螯合反应制备肽锌螯合物.螯合物的红外光谱和圆二色光谱表征结果表明,锌离子主要与多肽链上的羰基氧发生相互作用.与多肽的空间结构相比,螯合物的无规则卷曲结构减少,β转角增加而β折叠减少.由肽锌螯合物的分子模拟和二级质谱结果可知,多肽与锌离子螯合后有2种空间构象:一种通过六配位的方式螯合1个锌离子,其中主要的螯合位点为多肽Val-2和Pro-3或者Glu-5和Glu-6之间的羰基氧;另一种是通过四配位的方式螯合1个锌离子,主要的螯合位点为多肽Glu-5和Glu-6之间的羰基氧.

An oyster peptide modified by plastein reaction was isolated from the modified product using different methods in Gluding Sephadex G-15 gelcolumn chromatography and reversed-phase high-performance liquid chromalography（RP-HPLC）.The purified peptide was sequenced as EVPPEEH with a zinc chelate activity of 161 mg/g.The peptide sequence was used as template to synthesize the pure peptide which was used to prepare peptide-zinc complexes.The results of infrared spectroscopy and circular dichroism of the complexes showed that the carbonyl oxygen belonging to the peptide chain was the primary binding site for Zn^（2＋）.Compared with the pure peptide,the irregular curl and β fold of complexes was reduced while β angle increases.Molecular mechanics simulation experiment and secondary spectrum of complexes showed that there were two kinds of spatial conformation of complexes.One way was to chelate a zinc ion by six coordination where the main chelating site was the carbonyl oxygen between Val-2 and Pro-3 or Glu-5 and Glu-6.The other was to chelate a zinc ion by four coordination where the main chelating site was the carbonyl oxygen between Glu-5 and Glu-6.