一组新的沙蚕蛋白酶同工酶的分离纯化与鉴定

Purification and Identification of New Isoenzymes of Nereid Proteinase

分离纯化一组新的沙蚕蛋白酶同工酶，并对其性质进行鉴定．用硫酸铵盐析、凝胶过滤层析和疏水层析技术从沙蚕中分离得到了沙蚕蛋白酶，经聚丙烯酰胺凝胶电泳（PAGE）、高效液相色谱（HPLC）、等电聚焦电泳（IEF）和质谱分析（MS）鉴定，发现沙蚕蛋白酶由3个组分组成，均有纤溶活性，其等电点为3．5～4．5，分子量分别为29248．75、29007．66、28954．17，肽指纹图谱分析发现，它们均为未知的新蛋白质，其中2个组分结构相似，具有较高的同源性，与另1个有不同．用抗原抗体反应鉴定其免疫原性，发现3个组分中有2个组分具有相同的免疫原性，另1个组分与2者不同．但3者具有相同的抗原决定簇，证明沙蚕蛋白酶由2个同工酶组成．以4种专一性的抑制剂对其进行抑制，检测酶的活性确定其酶学性质，发现其反应的适宜温度为40℃～50℃、适宜pH值为8～9．沙蚕蛋白酶属于丝氨酸蛋白酶．

The native Nereid proteinase is a serine proteinase with two known isoenzymes, which have the optimal temperature 40℃ - 50℃ and the optimal pH 8 - 9. We have identified several new Nereid proteinase isoenzymes following a combination of purification procedures of ammonium sulfate salting out, column filtration and hydrophobic chromatography. The major three proteinase identification possess fibrinolytic activities and are responsive to four narrow spectrum inhibitors in enzymological activities , and have the biochemical properties of isoelectric points between 3.5 and 4.5 determined by isoelectric focusing electrophoresis. The molecular weights are 29.2 kD, 29.0 kD and 28.9 kD, respectively, as determined and confirmed by SDSPAGE, high performance liquid chromatogram, and mass spectrum. The peptide finger printing revealed that the three isoenzymes appeared to be novel proteins, two of which have fairly high homologies. Although all three new enzymes have the same antigen determinant group, only the highly homologous two have the identical immunogenicity determined by antigen-antibody tittering reactions.