平颏海蛇蛇毒磷脂酶A_2的纯化及性质(英文)

PURIFICATION AND CHARACTERIZATION OF A PHOSPHOLIPASE A_2 FROM THE VENOM OF THE SEA SNAKE, LAPIMIS HARDWICKII

前文报导平頦海蛇L.handwickii的毒液含有丰富的磷脂酶A_2(PLA_2,EC3.1.1.4)现采用sephadex G—75凝胶过滤,CM—纤维素及DEAE—纤维素柱层析等步骤,将此酶提纯至SDS —PAGE电泳中仅显一条区带并测出其相应分子量为12000,在聚丙烯酰胺凝胶IBF中测得此酶PI为5.5,氨基酸组份分析结果表明酶分子中含有较多的半胱氨酸和门冬氨酸残基。此酶水解卵磷脂的产物经TLC鉴定为溶血卵磷脂,平颌海蛇蛇毒PLA_2在pH5.0～8.0范围稳定,最适pH为7.0;在pH6.0条件下,经90℃保温10min仍保持大部价活力,此酶可被Ca^(2+),Na^+,Mg^(2+)等阳离子激活,而受EDTA—Na_2,Zn^(3+),Cu^(2+)和柠檬酸、草酸等阴离子抑制。通过紫外吸收光谱及喇曼光谱的观察,初步研究了此酶的分子构型及某些基团的性质,最后,对已被研究的三种海蛇(平頦海蛇和L.Semifa—rciata及E.Schistosa)的PLA_2的某些性质进行了比较并作了简单的讨论。

A phospholipase A2 （E C 3.1.1.4.）from sea snake Lapimis hardwickii crude venom containing a large amount of this enzyme was isolated by column chromatography of Sephadex G-75, CM-cellulose and DEAE-cellulose. On pore gradient SDS-PAGE, this component gave a single band with a Mr of about 12000 and it contained a major band with an apparent PI’ of about 5.5 on poly- acrylamide gel IEF. Amino acid analysis indicated that the enzyme contained re- latively large number of cystein and aspartic acid residues. The hydrolysis prod- uct of lecithin by this enzyme was identified as lysolecithin by TLC. This enzy- me was stable at the pH ranging from 5.5 to 8.0 with the optimum pH of 7.0 and when pH <6 or>9, its activity was reduced dramatically. In the pH 6.0 buffer, after incubation at 90℃ for 10’, it retained most of its activity. The enzyme was activated by Ca2+, Na+, Mg2+ and inhibited by Zn2+, Cu2+, citric acid, C2O42- and EDTA-Na2. The molecular conformation of this enzyme was observed by UV absorption spectrum and Raman spectroscopy. Some properties of this PLA2 were compared with those of the PLA2s from the vonom of other two sea snake, L. semifarciata and E. schistosa.