五步蛇蛇毒磷脂酶A_2的纯化及部分性质

经Sephadex G-75和QAE-Sephadex A-50离子交换层析等方法,从湖南产五步蛇(Agkistrodon acutus)蛇毒中纯化一种均一的酸性磷脂酶A_2。SDS-PAGE测得分子量为15.8kD,按氨基酸残基计算其分子量为14.352kD,IEF-PAGE测得等电点为5.32。氨基酸组份分析表明磷脂酶A_2分子由128个氨基酸残基组成,富含Asp和Glu,不含中性糖。PLA_2酶活性的最适温度为45℃,最适pH为8.5左右,没有抗胰蛋白酶的活性,具一定的热稳定性。K~+、Ca~(++)和Na~+离子激活,而Cd~(++)、Sn~(++)、Cu~(++)、Li~+、Hg(++)、Zn~(++)、Fe~(++)和Co~(++)离子可抑制或完全丧失酶活力。手工微量顺序分析测得PLA_2分子N-末端氨基酸为Leu。此酶对小白鼠的LD_(50)至少大于10mg/kg(ip)。

Purification and Characterization of Phospholipase A_2 from the Venom of Agkistrodon acutus

An acidic phospholipase A2 was purified by Sephadex G-75 gel filtration and QAE-Sephadex A-50 ion exchange chromatography from Agkistrodon acutus venom in Hunan province, China.There were two peaks with phospholipase A2 activity on QAE-Sephadex A-50 chromatography, one showing one band on polya-crylamide gel electrophoresis.The molecular weight was estimated to be 15.8kD by SDS-polyacrylamide gel electrophoresis and isoelectric point determined by isoele- etric focusing electrophoresis was pH5.32 Analysis of amino acid composition indicated that the phospholipase A2 consists of 128 amino acid residues with 14 half cystine, being rich in aspartic acid and glutamic acid.The enzyme exhibits considerable stability toward heat.The optmum temperature for the phospholipase A2 activity was observed at 45***********C,and it has maximum enzymatic activity at pH 8.5.In contrast to some acidic phospholipases A2 from snake venoms, the enzyme was not resistant to trypsin.Metal ions such as Ca++ , Na+, and K+ activated the enzyme while Cd++, Cu++, Co++, and Fe++ inhibited the enzyme activity, the N-terminal amino acid detected by 4-N, N-diraethylaminoazobenzene 4'-isothiocyanate was found to be leucine.Toxic test of this enzyme in mice (i.p.) showed that LD50 was at leasl more than 10mg/kg.