重组水蛭素的突变及突变体部分性质研究  

Use of Site Directed Mutagenesis to Investigate the Properties of Recombinant Hirudin HV2

作　　者：鹿峪峰[1] 王朝晖[1] 韩玉珉[1] 

机构地区：[1]中国科学院生物物理研究所

出　　处：《中国生物化学与分子生物学报》1998年第1期32-37,共6页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：以基因突变结合动力学分析的方法研究了水蛭素空间结构及其与凝血酶的相互作用．采用基因定点突变和随机突变的方法得到两个重组水蛭素突变体，并从抗酰胺水解活性，抗凝血酶活力和稳定性三个方面，比较研究了重组水蛭素ｒＨＶ２中４７位和１１位两个氨基酸残基对其稳定性和抑制能力的影响．将ｒＨＶ２中Ｇｌｎ１１和Ａｓｎ４７分别突变为Ｈｉｓ１１和Ｌｙｓ４７后，ｒＨＶ２－Ｈ１１生物活力降低３０％，ｒＨＶ２－Ｋ４７生物活力提高６１％．测定抑制常数Ｋｉ表明，ｒＨＶ２－Ｈ１１突变体Ｋｉ值升高１４倍，ｒＨＶ２－Ｋ４７突变体Ｋｉ值降低１４倍，两个突变体的热稳定性均有所增强，ｒＨＶ２－Ｈ１１在酸性和碱性条件的稳定性降低．分析实验结果，可以认为：①４７位的Ｌｙｓ可能是通过氢键和静电两种作用力同时影响着水蛭素的三维结构和其与凝血酶的结合．②１１位氨基酸可能是水蛭素分子中另一个重要位点．Hirudin HV2 is a potent and highly specific inhibitor of thrombin.The purpose of this research was to study the kinetics of complex formation between recombinant hirudin(rHV2) or recombinant hirudin mutants and thrombin.In order to elucidate the reactive site of the inhibitor,predetermined amino acid substitutions were introduced by means of site directed mutagenesis and random mutagenesis of a hirudin gene.The antiamidolytic activity,bioactivity and stability of rHV2 and its two mutants were compared.In comparison with recombinant hirudin( K i=1 9×10 -9 mol/L),mutant rHV2 K47 showed a lower K i value (1 4×10 -10 mol/L) and mutant rHV2 H11 showed a higher K i value (2 6×10 -8 mol/L).Bioactivity of mutant rHV2 K47 increased by 61% and those of mutant rHV2 H11 decreased by 30%.The stability of recombinant hirudin and its mutants were tested at various extreme conditions.In comparison with recombinant hirudin,the increase in stability of the two mutants were observed when the alkaline solution was heated at 70℃.There was a decrease in stability of rHV2 H11 when the acidic or alkaline solution was incubated at 25℃ for 2 hours.According to above results,the structure of hirudin and its interaction with thrombin was affected by both the hydrogen bond and static charge of Lys 47.It also suggested that Gln 11 may be another important amino acid in hirudin.

关 键 词：重组 水蛭素 基因突变 动力学 稳定性 

分 类 号：R973.2[医药卫生—药品]