摘要
目的对重组突变巴曲酶进行酶动力学初步研究,并建立一种蛇毒类凝血酶活性测定方法。方法使用可控温(37℃)紫外分光光度计,采用酶动力学测定法,测定重组突变巴曲酶的米氏常数,优化活性测定方法的反应时间和线性范围,验证此方法精密度,并用发色底物法与血浆凝集法同时测定不同蛇种来源的类凝血酶活性。结果重组突变巴曲酶的米氏常数(37℃)Km值为256.28μmol/L、Vm值为0.005 077μmol/min。该法呈现相应的剂量关系曲线,反应时间优选为0～20 min,线性范围优选为1～16.8 nmol/L,拟合度R^2≥0.99,方法精密度的变异系数≤5.0%。两种不同方法测定不同蛇种来源类凝血酶的活性单位比例范围为3.3～3.7(发色底物法/血浆凝集法),比例关系一致。结论建立的蛇毒类凝血酶活性测定方法拟合度优,精密度高,可测定不同来源的类凝血酶活性,并可替代传统血浆凝集活性测定法。
Objective The enzyme kinetics of recombinant mutant batroxobin was studied,and an activity analysis method for snake venom thrombin-like enzymes was established.Methods The Michaelis constants of recombinant mutant batroxobin were determined by using enzyme kinetics assay with ultraviolet spectrophotometer at controlled temperature (37℃).The reaction time and linear range were optimized,and the activity analysis method was verified.Chromogenic assay method and plasma coagulation assay method were compared and used to test thrombin-like enzymes purified from venom of different snake specie.Results The Michaelis constant Km value of recombinant mutant batroxobin at 37 degree was 256.28 μmol/L,Vm value was 0.005077 μmol/min.The standard curve presented the corresponding dose relationship,and the optima reaction time was 0-20 min,linear range was 1-16.8 nmol/L,the curve fitting was R2≥0.99,precision was CV≤5.0%.The ratio range of the active units of thrombin-like enzymes from different snake species was 3.3-3.7(chromogenic assay/plasma coagulation assay)by two different methods,and was reasonably consistent.Conclusion The chromogenic assay method,has advantages of better degree fitting and high precision,can be used to determine the activity of thrombin-like enzymes purified from venoms of different snake species,especially can replace the traditional plasma coagulation method.
作者
李娜
兰海英
康乐
陈宏超
徐梅
LI Na;LAN Hai-ying;KANG Le;CHEN Hong-chao;XU Mei(Liaoning Yuanda Nuokang Biopharmaceutical Co,Ltd,Shenyang City,Liaoning Province,110171,China)
出处
《蛇志》
2019年第1期10-12,31,共4页
Journal of Snake
关键词
蛇毒类凝血酶
巴曲酶
酶动力学
活性分析
发色底物法
血浆凝集法
Venom thrombin-like enzymes
Batroxobin
Enzymes kinetics
Activity analysis
Chromogenic assay
Plasma coagulation assay
