S-同型半胱氨酸甲基转移酶活性检测和保存方法的建立

摘要

Objective To construct a simple method for the measurement of activity of S-homocysteine methyltransferase (HMT),and explore the best processing condition for HMT and the preservation of HMT.Methods HMT was expressed in pro-karyotic system by using genetic engineering technology,then was purified by using affinity and Sephadex G1 5 chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed to identify the physicochemical and bio-logical properties of target protein.Based on the principle of 5 ,5′-disulfide-double (2- nitro benzoic acid)(DTNB)could react with sulfydryl compounds rapidly,the reduction of homocysteine was detect to evaluate the activity of enzyme,then the best processing conditions of HTM were determined.Activity of enzyme,preserved in preservation solution with or without glycerol and preserved under different temperatures,was detected.Activity remaining ratios were detected and compared between HMT preserved in pres-ervation solution with different protective agents of different concentration and preserved by cryodesiccation.Results The purity of recombined HMT was above 95%,with molecular weight of 36 000 and excellent catalytic activity,and the catalytic activity was 2 000 U/mg.The optimum condition for the detection of biological activity was using HEPES buffer of pH 7.4 at 37 ℃ and reac-tion for 25 min.Glycerol could significantly prolong the preserving time of HMT,and half activity of HMT could be remained for six months.The reservation rates of activity of HMT,preserved in preservation solution with mannitol and trehalose,were 104%and 100%,respectively.Conclusion HMT could be obtained through genetic engineering.A simple test method of HMT was es-tablished,and the best processing conditions and preserving methods of HMT were determined,which laid a foundation for clinical application.%目的：建立一种简单的S-同型半胱氨酸甲基转移酶（H MT）活性测定方法，确定 H MT的最佳使用条件，并初步探索HMT的保存方法。方法采用原核表达系统制备 HMT，经镍亲和层析和Sephadex G15两步纯化。SDS-PAGE 对目的蛋白进行理化性质鉴定。基于5，5′-二硫基-双（2-硝基苯甲酸）（DTNB）可以特异性地与含有巯基的化合物快速发生化学反应的原理，通过检测同型半胱氨酸的减少量来计算酶活性，确定 HMT最佳活性测定条件。比较添加和不添加甘油的两组 HMT 酶液置于不同温度下保存，定期测定活力；在 HMT酶液中加入不同浓度的不同保护剂，比较冷冻干燥后的活性保留率。结果重组表达的酶纯度高达95％以上，相对分子质量为36000，具有良好的催化活性，比活为2000 U/mg。在37℃、pH7．4的HEPES缓冲液中反应25 min为酶活性测定最佳条件。加入甘油能明显延长酶的保存时间并且酶液在6个月内的活力保留一半。冷冻保护剂甘露醇和海藻糖的添加对 HMT酶冻干品的酶活保留率分别为104．0％和100．0％。结论通过基因工程技术获得 HMT，建立了简单的 HMT活性测定方法，并确定了该酶的最佳保存方法，为临床应用奠定了基础。