Activity assay and preservation of S-homocysteine methyltransferase / 国际检验医学杂志

ABSTRACT

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.