目的：建立一种快速简便测定谷胱甘肽的新方法。方法：样品1式2份，pH 8.0条件下分别和甲醛反应2 min和60 min，各取1 mL加入5 mL DTNB溶液，25 ℃反应5 min后分别测定在波长412 nm的吸光度，算出2者的差值ΔA，代入标准曲线计算得出谷胱甘肽含量。结果：谷胱甘肽浓度在0.19～0.95 g.L-1之间线性关系良好，回归方程为：Y=0.715 4X-0.000 4，r=0.999 9，最大误差不超过6%。结论：方法成本低廉，简单易行，特别适合于有干扰物质存在时还原型谷胱甘肽浓度的分析。关键词：谷胱甘肽　甲醛　半胱氨酸　DTNBA Simple Method for Rapid Determination of Reduced GlutathioneZhao Xudong　Wei Dongzhi　Wan Qun　Yu Juntang（State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry,East China University of Science and Technology, Shanghai　200237）Abstract：Objective：A new simple method for rapid determination of reduced glutathione in the presence of other thiols is developed based on the reactions of formaldehyde with glutathione and interefering substances. Method:After each aliquot of two same samples reacts with formaldehyde for 2 and 60 min respectively, 1 mL of each solution is added to 5 mL DTNB and reacts for 5 min at 25 ℃. The absorbance is measured immediately at wavelength 412 nm. The difference between two absorbance(ΔA=A2 min-A60 min)is calculated, concentration of glutathione can be obtained from standard curve. Result:There is a good linearity between 0.19 to 0.95 g.L-1 glutathione. Regression equation:Y=0.715 4X-0.000 4，r=0.999 9，maximum deviation is less than 6%. Conclusion:The method is cheap, simple, practicable and is applicable to assay of reduced glutathione in the presence of other interefering substances.Key words：glutathione, formaldehyde, cysteine, DTNB▲　　谷胱甘肽(GSH)是由谷氨酸、半胱氨酸(Cys)和甘氨酸组成的天然三肽，用途广泛。利用基因工程微生物生物合成方法是生产谷胱甘肽的主要方式。作为前体之一，半胱氨酸的浓度较高，对谷胱甘肽的测定有干扰。已有几种方法用于半胱氨酸存在时谷胱甘肽的测定：酶分析方法［1，2］可测高达1 000倍半胱氨酸存在时谷胱甘肽的含量，结果较准确，但需价格昂贵的辅酶Ⅱ和谷胱甘肽还原酶，并且后者活力变化严重影响测量结果；色谱法［3，4］冗长费时；Jocelyn［5］采用将样品在硫酸中煮沸1 h的方法，不太安全；Wronski［6，7］方法效果较好，但需用对羟基汞苯甲酸和萤光黄；荧光法［8］容易受干扰，误差较大；乙二醛酶法［9］需要复杂的技术和设备。针对上述方法的不足，本文在研究甲醛同谷胱甘肽和常见含巯基物质反应特点的基础上结合巯基的测定方法［10，11］提出一种简单，快速的新方法，应用于生物合成反应溶液中谷胱甘肽含量的分析取得满意结果。作者单位：赵旭东（华东理工大学生物反应器工程国家重点实验室　上海　200237）　　　　　魏东芝（华东理工大学生物反应器工程国家重点实验室　上海　200237）　　　　　万群（华东理工大学生物反应器工程国家重点实验室　上海　200237）　　　　　俞俊棠（华东理工大学生物反应器工程国家重点实验室　上海　200237）参考文献：［1］Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal Biochem, 1969, 27(3):502［2］Griffith O W. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem, 1980, 106(1):207［3］Newton G L, Dorian R, Fahey R C. Analysis of biological thiols: derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatograph. Anal Biochem, 1981, 114(2):383［4］Anderson M E, Meister A. Transport and direct utilization of γ-glutamylcysteine for glutathione synthesis. Proc Natl Acad Sci U S A, 1983, 80(3):707［5］Jocelyn P C. An assay for glutathione in acid solution. Anal Biochem, 1967,18(3):493［6］Wronski M. The micro-determination of glutathione and cysteine in the presence of each other. Analyst, 1965, 90(11):697［7］Wronski M. Hydrolysis of cystine-n-peptide bonds in keratin by hydrochloric acid. Aust J Chem, 1966, 19(2):341［8］Cohn V H, Lyle J. A fluorometric assay for glutathione. Anal Biochem, 1966, 14(1):434［9］Woodward G E. Glyoxalase　Ⅲ, glyoxalase as a reagent for the quantitative microestimation of glutathione. J Biol Chem, 1935, 109(1):1［10］Kortt A A, Liu T Y. On the mechanism of action streptococcal Proteinase I, Active-site titration. Biochemistry, 1973, 12(2):320［11］Ellman G L. Tissue sulfhydryl groups. Arch Biochem Biophys, 1959, 82(1):70［12］蔡武城，袁厚积.生物物质常用化学分析法.北京：科学出版社，1982.88