分光光度法结合抗干扰补偿检测谷胱甘肽

Glutathione Detection by Spectrophotometry Coupled with Anti-interference Compensation

分光光度法应用于酶法合成谷胱甘肽（GSH）的跟踪检测时,前体半胱氨酸（Cys）中的巯基对GSH的分析结果会产生显著干扰。该文通过多组干扰实验,统计建立了干扰模型和补偿算法,实现了不分离干扰物质即可定量检测GSH。通过干扰分析,引入干扰系数和补偿系数,利用干扰系数、干扰倍数、补偿系数之间的关系建立抗干扰机制。检测样品320 nm处的吸光度和导数,代入标准流程,即可判断干扰倍数和补偿系数,从而计算GSH及Cys含量。结果显示：四氧嘧啶补偿法的GSH浓度-吸光度标准方程为y=3.062 9x＋0.000 1,r2=0.999 9。当Cys浓度为GSH浓度的4倍以下时,GSH的回收率为95%～100%,Cys的回收率为78%～120%。相比四氧嘧啶法,四氧嘧啶补偿法显著增强了分析检测的抗干扰特性,达到快速跟踪检测GSH含量并依据干扰倍数半定量检测Cys的目的。

Spectrophotometry is a general method used in enzymatic synthesis of glutathione（ GSH）for tracking detection of reaction process. However,- SH from cysteine（ Cys） could significantly interfere the results of GSH analysis. By multiple interference experiments and that of data processing and principle analysis, an anti-interference compensation method for elimination of interference caused by the reaction of alloxan and Cys was established. Anti-interference mechanism was set by introducing interference coefficient and compensation coefficient, building interference model and compensation algorithm. The absorbance and derivative of unknown sample were detected at 320 nm,then the data detected were taken into the flow chart of anti-interference compensation,the interference ratio and compensation coefficient were obtained,and the contents of GSH and Cys could be calculated. The detection results of new method showed that when the concentration of Cys in reaction liquid was not more than 4 times of that of GSH,the recoveries of GSH and Cys were in the ranges of95%- 100% and 78%- 120%,respectively. The disturbance of Cys to the detection results could be eliminated. The standard equation for GSH concentration and absorbance is y = 3. 062 9x ＋ 0. 000 1,r2= 0. 999 9. And compared with alloxan method,it has a significant enhancement in the anti-interference characteristics for detection of the contents of GSH and semi-quantitation of Cys on the basis of multiple relationships. It has a practical application value to the quantitative detection of GSH with non-separating cysteine in enzymatic synthesis of GSH.