Kinetic method for enzymatic analysis by predicting background with uricase reaction as model  

作　　者：廖飞 赵运胜 赵利娜 陶佳 朱小云 王咏梅 左渝平 

机构地区：[1]Laboratory of Bioinformatics & Molecular Engineering, Chongqing University of Medical Sciences, Chongqing 400016, China,Department of Biochemistry & Molecular Biology, Chongqing University of Medical Sciences, Chongqing 400016, China [2]Laboratory of Bioinformatics & Molecular Engineering, Chongqing University of Medical Sciences, Chongqing 400016, China [3]Department of Biochemistry & Molecular Biology, Chongqing University of Medical Sciences, Chongqing 400016, China

出　　处：《Journal of Medical Colleges of PLA(China)》2005年第6期338-344,共7页中国人民解放军军医大学学报（英文版）

基　　金：SupportedbytheNationalNaturalScienceFoundationofChina(No.30200266)

摘　　要：Objective：To investigate the reliability for kinetic assay of substance with background predicted by the integrated method using uricase reaction as model. Methods： Absorbance before uricase action （Δ0） was estimated by extrapolation with given lag time of steady-state reaction. With Km fixed at 12.5μmol/L, background absorbance （Δb） was predicted by nonlinearly fitting integrated Michaelis-Menten equation to Candida utilis uricase reaction curve. Uric acid in reaction solution was determined by the difference （ΔA） between Δ0 and Δb. Results .Ab usually showed deviation 〈3% from direct assay with residual substrate done fifth of initial substrate for analysis. ΔA showed CV 〈5% with resistance to common interferences except xanthine, and it linearly responded to uric acid with slope consistent to the absorptivity of uric acid. The lower limit was 2.0 μmol/L and upper limit reached 30 μmol/L in reaction solution with data monitored within 8 min reaction at 0. 015 U/ml uricase. Preliminary application to serum and urine gave better precision than the direct equilibrium method without the removal of proteins before analysis. Conclusion .This kinetic method with background predicted by the integrated method was reliable for enzymatic analysis, and it showed resistance to common interferences and enhanced efficiency at much lower cost.Objective:To investigate the reliability for kinetic assay of substance with background predicted by the integrated method using uricase reaction as model. Methods: Absorbance before uricase action （A0） was estimated by extrapolation with given lag time of steady-state reaction. With Km fixed at 12.5 μmol/L, background absorbance （Ab） was predicted by nonlinearly fitting integrated Michaelis-Menten equation to Candida utilis uricase reaction curve. Uric acid in reaction solution was determined by the difference （ΔA） between A0 and Ab. Results:Ab usually showed deviation <3% from direct assay with residual substrate <one-fifth of initial substrate for analysis. ΔA showed CV <5% with resistance to common interferences except xanthine, and it linearly responded to uric acid with slope consistent to the absorptivity of uric acid. The lower limit was 2.0 μmol/L and upper limit reached 30 μmol/L in reaction solution with data monitored within 8 min reaction at 0.015 U/ml uricase. Preliminary application to serum and urine gave better precision than the direct equilibrium method without the removal of proteins before analysis. Conclusion:This kinetic method with background predicted by the integrated method was reliable for enzymatic analysis, and it showed resistance to common interferences and enhanced efficiency at much lower cost.

关 键 词：kinetic method enzymatic methods PREDICTION reaction curve fitting URICASE 

分 类 号：R341[医药卫生—基础医学]