A Systematic Approach to Enzyme Assay Optimization, Illustrated by Aminotransferase Assays

[摘要] We have developed a systematic approach to optimization of reagent concentrations for assays of alanine aminotransferase and aspartate aminotransferase: ( a ) Michaelis constants describing the initial-velocity kinetics of the coupled enzyme reactions were evaluated by a nonlinear least-squares fit of the appropriate equation to measured enzyme activities. Activities of more than 50 normal and pathological sera were measured at 30 °C. ( b ) These kinetic equations are used to calculate the set of reagent amino- and keto-acid concentrations that all yield a selected fraction of the theoretical maximum enzyme velocity. An optimal pair is determined by defining an additional criterion, such as minimal reagent cost or minimal concentration to K m ratio. ( c ) The optimum amounts of reagent NADH and coupling enzyme, being a function of desired pre-incubation and measurement intervals, maximum aminotransferase activity to be measured, and endogenous keto-acid concentration, are determined by computer simulation. An approximate relationship and an exact method for computing assay lag time are presented, along with experimentally measured endogenous keto-acid concentrations in serum. All procedures may be applied to other enzyme assays if appropriately modified.

[学科分类] 过敏症与临床免疫学