Computerized analysis of enzyme cascade reactions using continuous rate data obtained with an ELISA reader

Two programs have been written which permit analysis of multiple continuous-rate enzyme-cascade assays conducted with the use of an ELISA spectrophotometer and a synthetic chromogenic substrate. Because the product of the first reaction functions as the enzyme in the second reaction, production of chromophore continuously accelerates and it is the rate of acceleration which serves to measure the rate of the initial reaction in the system. The first program determines the rate of acceleration using linear regression to analyze the reaction curves as a function of the square of time. The second program, using a Simplex algorithm, determines the parameters which establish the assay standard curve by fitting the rate data to the Hill equation. Used together, these programs facilitate the analysis of many kinetic experiments conducted simultaneously.