Minimizing errors in differential kinetic analyses of isoenzymes: Determination of aspartate aminotransferase (GOT) using differential substrate levels

Determination of isoenzymes by a method of differential substrate levels is fast and simple and usually does not require prior separations. It can often be adapted to existing total enzyme assays. Application has been made to mixtures of purified tissue fractions of human l-aspartate aminotransferase using a slightly modified commercial assay kit. Colorimetric rate measurements, accurate to 2–3%, allowed resolution of components to an accuracy of 6–9%. A generalized calibration scheme was devised which allowed standardization either by mixed or pure isoenzyme standards. The concept of an “error multiplier” has been developed for prediction of the most favorable operating conditions and for estimating the expected accuracy of the simultaneous determination. Curves are presented that provide a direct means of selecting optimum conditions for two-component isoenzyme systems. The computations are well suited to computer methods. However, in the event a manual data-reduction procedure is desired, a simple graphic scheme has been presented.