Regression data processing methods for parallel zero- and first-order processes applied to lactate dehydrogenase subunit determinations

Multipoint data-processing methods developed for parallel zero-order and first-order processes have been evaluated for the simultaneous determination of the heart and muscle subunits of lactate dehydrogenase. Conditions are adjusted such that pyruvate inhibition of heart and muscle subunits of the enzyme follows pseudo-first-order kinetics with apparent rate constants of k_H? 0.7 s^-1 and k_M? 1.4 s^-1 for the heart and muscle subunits, respectively. The influence of these first-order inhibition processes on the zero-order catalytic reaction is used to determine the two subunits in single- and two-component samples. Stopped-flow mixing is used and 250 data points collected during the early part of the reaction are fitted to four different mathematical models for the parallel process in order to determine initial velocity components resulting from each subunit present, and these velocity components are related to enzyme concentration. Enzymes in synthetic samples are determined in the 0–80 nmol l^-1 range with uncertainties of about 1 nmol l^-1 for single- and two-component samples prepared from purified preparations for the enzymes.