DIFFERENTIAL RATE EVALUATION OF PARALLEL FIRST AND ZERO ORDER REACTIONS AND ITS APPLICATION TO LACTATE DEHYDROGENASE SUBUNIT DETERMINATION
Abstract
A multipoint multicomponent kinetic method based on inhibition reactions of similar catalytic species has been developed and applied to the simultaneous determination of lactate dehydrogenase subunit activities from a single rapid experiment. An uncompetitive substrate inhibition mechanism is used to derive a rate equation for the oxidation of reduced nicotinamide-adenine dinucleotide and a time dependent absorbance equation for the concommitant decrease in absorbance at 340 nm for reactions of pure subunit (H or M) preparations. Two distinct approaches are utilized to extract pertinent kinetic parameters from pure subunit data. In a derivative approach a quadratic first derivative convolute is generated and represents instantaneous reaction velocities over the course of inhibition. First-order regression of these rate data is used to estimate the initial and steady state velocities. The second approach involves fitting the absorbance response to integrated rate equations which describe parallel first- and zero-order processes corresponding to the partial inhibition of either subunit. This integrated approach has been expanded to include simultaneous subunit reactions. Our studies confirm that the extent of inhibition is strongly dependent on pyruvate concentration, but pseudo-first-order rate constants which describe the process by which the reaction velocity approaches the steady state level are virtually independent of pyruvate concentration. These constants are estimated in the range of 0.6 to 0.8 s('-1) for the H subunit and 1.4 to 1.8 s('-1) for the M subunit. Substrate profiles of initial and steady state velocities confirm that the uncompetitive mechanism only partially accounts for the observed pyruvate inhibition. Results are reported for single and two component synthetic samples of H and M subunits. Regression equations show good linearity between subunit concentration and estimated initial and steady state velocities for single component samples, good agreement between added and found values of subunit concentration for two component systems, and good correlation between added and found total enzyme concentration in mixtures. Regression slopes for the H subunit, M subunit, and total enzyme concentration in mixtures are 0.97 (+OR-) 0.02, 0.98 (+OR-) 0.01, and 1.01 (+OR-) 0.01 respectively, with intercepts of 0.21 (+OR-) 0.22, 0.01 (+OR-) 0.14, and 0.66 (+OR-) 0.24 nmol/L and standard errors of estimate 0.74, 0.46, and 0.44 nmol/L. These data suggest unbiased performance for simultaneous or total enzyme determinations.
Degree
Ph.D.
Subject Area
Analytical chemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.