Spectrophotometric-Dual-Enzyme-Simultaneous Assay in One Reaction Solution: Chemometrics and Experimental Models

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (λ_A) much larger than that of Product B (λ_B) formed by Enzyme B action on Substrate B; λ_B is close to the longest isoabsorbance wavelength of Product A and Substrate A (λ₀); (b) absorbance at λ_A and λ₀ is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with γ-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. γ-Glutamyltransferase releases <i>p</i>-nitroaniline from γ-glutamyl-<i>p</i>-nitroaniline with λ₀ at 344 nm and λ_A close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing λ_B at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by <i>p</i>-nitroaniline; the linear range of initial rates of γ-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and β-d-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with λ₀ at 405 nm and λ_A at 458 nm, β-d-galactosidase releases 4-nitrophenol from β-d-(4-nitrophenyl)-galactoside with λ_B at 405 nm. No interference from substrates/products made SDESA of β-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising