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

• Published in: Analytical chemistry (Washington) Vol. 85; no. 4; pp. 2143 - 2154
• Main Authors: Liu, Hongbo, Yang, Xiaolan, Liu, Lin, Dang, Jizheng, Xie, Yanling, Zhang, Yi, Pu, Jun, Long, Gaobo, Li, Yuanli, Yuan, Yonghua, Liao, Juan, Liao, Fei
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.02.2013
• Subjects: Alkaline Phosphatase - metabolism; Analytical chemistry; Aniline Compounds - metabolism; Animals; beta-Galactosidase - metabolism; Cattle; Clenbuterol - metabolism; Enzyme Assays; Enzyme kinetics; Enzyme-Linked Immunosorbent Assay; gamma-Glutamyltransferase - metabolism; Kinetics; L-Lactate Dehydrogenase - metabolism; Milk - chemistry; NAD - metabolism; Naphthalenes - metabolism; Penicillin; Penicillin G - metabolism; Phosphates - metabolism; Spectrophotometry; Substrate Specificity; Substrates
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac302786p

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 (λA) much larger than that of Product B (λB) formed by Enzyme B action on Substrate B; λB is close to the longest isoabsorbance wavelength of Product A and Substrate A (λ0); (b) absorbance at λA 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 p-nitroaniline from γ-glutamyl-p-nitroaniline with λ0 at 344 nm and λA close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing λB at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-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 λA at 458 nm, β-d-galactosidase releases 4-nitrophenol from β-d-(4-nitrophenyl)-galactoside with λB 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.