title: Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution: chemometrics and experimental models.
abstract: 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 (λ(0)); (b) absorbance at λ(A) and λ(0) 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 λ(0) 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 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 λ(0) 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.
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Liu
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yang
authors.forename: Lin
authors.initials: L
authors.lastname: Liu
authors.forename: Jizheng
authors.initials: J
authors.lastname: Dang
authors.forename: Yanling
authors.initials: Y
authors.lastname: Xie
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Pu
authors.forename: Gaobo
authors.initials: G
authors.lastname: Long
authors.forename: Yuanli
authors.initials: Y
authors.lastname: Li
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Yuan
authors.forename: Juan
authors.initials: J
authors.lastname: Liao
authors.forename: Fei
authors.initials: F
authors.lastname: Liao
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002976:Clenbuterol / Q000378:metabolism; D057075:Enzyme Assays; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008892:Milk / Q000737:chemistry; D009243:NAD / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D010400:Penicillin G / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
chemical_list: C474534:4-nitronaphthyl-1-phosphate; D000814:Aniline Compounds; D009281:Naphthalenes; D010710:Phosphates; D009243:NAD; C008625:nitroaniline; D007770:L-Lactate Dehydrogenase; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D010400:Penicillin G; D002976:Clenbuterol