title: Fluorometric enzymatic assay of l-arginine.
abstract: The enzymes of l-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415nm under excitation at 360nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100nM to 6μМ with a limit of detection of 34nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.
authors.affiliation: Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Str. 14/16, 79005 Lviv, Ukraine.
authors.forename: Nataliya
authors.initials: N
authors.lastname: Stasyuk
authors.affiliation: Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Str. 14/16, 79005 Lviv, Ukraine. Electronic address: galina.gayda@gmail.com.
authors.forename: Galina
authors.initials: G
authors.lastname: Gayda
authors.affiliation: Scientific and Production Center "Armbiotechnology", National Academy of Sciences of Armenia, Armenia.
authors.forename: Hasmik
authors.initials: H
authors.lastname: Yepremyan
authors.affiliation: Department of Dietetics, Institute of Nursing and Health Sciences, Medical Faculty, University of Rzeszow, Al. mjr.W.Kopisto 2 a, 35-310 Rzeszów, Poland; Centre for Innovative Research in Medical and Natural Sciences, Medical Faculty of the University of Rzeszow, Warzywna 1A, 35-310 Rzeszów, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Stepien
authors.affiliation: Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Str. 14/16, 79005 Lviv, Ukraine.
authors.forename: Mykhailo
authors.initials: M
authors.lastname: Gonchar
mesh_terms: D000641:Ammonia / Q000097:blood; D001119:Arginase / Q000378:metabolism; D001120:Arginine / Q000097:blood / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000097:blood; D009764:o-Phthalaldehyde / Q000737:chemistry
chemical_list: D009764:o-Phthalaldehyde; D000641:Ammonia; D014508:Urea; D001120:Arginine; C574619:ARG1 protein, human; D001119:Arginase
keywords: Enzymatic assay; Recombinant arginase I; Sulfite; Urease; l-arginine; o-Phthalaldehyde