Rapid enzymatic assays for l-citrulline and l-arginine based on the platform of pyrophosphate detection

Abstract

Rapid determination of L-citrulline and L-arginine, physiologically important amino acids, is a beneficial technique from the scientific and medical viewpoints. In this study, enzymatic assays for L-citrulline and L-arginine were established and evaluated. L-Citrulline assay was constructed by coupling argininosuccinate synthetase to a pyrophosphate detection system, in which pyruvate phosphate dikinase was employed, so that the citrulline-dependent production of pyrophosphate could be determined. Furthermore, the L-arginine assay was developed by coupling arginine deiminase to the L-citrulline assay. Both assays exhibited high selectivity to L-citrulline and L-arginine without any significant reactivity to other proteinaceous amino acids. These assays were also resistant to various contaminants that interfered with the conventional L-citrulline and L-arginine assays. The high accuracy of these assays was demonstrated by measurements in the presence of human plasma. Because these assays can be conducted under the neutral pH without terminating the reaction progress, they allow not only measurements in static analyte solutions, but also real-time monitoring of L-citrulline and L-arginine synthesis in the reaction mixture. The features of these assays also demonstrated that the pyrophosphate detection system served as a useful platform to develop selective and robust enzymatic assays by being coupled to a pyrophosphate-producing enzyme.

Fruit Morphology, Citrulline, and Arginine Levels in Diverse Watermelon (Citrullus lanatus) Germplasm Collections

“…Colorimetric methods for the quantification of citrulline that is developed earlier [ 39 ] could overestimate the actual citrulline levels, and in thin layer chromatography and amino acid analyzers, the presence of glutamine could complicate quantification [ 6 , 23 ]. Other colorimetric citrulline analysis methods that have been mainly developed for animal and human tissues use strong oxidizers to hydrolyze tissues completely [ 40 , 41 , 42 ]. These strong sample treatments could cause the oxidation of citrulline in watermelon tissues and result in erroneously low values.…”

Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

“…The method is applicable to many food and clinical analyses because L-arginine is an intermediate in the human urea-cycle and is an essential amino acid in humans. Although a colorimetric method for assaying L-arginine using five different enzymes has been reported [12], our simple method for assaying L-arginine may be much more attractive for industrial and clinical uses.…”

Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

“…The method is applicable to many food and clinical analyses because L-arginine is an intermediate in the human urea-cycle and is an essential amino acid in humans. Although a colorimetric method for assaying L-arginine using five different enzymes has been reported (Kameya and Asano, 2014), our simple method for assaying L-arginine may be much more attractive for industrial and clinical use. Wendel U, Hummel W, Langenbech U (1989) Monitoring of phenylketonuria: A colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase.…”