Rapid and selective enzymatic assay for L-methionine based on a pyrophosphate detection system.
An enzymatic assay for L-methionine was developed by coupling adenosylmethionine synthetase (AdoMetS) to a pyrophosphate (PP(i)) detection system, which was constructed using pyruvate, phosphate dikinase. To expand the use of this assay, the PP(i) detection system was embodied as three different forms, which allowed PP(i) to be measured by UV, visible, and fluorescent light detectors. The assay system was robust and could tolerate the addition of inorganic phosphate and ATP to the assay mixtures. L-Methionine could be accurately determined by coupling the PP(i) detection system and AdoMetS. This AdoMetS coupling assay was highly selective to L-methionine and exhibited no significant activity to other proteinaceous amino acids, ammonia, or urea, unlike conventional enzymatic assays for L-methionine. Spike and recovery tests showed that the AdoMetS assay could accurately and reproducibly determine increases in L-methionine in human plasma samples without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. The high selectivity and robustness of the AdoMetS assay provide rapid and high-throughput analysis of L-methionine in various kinds of analytes.
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan; Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Masafumi
M
Kameya
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Mariko
M
Himi
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan; Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan. Electronic address: asano@pu-toyama.ac.jp.
Yasuhisa
Y
Asano
D000255:Adenosine Triphosphate / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D011756:Diphosphates / Q000378:metabolism*; D006801:Humans; D008715:Methionine / Q000032:analysis* / Q000097:blood; D008716:Methionine Adenosyltransferase / Q000378:metabolism; D011424:Propionibacterium / Q000201:enzymology; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism; D013997:Time Factors
D011756:Diphosphates; C107241:diphosphoric acid; D000255:Adenosine Triphosphate; D008715:Methionine; D008716:Methionine Adenosyltransferase; D011772:Pyruvate, Orthophosphate Dikinase
Adenosylmethionine synthetase; Pyrophosphate; Pyruvate phosphate dikinase; l-Methionine