Novel method for l-methionine determination using l-methionine decarboxylase and application of the enzyme for l-homocysteine determination

Abstract

Abstract For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10–200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC. Abbreviations DTT: dithiothreitol; IPTG: isopropyl-β-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5ʹ-phosphate