Homogeneous Enzymatic Colorimetric Assay for Total Cysteine

Patients with vascular disease have significantly higher concentrations of plasma total cysteine (tCYS) than do healthy individuals (1)(2)(3)(4)(5)(6)(7). The present method is a new, rapid, and sensitive enzymatic colorimetric assay for tCYS in plasma samples and is homogeneous in that it avoids separation methods. The tCYS assay uses only the recombinant enzymes methionine α,γ-lyase (rMETase) and S-adenosylhomocysteine hydrolase (rSAHH) cloned from Pseudomonas putida and Trichomonas vaginalis , respectively. We have also developed enzymatic assay methods for total homocysteine (tHCY) (8)(9) and vitamin B6 (10) in plasma that use the same analyte, H2S, that is used for tCYS in the present report. Simultaneous assay of tHCY, vitamin B6, and tCYS may be relevant to the study for the occurrence and prevalence of cardiovascular disease (11).The instrumentation included a Hitachi U-2000 Spectrophotometer (Hitachi, Ltd.) and Fl-1000 fluorescence spectrophotometer and a Hitachi HPLC (L-6200A Intelligent Pump) equipped with a Supelcosil LC-18DB column [25 cm × 4.8 mm (i.d.); particle size, 5 μm; Supelco].The chemicals used were l-cysteine, d,l-homocysteine, l-methionine, adenosine (ADO), l-dithiothreitol, Triton X-100, pyridoxal 5-phosphate, and potassium ferricyanide and were purchased from Sigma Chemical Co. N,N -Dibutylphenylenediamine (DBPDA) was synthesized in our laboratory (8) …