Homogeneous enzymatic colorimetric assay for total cysteine.

نویسندگان

  • Qinghong Han
  • Mingxu Xu
  • Li Tang
  • Xinghua Sun
  • Nan Zhang
  • Xuezhong Tan
  • Xiuying Tan
  • Yuying Tan
  • Robert M Hoffman
چکیده

Patients with vascular disease have significantly higher concentrations of plasma total cysteine (tCYS) than do healthy individuals (1–7). 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 , -lyase (rMETase) and Sadenosylhomocysteine 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 m; 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,NDibutylphenylenediamine (DBPDA) was synthesized in our laboratory (8, 9, 12). rMETase and SAHH were produced in our laboratory (13, 14). Four reagents were used for the assay. Reagent 1 contained 20 mmol/L potassium phosphate (pH 8.3), 150 mmol/L NaCl, 9 mg/L rSAHH, 2 mL/L Triton X-100, 1.0 mmol/L l-dithiothreitol, and 100 mol/L ADO. Reagent 2 contained rMETase (1.08 g/L protein) in 20 mmol/L potassium phosphate (pH 7.2). Reagent 3 contained 40 mmol/L DBPDA in 3 mol/L HCl. Reagent 4 contained 15 mmol/L potassium ferricyanide in 20 mmol/L potassium phosphate (pH 7.2). Whole blood was collected in evacuated blood-collection tubes without additive or containing EDTA, heparin, or citrate as anticoagulants. Centrifugation was carried out as soon as possible at 2000g for 10 min. The plasma was collected and stored at 70 °C until analysis. The principle of the assay is as follows: In step 1, samples are reduced by l-dithiothreitol to generate free reduced CYS and HCY. Simultaneous use of rSAHH with excess ADO converts the reduced HCY to S-adenosylhomocysteine (SAH).

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عنوان ژورنال:
  • Clinical chemistry

دوره 50 7  شماره 

صفحات  -

تاریخ انتشار 2004