L hcy thiolactone hcl

HUVECs, purchased from Lonza, were seeded into 75-cm² flasks coated with 1 % gelatin and cultured in EGM2 medium (Lonza) at 37 °C/5 % CO₂, supplemented with 2 % fetal bovine serum (FBS; Sigma–Aldrich), bovine endothelial cell growth factor, and antibiotics: 50 μg/mL penicillin and 100 μg/mL streptomycin (Sigma–Aldrich). Cells between 4 and 10 passage were used in the experiments. After reaching 80–90 % confluence, the cells were treated for 24 h with 0, 10, or 1000 μM D, L-Hcy, or L-Hcy-thiolactone HCl (Sigma–Aldrich), 10 or 40 μM N-Hcy-protein in the EGM2/FBS medium without antibiotics.

N-Hcy-protein was prepared by the modification of FBS with Hcy-thiolactone (Jakubowski 1999 (link)). Such preparation mimics N-Hcy-protein that was present in the plasma of cystathionine β-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR)-deficient patients (Jakubowski et al. 2008 (link)). FBS (0.2 mL, 50 mg protein/ml) was incubated with 5 or 25 mM L-Hcy-thiolactone∙HCl (Sigma–Aldrich) in 0.1 M phosphate buffer pH 7.4, 1 mM EDTA (buffer A) at 37 °C for 16 h. Hcy and unreacted Hcy-thiolactone were removed by four cycles of dilution with buffer A containing 2 mM dithiothreitol (DTT) and ultrafiltration using a 10-kDa cutoff Millipore device (10,000xg, 4 °C, 30 min). DTT was then removed by three cycles of dilution with 0.2 mL buffer A and ultrafiltration, and the N-Hcy-protein preparation was diluted to 50 mg protein/mL with buffer A. The thiol content in N-Hcy-protein was quantified according to Ellman (1959 (link)). These N-Hcy-protein preparations were added to cell culture media to constitute 2 % of the medium volume, resulting in N-Hcy-protein concentrations of 10.1 and 41.1 μM, respectively, similar to the range of N-Hcy-protein concentrations observed in disorders of Hcy/folate metabolism in humans (Jakubowski et al. 2008 (link)) and mice (Jakubowski et al. 2009 (link)).

l-Hcy-thiolactone·HCl, d,l-Hcy, l-methionine, N-acetyl-l-cysteine (NAC), horse spleen ferritin, dithiothreitol (DTT), tris-(2-carboxyethyl)phosphine (TCEP), o-phthaldialdehyde (OPA), monosodium phosphate, NaOH, sodium citrate, NaCl, CuCl₂, FeCl₂, FeCl₃, vitamin C, sodium dodecyl sulfate (SDS), chloroform, methanol, HCl, trichloroacetic acid, were purchased from Sigma-Aldrich. Suprapure nitric acid, 65% and perchloric acid, 60% were from Merck. Reagents were prepared in MiliQ purified water.