Myeloperoxidase-oxidase oxidation of cysteamine

Abstract

Cysteamine oxidation was shown to be catalysed by nanomolar concentrations of myeloperoxidase in a peroxidase-oxidase reaction, i.e. an O2-consuming oxidation of a compound catalysed by peroxidase without H2O2 addition. When auto-oxidation of the thiol was prevented by the metal-ion chelator diethylenetriaminepenta-acetic acid, native, but not heat-inactivated, myeloperoxidase induced changes in the u.v.-light-absorption spectrum of cysteamine. These changes were consistent with disulphide (cystamine) formation. Concomitantly, O2 was consumed and superoxide radical anion formation could be detected by Nitro Blue Tetrazolium reduction. Both superoxide dismutase and catalase inhibited the reaction, whereas the hydroxyl-radical scavengers mannitol and ethanol did not. O2 consumption increased with increasing pH (between pH 6.0 and 8.0), and 50% inhibition was exhibited by about 3 mM-NaCl at pH 7.0 and by about 100 mM-NaCl at pH 8.0. Cysteamine was about 5 times as active (in terms of increased O2 consumption at pH 7.5) as the previously reported peroxidase-oxidase substrates NADPH, dihydroxyfumaric acid and indol-3-ylacetic acid. A possible reaction pathway for the myeloperoxidase-oxidase oxidation of cysteamine is discussed. These results indicate that cysteamine is a very useful substrate for studies on myeloperoxidase-oxidase activity.

Abilities of peroxidases to catalyse peroxidase-oxidase oxidation of thiols

“…Thiols such as cysteine and glutathione have been reported to be HRP-oxidase substrates (Olsen & Davis, 1976;Harman et al, 1984;Wefers et al, 1985). The thiols cysteamine and cysteine esters, but not cysteine and glutathione, have been reported to be MPO-oxidase substrates (Svensson, 1988a;Svensson & Lindvall, 1988 (Agner, 1958)] have previously been described (Svensson et al, 1987). Cationexchange peak IV from chronic-myeloid-leukaemia MPO (dialysed; 677 kat/mol; A430/A480 = 0.83) was used in Table 1 and Fig.…”

“…Cationexchange peak IV from chronic-myeloid-leukaemia MPO (dialysed; 677 kat/mol; A430/A480 = 0.83) was used in Table 1 and Fig. 1 (below) and cation-exchange peak II from normal MPO (without dialysis; 734 kat/ mol; A430/A280 = 0.82) was used in (Sawada & Yamazaki, 1973)], bovine catalase [H202 : H202 oxidoreductase; EC 1.11.1.6; dialysed; 31 000 units/mg (Svensson & Lindvall, 1988)], human superoxide dismutase (02-:02-oxidoreductase; EC 1.15.1.1; 2900 units/mg as assayed by the manufacturer) and human haemoglobin [type IV; dialysed; 6406 = 1.20 x 1 M-l cm- (Lemberg & Legge, 1949)] were bought from Sigma. The MPO contamination of the EPO preparation was less than 0.5% as analysed by radioimmunoassay, which was kindly carried out by Dr. P. Venge, Department of Clinical Chemistry, University Hospital, Uppsala, Sweden (Carlson et al, 1985).…”

“…Cysteamine oxidation was measured by u.v. spectroscopy at 37°C by recording the increase at 270 nm or the decrease at 235 nm (Svensson & Lindvall, 1988), and the initial cysteamine oxidation rate was calculated by using £270 = 91 M-1cm-1 [difference in the molar absorption coefficient between cysteamine in its oxidized form (cystamine) and reduced form] or £235 = 283 M-1 cm-'. Nitro Blue Tetrazolium reduction was measured as previously described (Svensson, 1988a).…”

“…After dialysis the haemoglobin was mainly in its methaemoglobin form, as judged by its visible-lightabsorption spectrum. Methods 02 consumption was measured at 37°C with a Clarktype oxygen electrode (Svensson & Lindvall, 1988), and (4.4-6.7) (4) 1.2 (1.0-1.4) (4) 0.1…”

Synthesis of diacylated γ-glutamyl-cysteamine prodrugs, and in vitro evaluation of their cytotoxicity and intracellular delivery of cysteamine

“…When exposed to air, cysteamine is rapidly oxidized to its disulfide form, cystamine [39], particularly when in dilute aqueous solution. The rate of oxidation of cysteamine hydrochloride in aqueous solution was investigated by HPLC with UV detection, using pre-analysis derivatisation by CMQT.…”