[Determining role of media in peroxide oxidation of aromatic type antioxidants with participation of methemalbumins]. / Opredeliaiushchaia rol' sredy v peroksidnom okislenii antioksidantov aromaticheskiogo riada s uchastiem metgemal'buminov.

Participation of the complexes of hemin and albumins (or delipidated albumins) in peroxidation of aromatic free radical scavengers and antioxidants was studied at varying hemin/albumin ratios. The radical-scavenging amines included o-phenylenediamine (OPD) and tetramethylbenzidine (TMB); the antioxidants, gallic acid (GA) and GA polydisulfide (GAPD). Peroxidation reactions were carried out in buffered physiological saline (BPS) supplemented with 2% dimethylsulfoxide(DMSO), pH 7.4 (medium A), or in 40% aqueous dimethylformamide (DMF), pH 7.4 (medium B). In all systems involving methemalbumins, kinetic constants (kcat), Michaelis constants (kM), and the ratios thereof (kcat/kM) were determined for OPD oxidation in medium A and TMB oxidation in medium B. Oxidation of OPD, GA, and GAPD in medium A was characterized by a decrease in the catalytic activity of hemin after the formation of hemin-albumin complexes. Conversely, oxidation of TMB and OPD in medium B was distinguished by pronounced activation of hemin present within methemalbumins.

[Spectrophotometric and fluorometric study of albumins and hemin interactions with aromatic antioxidants]. / Spektrofotometricheskoe i fluorimetricheskoe issledovanie vzaimodeistviia al'buminov i gemina s antioksidantami aromaticheskogo riada.

Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants (Ka) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4). Ka values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes with o-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media; Ka values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically; Ka values were of order of approximately 10(-5) M-1 and decreased with the ligand hydrophobicity.

Inhibition of oxidation of aromatic amines in heme-containing hydrogen peroxide systems by substituted 4,6-di-tert-butyl-pyrocatechols.

The substituted pyrocatechols sodium (2,3-dihydroxy-4,6-di-tert-butyl-phenyl)-S-thiosulfate (InH1) and 3-(S-glutathionyl)-4,6-di-tert-butyl-pyrocatechol (InH2) effectively inhibited oxidation of o-phenyle-nediamine and tetramethylbenzidine (TMB) catalyzed by hemin or its complex with BSA (methemalbumin). The method of competitive reactions of aromatic amines (PDA, TMB) and pyrocatechols (InH1, InH2) with active radicals was used for the quantitative determination of rates of radical initiation in systems containing hemin (methemalbumin) and H2O2 (TBHP). The use of the inhibitor method for heme-protein-peroxide systems is complicated by heterogeneity of the radicals generated (HO., RO., HO2., RO2., peroxide-like compounds I and II), formation of complexes of the inhibitors with protein component, and possible interaction of active radicals with organic co-solvents (DMF or DMSO). The system hemin (methemalbumin)-H2O2 (TBHP)-aromatic amines can be used for testing the efficacy of potential bioantioxidants.

[Methemalbumin--a biocatalyst of aromatic amine oxidation by hydrogen peroxide]. / Metgemal'bumin--biokatalizator okisleniia aromaticheskikh aminov perekis'iu vodoroda.

The kinetics of hydrogen peroxide-dependent oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine catalyzed by hemin and hemin-bovine serum albumin (BSA) complex (methemalbumin) was studied in buffered physiological solution containing dimethylformamide (40%) and dimethyl sulfoxide (2%), respectively. The formation of hemin-BSA complex enhanced hemin catalytic activity in oxidation of both amines. The reaction follows first-order kinetics in biocatalyst concentrations, H2O2, and H+ ions from pH 6.5 to 9.0. The catalytic constants, Michaelis constants, and kcat/K(m) ratios for both substrates in hemin- and methemalbumin-catalyzed reactions were calculated using double reciprocal plots of the effect of the initial TMB and PDA concentrations on the initial reaction rates. Mechanism of radical oxidation of amines in hemin-H2O2 and hemin-BSA-H2O2 systems is discussed. Both systems can effectively initiate radicals free radicals formation; their activity is similar to the previously studied ferritin-H2O2 system.