Oxidation of benzidine and its derivatives by thyroid peroxidase.

Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3',5,5'-tetramethylbenzidine (TMB). The kinetic characteristics (k(cat) and K(m)) of benzidine (BD), 3,3'-dimethylbenzidine (o-tolidine), 3,3'-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25 degrees C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K(m) values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3', 5, and 5' positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k(cat)/K(m) was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.

Peroxidase-catalyzed co-oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of substituted phenols and their polydisulfides.

The steady-state kinetics of the horseradish peroxidase (HRP)-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) has been studied in the presence of 2-amino-4-nitrophenol (ANP), gallic acid (GA) or 4,4'-dihydroxydiphenylsulfone (DDS) and their polydisulfides poly(ADSNP), poly(DSGA), poly(DSDDS) at 20 degrees C in 10 mM phosphate buffer, pH 6.4, supplemented with 5-10% dimethylformamide. The second-order rate constants for the reactions of ANP, GA, poly(DSGA) and poly(DSDDS) with HRP-Compound I (k2) and Compound II (k3) have been determined at 25 degrees C in 10 mM phosphate buffer, pH 6.0 by stopped-flow spectrophotometry. ANP, GA and their polydisulfides strongly inhibited HRP-catalyzed TMB oxidation. Inhibition constants (Ki) and stoichiometric coefficients of inhibition (f) have been determined for these reactions. The most effective inhibitor was poly(DSGA) (Ki=1.3 microM, f=35.6). The oxidation of substrate pairs by HRP, i.e., TMB-DDS and TMB-poly(DSDDS) at pH 7.2 resulted in a approximately 8- and approximately 12-fold stimulation of TMB oxidation rates, respectively. The mechanisms of the HRP-catalyzed co-oxidation of TMB-phenol pairs are discussed.

Polydisulfides of substituted phenols as effective protectors of peroxidase against inactivation by ultrasonic cavitation.

Kinetics of inactivation of horseradish peroxidase (HP) induced by low-frequency ultrasonic (US) treatment (27 kHz) with the specific power of 60 W/cm2 were studied in phosphate (pH 7.4) and acetate (pH 5.2) buffers within the temperature range of 36.0 to 50.0 degrees C and characterized by effective first-order rate constants of US inactivation k(in)(us) in min(-1). Values of k(in)(us) depend on the specific ultrasonic power within the range of 20-60 W/cm2, on the concentration of HP, and on pH and temperature of the solutions. The activation energy of US inactivation of HP is 9.4 kcal/mole. Scavengers of HO* radicals, mannitol and dimethylformamide, significantly inhibit the US inactivation of HP at 36.0 degrees C, whereas micromolar concentrations of polydisulfide of gallic acid (poly(DSG)) and of poly(2-aminodisulfide-4-nitrophenol) (poly(ADSNP)) virtually completely suppress the US inactivation of peroxidase at the ultrasonic power of 60 W/cm2 on the sonication of the enzyme solutions for more than 1 h at pH 5.2. Various complexes of poly(DSG) with human serum albumin effectively protect HP against the US inactivation in phosphate buffer (pH 7.4). The findings unambiguously confirm a free radical mechanism of the US inactivation of HP in aqueous solutions. Polydisulfides of substituted phenols are very effective protectors of peroxidase against inactivation caused by US cavitation.

[Inactivation of the human thyroid peroxidase by ultrasound cavitation]. / Inaktivatsiia tireoid-peroksidazy cheloveka ultrazvukovoi kavitatsiei.

The inactivation kinetics of a human thyroid peroxidase protein fraction upon sonication (ultrasound frequency 27 kHz, power 60 W/cm2) of the enzyme solution in 15 mM phosphate buffer, pH 7.5, was studied. To quantitatively characterize the dependence of the slowest stage of the human thyroid peroxidase inactivation on temperature (36.0-50.4) degrees C, an effective constant of ultrasound inactivation rate Kin(US) was used. From the temperature dependence of Kin(US) at temperatures below 43 degrees C, the activation energy was estimated to be 8.11 kcal/mol. It was shown that the rate of human thyroid peroxidase inactivation strongly depends on the concentration of total protein in solution: the kin(US) value decreases more than sixfold in the protein concentration range from 0.2 to 0.8 mg/ml. It was also shown that poly(2-aminodisulfide-4-nitrophenol), its complexes with human serum albumin as well as the complexes human serum albumin--poly(gallic acid disulfide) substantially inhibit the ultrasound-induced inactivation of the enzyme and can be its effective stabilizers in the ultrasound cavitation field. This confirms the suggestion that active free radicals HO., O2.- and HO2. play a key role in the inactivation of human thyroid peroxidase. A general scheme of the inactivation of human thyroid peroxidase is proposed, which represents a chain of successive and parallel reversible and irreversible elementary steps.

Propyl gallate inhibition of iodide and tetramethylbenzidine oxidation catalyzed by human thyroid peroxidase.

The kinetic characteristics (kcat, Km, and their ratio) for oxidation of iodide (I-) at 25 degrees C in 0.2 M acetate buffer, pH 5.2, and tetramethylbenzidine (TMB) at 20 degrees C in 0.05 M phosphate buffer, pH 6.0, with 10% DMF catalyzed by human thyroid peroxidase (HTP) and horseradish peroxidase (HRP) were determined. The catalytic activity of HRP in I- oxidation was about 20-fold higher than that of HTP. The kcat/Km ratio reflecting HTP efficiency was 35-fold higher in TMB oxidation than that in I- oxidation. Propyl gallate (PG) effectively inhibited all four peroxidase processes and its effects were characterized in terms of inhibition constants Ki and the inhibitor stoichiometric coefficient f. For both peroxidases, inhibition of I- oxidation by PG was characterized by mixed-type inhibition; Ki for HTP was 0.93 microM at 25 degrees C. However, in the case of TMB oxidation the mixed-type inhibition by PG was observed only with HTP (Ki = 3.9 microM at 20 degrees C), whereas for HRP it acted as a competitive inhibitor (Ki = 42 microM at 20 degrees C). A general scheme of inhibition of iodide peroxidation containing both enzymatic and non-enzymatic stages is proposed and discussed.