Search

Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), and/or cofactor, NADP+, has been studied within the range 20-40 degrees C in three media: (a) 0.04 M NaOH-glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, kin (s-1). In the case of G6PDH-NADP+ complexes, the values of kin were independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of kin for the complex G6PDH-GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of kin corresponded to the degree of hydration W0 = 16.7; at W0 > 16.7 and W0 < 16.7, kin increased. Within the range 20-40 degrees C, the values of kin measured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of kin were characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6 degrees C to 40 degrees C. In all media studied, NADP+ complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH-NADP+ melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.

Inhibition of soybean urease by polymeric substrate analogues, urea and thiourea polydisulfides (PDSU and PDSTU, respectively), or three thiophosphoric acid amides (TPAA), tri-(N-3-hydroxyphenyl)thiophosphamide (1), tri-(N-4,4'-aminodiphenyl)thiophosphamide, and di-oxy-(N-alpha-piridyl)thiophosphamide (3) was studied in aqueous solutions at various pH values. The inhibitory effects of all these substances were reversible and competitive with the lowest inhibition constant Ki 2.8 microM for TPAA-1 at pH 3.85. Above and below this pH value, Ki increased reaching 24 [mu]M at pH 7.2. All test substances inhibited urease comparably with known inhibitors such as thiols (cysteamine, etc.) and hydroxamic acid derivatives, but were less efficient than phosphorodiamidates. Structural features of possible urease inhibitors of higher efficiency were proposed.

A comparative kinetic study on the poly(gallic acid disulfide) (poly(DSGA)) inhibition of the iodide ion oxidation and on the 2-hydroxy-3,5-di-tert-butyl-N-phenylaniline (butaminophene) inhibition of 3,3',5,5'-tetramethylbenzidine (TMB) oxidation involving human thyroid peroxidase (hTPO) and horseradish peroxidase (HRP) was performed. The inhibition processes were characterized with the inhibition constants Ki and stoichiometric inhibition coefficients f, indicating the number of radical particles perishing on one inhibitor molecule. In the case of poly(DSGA), the Ki values for the I- oxidation were 0.60 and 0.04 microM, and the coefficients f were 13.6 and 16.5 for hTPO and HRP, respectively, which evidences the regeneration and high effectiveness of the polymeric inhibitor. In the case of butaminophene, the Ki values for TMB oxidation were 38 and 46 microM for hTPO and HRP, respectively. The coefficients f were 1.33 and 1.47, respectively, to reveal that butaminophene does not regenerate. The inhibition mechanisms for I- and TMB oxidation involving the two peroxidases are discussed.

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.

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.

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.