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Abstract

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.

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

“…Based on the results presented above and our previous work [1][2][3][4][5][6][7][8][9][10][11][12][13], we conclude that the HRP-catalyzed oxidation of TMB can be either inhibited or activated by the addition of phenolic compounds. The structure of the phenol determines whether inhibition or activation occurs.…”

“…The choice was also influenced by the high antioxidative efficiency of polydisulfides of substituted phenols and their successful use as bioantioxidants of lipid peroxidation in cranio-cerebral traumas and by phenolÕs ability to diminish free radical formation in biochemical systems [4][5][6][7][8][9][10][11][12].…”

“…We have previously shown that peroxidase-catalyzed co-oxidation of amines and phenols involves sequential and parallel enzymatic and non-enzymatic reactions [1][2][3][4][5][6][7][8][9][10][11][12][13]. The peroxidase-catalyzed oxidation of substrate pairs (i.e., 4-amino-antipyrine-phenols [1,2] and luminol-phenols [2,3]) is characterized by an enhanced rate of amine oxidation.…”

“…The peroxidase-catalyzed oxidation of substrate pairs (i.e., 4-amino-antipyrine-phenols [1,2] and luminol-phenols [2,3]) is characterized by an enhanced rate of amine oxidation. The co-oxidations of 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) with gallic acid (GA) [9], its polydisulfide poly(DSGA) [4,5,7], 2-amino-4-nitrophenol (ANP), and its polydisulfide (poly(ADSNP) [6,10], 1-amino-2-naphthol-4-sulfonic acid and its polydisulfide [11], 2,4-dinitrosoresorcinol and polydisulfide derivatives of resorcinol and 2,4-dinitrosoresorcinol [12] and propylgallate [8] are characterized by a pronounced inhibition of the rate of oxidation of the amine component. The degree of inhibition of TMB oxidation depends on the phenol type and is much higher for polydisulfides inhibitors compared to the corresponding monomeric forms [4][5][6][7][8][9][10][11][12].…”

“…(1) [14,15] Journal of Inorganic Biochemistry 98 (2004) [1][2][3][4][5][6][7][8][9] www.elsevier.com/locate/jinorgbio…”