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°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°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.     

Initiation and Inhibition of Free Radical Processes in H2O2–Metmyoglobin (Methemoglobin)–2,2"-Azino-bis-(3-Ethylbenzthiazoline-6-Sulfonic Acid) Systems

Rates of free radical initiation were determined at 20°C in 10 mM phosphate buffer (pH 7.4) in the systems metmyoglobin (methemoglobin)–H₂O₂ using 2,2"-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as the diammonium salt (ABTS). The catalytic activity of MetMb was 2-3-fold higher than that of MetHb. The process can be described by the Michaelis–Menten equation, from which effective values of K _m and V _max were calculated. Comparative kinetic studies on the inhibition of ABTS oxidation were carried out using Trolox, propylgallate (PG), polydisulfide of gallic acid (poly(DSG)), polydisulfide of (2-amino-4-nitrophenol) (poly(ADSNP)), and its conjugate with human serum albumin (HSA–poly(ADSNP)). The inhibitors were characterized by inhibition constants K _i and stoichiometric inhibition coefficients f (the number of radicals terminated by a single molecule of inhibitor). The minimum K _i and the maximum f values were obtained for poly(DSG), and in the system of MetHb–H₂O₂–ABTS they were 0.08 M and 27.5, respectively. According to their antiradical activities, the inhibitors can be arranged as follows: poly(DSG) > poly(ADSNP) > PG > Trolox. PG, poly(DSG), poly(ADSNP), and its conjugate with HSA are suggested as calibrators, i.e., inhibition standards for evaluation of antioxidant status of biological fluids in possible test systems based on the free radical-generating pair MetMb–H₂O₂ with ABTS as the acceptor of the active radicals.     

Intramolecular synergism of the inhibiting action of polydisulfide antioxidants in the system ferritin--H2O2--tetramethylbenzidine

Polydisulfides of urea (PDSU), thiourea (PDSTU), biuret (PDSB), and gallic acid (PDSG) and their monomer analogues (urea, biuret, and gallic acid) inhibited (in a competitive manner) tetramethylbenzidine (TMB) peroxidation catalyzed by ferritin in 0.1 M acetate buffer, pH 4.2, containing 10% dimethylformamide. Their efficiency characterized in terms of inhibition constants, Ki, increased in the following order PDSU < PDSB approximately PDSTU < PDSG. This order is determined by the reactivity of monomers with respect to HO* radicals which are the main oxidizing agents in the system ferritin--H2O2. Polydisulfide antioxidants exhibit the intramolecular synergism of the inhibiting action (non-additivity of antiradical activity relative to their monomers) that was quantitatively characterized by alpha = (Ki)pol/(Ki)mon x n, where n is the number of monomers in the polymeric inhibitors. The alpha values increased from 1.5 up to 5.18 in the following order: PDSG < PDSU < PDSB. Significantly higher inhibiting efficiency of polydisulfide antioxidants as compared to monomer forms and synergism of the inhibitory action offer promising opportunities of their use as quenchers of free radical processes in biochemical systems.     

Kinetics of Catalase Inactivation Induced by Ultrasonic Cavitation

Kinetic patterns of sonication-induced inactivation of bovine liver catalase (CAT) were studied in buffer solutions (pH 4.0–11.0) within the temperature range from 36 to 55^o. Solutions of CAT were exposed to LF (20.8 kHz) ultrasound (specific power, 48–62 W/cm²). The kinetics of CAT inactivation was characterized by effective first-order rate constants (s^–1) of total inactivation (k _in), thermal inactivation (*k _in), and ultrasonic inactivation (k _in(us)). In all cases, the following inequality was valid: k _in > *k _in. The value of k _in(us) increased with the ultrasound power (range, 48–62 W/cm²) and exhibited a strong dependence on the pH of the medium. On increasing initial concentration of CAT (0.4–4.0 nM), k _in(us) decreased. The three rate constants were minimum within the range pH 6.5–8.0; their values increased considerably at pH < 6.0 and pH > 9.0. At 36–55^o, the temperature dependence of k _in(us) was characterized by an activation energy (E _act) of 19.7 kcal/mol, whereas the value of E _act for CAT thermoinactivation was equal to 44.2 kcal/mol. Bovine and human serum albumins (BSA and HSA, respectively) inhibited sonication-induced CAT inactivation; complete prevention was observed at concentrations above 2.5 g/ml. Dimethyl formamide (DMFA), a scavenger of hydroxyl radicals (O ), prevented sonication-induced CAT inactivation at 10% (k _in and *k _in increased with the content of DMFA at concentrations in excess of 3%). The results obtained indicate that free radicals generated in the field of ultrasonic cavitation play a decisive role in the inactivation of CAT, which takes place when its solutions are exposed to low-frequency ultrasound. However, the efficiency of CAT inactivation by the radicals is determined by (1) the degree of association between the enzyme molecules in the reaction medium and (2) the composition thereof.     

Polyphenolic Antioxidants Efficiently Protect Urease from Inactivation by Ultrasonic Cavitation

Inactivation of urease (25 nM) in aqueous solutions (pH 5.0–6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm², 36–56°C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 W/cm², 36 or 56°C) has been characterized quantitatively, using first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(us), ultrasonic inactivation. Within the range from 1 nM to 10 M, propyl gallate (PG) decreases by approximately threefold the rate of LFUS-induced inactivation of urease (56°C), whereas resorcinol poly-2-disulfide stops this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36°C) or 10 nM (56°C). At 0.2–1.0 M, human serum albumin (HSA) increases the resistance of urease treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS–HSA), formed by conjugation of 1.0 nM GAPDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56°C).     

Activation of peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine with poly(salicylic acid 5-aminodisulfide)

5-Aminosalicylic acid (5-ASA) inhibited by a mixed mechanism the peroxidase catalyzed oxidation of tetramethylbenzidine (TMB) in 0.015 M phosphate-citrate buffer (pH 6.4) supplemented with 5% DMSO and 5% DMF. Poly(salicylic acid 5-aminodisulfide) (poly(SAADS)) in 0.01 M phosphate buffer (pH 6.2-7.4) supplemented with 5% DMSO and 5% DMF effectively activated the peroxidase-catalyzed oxidation of TMB. The activation was quantitatively characterized by coefficients (M^–1) determined at different pH values: increased linearly with increase in pH up to the maximal value of 2.44·10⁵ M^–1 at pH 7.0. The activating effect of poly(SAADS) on the peroxidase-catalyzed oxidation of TMB is explained by the activator properties of polyelectrolyte, with its anionic form interacting with peroxidase sites responsible for the acid-base catalysis.     

Initiation and inhibition of free-radical processes in biochemical peroxide systems: A review

The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase-and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Peroxidase-catalyzed co-oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (K _i, μmol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (α, M^?1), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).     

Activation of peroxidase-catalyzed oxidation of chromogenic substrates by tetrazole and its 5-substituted derivatives

Peroxidase-catalyzed oxidation of 2,2-azino-di(3-ethyl-benzthiazolydine-6-sulfonic acid) (ABTS) and 3,3,5,5-tetramethylbenzidine (TMB) is activated by tetrazole and its 5-substituted derivatives—5-amino-(AmT), 5-methyl-(MeT), 5-phenyl-(PhT), and 5-CF₃-(CF₃-T) tetrazoles. In phosphate-citrate or phosphate buffer (pH 6.4 or 7.2; 20°C), the activating effect of tetrazoles on TMB and ABTS oxidation decreased in the series AmT > MeT > T > PhT > CF₃-T and T > AmT > MeT > PhT, respectively. The coefficient (degree) of activation (), expressed in M^–1, determined for both substrates and all activators, depended on substrate type, buffer nature, and pH (it increased as pH increased from 6.4 to 7.2). For TMB oxidation, good correlation between log and the Hammet constants _meta for m-substituents in the benzene series NH₂, CH₃, C₆H₅, and CF₃ was found. It is suggested that AmT, MeT, and T can be used as activators of peroxidase-catalyzed oxidation of TMB and ABTS in enzyme immunoassay and designing peroxidase-based biosensors.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 148–157.Original Russian Text Copyright © 2005 by Karasyova, Gaponik, Metelitza.     

Ultrasonic and Thermal Inactivation of Catalases from Bovine Liver,the Methylotrophic Yeast Pichia pastoris,and the Fungus Penicillium piceum

The kinetics of inactivation of catalases from bovine liver (CAT), the fungus Penicillium piceum (CAT1), and the methylotrophic yeast Pichia pastoris (CAT2) was studied in phosphate buffer (pH 5.5 or 7.4) at 45 and 50°C or under the conditions of exposure to low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm²). The processes were characterized by effective first-order rate constants (s^?1): k _in (total inactivation), k ^*_in (thermal inactivation), and k ^*_in (us) (ultrasonic inactivation). The values of k _in and k ^*_in increased in the following order: CAT1 < CAT < CAT2. Circular dichroic spectra of the enzyme solutions were recorded in the course of inactivation by high temperatures (45 and 50°C ) and LFUS, and the contents of secondary structures were calculated. Processes of thermal and ultrasonic inactivation of catalases were associated with a decrease in the content of α helices and an increase in that of antiparallel β structures and irregular regions (CAT1 < CAT < CAT2). We conclude that the enzymes exhibit the following rank order of resistance: CAT1 > CAT > CAT2. Judging from the characteristics of CAT1, it appears to be an optimum component for antioxidant enzyme complexes.

     

Inactivation of Glucose-6-Phosphate Dehydrogenase in Solution by Low- and High-Frequency Ultrasound

We compared the kinetics of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) inactivation in 0.1 M phosphate buffer (pH 7.4) at 36–50° under conditions of exposure to low-frequency (LF, 27 kHz, 60 W/cm²) or high-frequency (HF, 880 kHz, 1.0 W/cm²) ultrasound (USD). The inactivation of G6PDH was characterized by effective first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(usd), ultrasonic inactivation. Dilution of the enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k _in > k _in ^*. The rate constants increased with temperature. The Arrhenius plots of the temperature dependences of k _in and k _in(usd) had an break point at 44°C. The activation energy ( _act) of the total inactivation of G6PDH was higher than _act for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: _act was higher in the case of inactivation with low-frequency ultrasound (LF-USD) than high-frequency ultrasound (HF-USD). The rate of the ultrasonic inactivation of this enzyme substantially decreased in the presence of low concentrations of HO^. radical scavengers (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in solutions exposed to USD.