Francisco García-Cánovas

An end-point method for estimation of the total antioxidant activity in plant material

The 2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase. This ABTS radical (ABTS ∞a ), a chromogen, is stable at room temperature but is unstable above 35°C and/or at pH values of above 7.5. Nevertheless, the most important factor in its stability is the ABTS/ABTS ∞a concentration ratio in the medium. The radical reacts with the antioxidant, L-ascorbic acid, with a high rate constant, the stoichiometry of the reaction being 1 mol of L-ascorbic acid per 2 mol of ABTS ∞a reduced. Based on these considerations, a spectrophotometric end-point method has been developed to evaluate L-ascorbic acid in aqueous media, and this represents an improvement over the lag-method previously reported. Under optimal conditions of temperature, pH and reagent concentration, the end-point method was capable of determining L-ascorbic acid with a limit of quantification of 0.38 nmol. In the assay described here, this ability is used to evaluate the total antioxidant activity of commercial citrus juices, in which ascorbic acid is a principal component. In our opinion this procedure can quickly provide useful information on the antioxidant content of foods and plant extracts. # 1998 John Wiley & Sons, Ltd. Phytochem. Anal. 9, 196‐202, 1998

A kinetic study on the suicide inactivation of peroxidase by hydrogen peroxide

In the absence of reductant substrates, and with excess H2O2, peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) shows the kinetic behaviour of a suicide inactivation, H2O2 being the suicide substrate. From the complex (compound I-H2O2), a competition is established between two catalytic pathways (the catalase pathway and the compound III-forming pathway), and the suicide inactivation pathway (formation of inactive enzyme). A kinetic analysis of this system allows us to obtain a value for the inactivation constant, ki = (3.92 +/- 0.06) x 10(-3) x s-1. Two partition ratios (r), defined as the number of turnovers given by one mol of enzyme before its inactivation, can be calculated: (a) one for the catalase pathway, rc = 449 +/- 47; (b) the other for the compound III-forming pathway, rCoIII = 2.00 +/- 0.07. Thus, the catalase activity of the enzyme and, also, the protective role of compound III against an H2O2-dependent peroxidase inactivation are both shown to be important.

Inactivation of peroxidase by hydrogen peroxide and its protection by a reductant agent

Hydrogen peroxide, the oxidant substrate of peroxidase, is also an inactivating agent of this enzyme. The reductant substrates protect the enzyme from the inactivating process. A reaction mechanism is proposed, in which two competitive routes exist for Compound I of peroxidase; one catalytic and one inactivating. The analytical solution produced at the end of the reaction supports the proposed mechanism and shows the dependence between the number of turnovers of the enzyme (r) and the ratio of both substrates.

Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2.

H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K(2) (affinity of the first intermediate, compound I, for H2O2) and k(3) (apparent rate constant controlling catalase activity) of 4.0 +/- 0.6 mM and 1.78 +/- 0.12 s(-1) respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation.

Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones.

Tyrosinase can act on monophenols because of the mixture of met- (E(m)) and oxy-tyrosinase (E(ox)) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D(0)](ss). Hence, the system reaches an initial pseudo-steady state when t-->0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D(f)](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, E(m)D, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E(ox) on monophenols, E(m)D, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D(f)](ss)) necessary for the final steady state to be reached after the lag period.

A kinetic study of the melanization pathway between L-tyrosine and dopachrome

In the pathway of melanin biosynthesis originating from L-tyrosine, the dopachrome accumulation at physiological pH is produced with a pronounced lag period, during which the level of L-dopa increases, following a sigmoidal kinetics to reach a steady-state. A kinetic model has been proposed for the overall pathway of melanization from L-tyrosine to dopachrome; it explains the lag period present during the dopachrome accumulation as well as the influence of L-tyrosine and tyrosinase over this lag period. Use of this model is also valid to explain the kinetics of L-dopa accumulation in the reaction medium, as has been tested by simulation.

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase.

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.

Mechanisms of compound I formation in heme peroxidases

The formation of compound I is the first step in the reaction mechanism of plant heme peroxidases. This intermediate stores two oxidizing equivalents from hydrogen peroxide as an oxyferryl iron center and a radical, either on the porphyrin ring or on a tryptophan residue. Site-directed mutagenesis has proved to be a most useful tool for the identification of the intermediates involved and the resulting nature of the compound I formed. Although there is no doubt that an acid-base mechanism operates in heme peroxidase during the formation of compound I, the roles of several distal pocket residues are currently the subject of intensive research. It is now generally accepted that the conserved distal histidine in the active site of heme peroxidases is the acid-base catalyst that promotes the heterolytic cleavage of hydrogen peroxide. Other residues, such as the distal arginine and asparagine, participate in a range of roles assisting catalysis by the distal histidine. Recent advances in the elucidation of the mechanism at the molecular level are discussed. Another aspect related to the nature of compound I is the location of the radical center. Novel radical species have been detected in the reactions of ascorbate peroxidase, lignin peroxidase and several mutants of horseradish peroxidase. Detailed kinetic and spectroscopic studies of these radical species have provided important insights about the factors that control porphyrin-protein radical exchange. The wide range of data being obtained on compound I will lead to an understanding of its vital function in peroxidase catalysis and the physiological roles played by these enzymes.

Kinetic study of the pathway of melanizationn between l-dopa and dopachrome

Abstract The first part of the melanization pathway from l -dopa to dopachrome has been studied as a system of various chemical reactions coupled by an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system. Rate constants for the implicated chemical steps at different pH and temperature values can be evaluated from measurement of the lag period arising from the accumulation of dopachrome that takes place when l -Dopa was oxidized at acid pH. The thermodynamic parameters of the chemical steps, the deprotonation of dopaquinone-H + into dopaquinone and the internal cyclization of dopaquinone into leukodopachrome, have been obtained. From the results presented, an alternative series of chemical reactions to the Raper-Mason scheme are proposed and discussed.

A comparative study of the purity, enzyme activity, and inactivation by hydrogen peroxide of commercially available horseradish peroxidase isoenzymes A and C

Horseradish peroxidase (HRP) is a commercially important enzyme that is available from a number of supply houses in a variety of grades of purity and isoenzymic combinations. The present article describes a comparative study made on nine HRP preparations. Six of these samples were predominantly composed of basic HRP, pl 8.5, and three of acidic HRP, pl 3.5. Two of the basic preparations were of lower purity than the others. The apparent molar catalytic activity of basic HRP with 0.5 mMABTS and 0.2 mM H2O2 was around 950 s−1 (about 770 s−1 for the less pure samples) and with a 5 mM guaiacol and 0.6 mM H2O2 was about 180 s−1 for all the samples. A similar value (approximately 1000 s−1) was observed for acidic HRP but only at higher concentrations of ABTS (20 mM). With 20 mM guaiacol the molar catalytic activity of the acid isoenzyme was 65 s−1. The apparent KM for ABTS of the acidic isoenzyme was 4 mM whereas for the basic isoenzyme it was 0.1 mM. All the enzymes were inactivated by H2O2 when it was supplied as the only substrate. Under these conditions the partition ratio (r = number of catalytic cycles given by the enzyme before its inactivation), apparent dissociation constant (Kl), and apparent rate constant of inactivation (kinact) were about twice as large for the acidic samples (1350, 2.6 mM, 9 · 10−3 s−1) as for the basic (650, 1.3 mM, 5 · 10−3 s−1). The apparent catalytic constant (kcat) was 3–4 times larger, and the efficiency of catalysis (kcat/Kl) was double for the acidic isoenzyme, but the efficiency of inactivation (kinact/Kl) was similar. The data obtained provide useful information for those using HRP isoenzymes for biotechnological applications (e.g., biosensors, bioreactors, or assays).