Francisco Martínez-Ortiz

Tyrosinase action on monophenols: evidence for direct enzymatic release of o-diphenol

Using gas chromatography-mass spectrometry, the direct enzymatic release of o-diphenol (4-tert-butylcatechol) during the action of tyrosinase on a monophenol (4-tert-butylphenol) has been demonstrated for the first time in the literature. The findings confirm the previously proposed mechanism to explain the action of tyrosinase on monophenols (J.N. Rodríguez-López, J. Tudela, R. Varón, F. García-Carmona, F. García-Cánovas, J. Biol. Chem. 267 (1992)). Oxytyrosinase, the oxidized form of the enzyme with a peroxide group, is the only form capable of catalysing the transformation of monophenols into diphenols, giving rise to an enzyme-substrate complex in the process. The o-diphenol formed is then released from the enzyme-substrate complex or oxidized to the corresponding o-quinone. In order to detect the enzymatic release of o-diphenol, the non-enzymatic evolution of the o-quinone to generate o-diphenol by weak nucleophilic attack reactions and subsequent oxidation-reduction was blocked by the nucleophilic attack of an excess of cysteine. Furthermore, the addition of catalytic quantities of an auxiliary o-diphenol (e.g. catechol) considerably increases the accumulation of 4-tert-butylcatechol. The enzyme acting on 4-tert-butylphenol generates the enzyme-4-tert-butylcatechol complex and 4-tert-butylcatechol is then released (with k(-2)) generating mettyrosinase. The auxiliary o-diphenol added (catechol) and the 4-tert-butylcatechol generated by the enzyme then enter into competition. When [catechol] >> [4-tert-butylcatechol], the enzyme preferentially binds with the catechol to close the catalytic cycle, while 4-tert-butylcatechol is accumulated in the medium. In conclusion, we demonstrate that the enzyme produces 4-tert-butylcatechol from 4-tert-butylphenol, the concentration of which increases considerably in the presence of an auxiliary o-diphenol such as catechol.

Effect of pH on the oxidation pathway of dopamine catalyzed by tyrosinase

The oxidation of 3,4-dihydroxyphenylethylamine (dopamine) by O2 catalyzed by tyrosinase yields 4-(2-aminoethyl)-1, 2-benzoquinone (o-dopaminequinone), which evolves nonenzymatically through two branches or sequences of reactions, whose respective operations are determined by the pH of the medium. The cyclization branch of o-dopaminequinone takes place in the entire range of pH and is the only significant branch at pH greater than or equal to 6. The hydroxylation branch of o-dopaminequinone only operates significantly at pH less than 6, and involves the accumulation of 2,4,5-trihydroxyphenylethylamine (6-hydroxydopamine) and 5-(2-aminoethyl)-2-hydroxy-1,4-benzoquinone (p-topaminequinone), identified from cyclic voltammetry assays. The kinetic characterization of the hydroxylation branch of o-dopaminequinone has been carried out by spectrophotometric and oxymetric assays. The successful fitting of data to the kinetic behavior predicted by the kinetic analysis at both pH greater than or equal to 6 and pH less than 6 confirms the overall oxidation pathway proposed for the dopamine oxidation catalyzed by tyrosinase. The antitumoral power of dopamine is possibly enhanced by the high cytotoxicity of 6-hydroxydopamine and p-topaminequinone, accumulated at the acidic pH characteristic of melanosomes and melanome cells.

Catalytic oxidation of 2,4,5-trihydroxyphenylalanine by tyrosinase: identification and evolution of intermediates.

The oxidation of 3,4-dihydroxyphenylalanine (dopa) by O2 catalyzed by tyrosinase yields 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaquinone-H+). This evolves non-enzymatically through two branches (cyclization and/or hydroxylation), whose respective operations are determined by pH. The hydroxylation branch of o-dopaquinone-H+ only operates significantly at pH < or = 5.0 and involves the accumulation of 2,4,5-trihydroxyphenylalanine (topa), which has been detected by high-performance liquid chromatography (HPLC). This last compound is also a substrate of tyrosinase. The oxidation of topa by both tyrosinase and periodate yields 5-(2-carboxy-2-aminoethyl)-4-hydroxy-1,2-benzoquinone, with its amino group protonated (o-topaquinone-H+), which is red (RTQH) (lambda max 272-485 nm) at pH 7.0 and yellow (TTQH) (lambda max 265-390 nm) at pH 3.0. This is based on pKa 4.5 of the 2-OH group of the benzene ring of o-topaquinone-H+, as derived from spectrophotometric and HPLC assays. At physiological pH, RTQH undergoes deprotonation of the ammonium group of the side chain to yields RTQ, which cyclize into 2-carboxy-2,3-dihydroxyindolen-5,6-quinone (dopachrome), with a 1:1 stoichiometry and first-order kinetics. The evolution of RTQH has been analyzed by spectrophotometry, HPLC, cyclic voltammetry and constant potential electrolytic assays. From HPLC assays, the value of the first-order constant for the evolution of RTQH at pH 7.0 (kRTQHapp 4.83 x 10(-5) s-1), as well as of the rate constant for the cyclization step of RTQ (kRTQc 2.53 x 10(-3) s-1) were determined.

Tyrosinase-catalyzed hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: A kinetic study

Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone (HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex met-tyrosinase-HHQ, which can evolve in two different ways, forming deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone or on the other way generating met-tyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot close the enzyme catalytic cycle for the lack of reductant (HHQ). However, in the presence of hydrogen peroxide, met-tyrosinase (inactive on hydroquinone) is transformed into oxy-tyrosinase, which is active on HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into 2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which after binding to oxygen, originates oxy-tyrosinase. This enzymatic form is now capable of reacting with HQ to generate p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of HHQ during the action of tyrosinase on HQ is demonstrated by means of high performance liquid chromatography mass spectrometry (HPLC-MS) by using hydrogen peroxide and high ascorbic acid concentrations. We propose a kinetic mechanism for the tyrosinase oxidation of HQ which allows us the kinetic characterization of the process. A possible explanation of the cytotoxic effect of HQ is discussed.

Chronopotentiometry with non-linear perturbation functions at the DME with a preceding blank period

Abstract A theoretical study on the use of non-linear perturbation functions of the form ( I ( t )= I 0 t w (w≥0) after a blank period, t 1 , at the DME, is presented. Equations for the potential-time curves are derived and discussed. The general equation obtained here becomes Galvezs equation when t 1 →0 and also the generalized Sand equation when t 1 ⪢ t .

Cyclic chronopotentiometry with non-linear current-time functions at an SMDE. Amalgamation and reversibility effects and experimental verification

Abstract The general analytical equations corresponding to the potential-time response obtained for a charge transfer reaction in cyclic chronopotentiometry with power current-time functions at a static mercury drop electrode are presented. The superposition principle can be applied in this case in spite of the fact that the successive applied currents are non-linear functions of time. These theoretical equations have been tested experimentally in the following cases: (a) for reversible systems in order to quantify the amalgamation product effects; (b) for non-reversible systems pointing out that a given electrode process becomes more irreversible the lower the power of time in the applied current; (c) to analyse the influence of the electrode kinetics on the potential-time curves by using solutions of Cd 2+ with different concentrations of n -pentanol by determining kinetic parameters of tie charge transfer reaction. The sensitivity obtained in the characterisation of amalgamation processes is similar to that of voltammetric stripping techniques. Moreover, the analysis of the different cycles of the potential-time response allows us to obtain accurate values of kinetic parameters, as well as to detect possible complications in the charge transfer reaction.

Chronopotentiometry with non-linear perturbation functions at the DME with a preceding blank period: Electrode curvature effects and experimental verification

The effect of electrode curvature on E−t curves obtained in Chronopotentiometry with non-linear perturbation of the form I(itt) = I0tw(w ⩾ −12) after a blank period, t1, at the DME, is presented. The cases where the reduction product dissolves both in the electrolyte solution and in the electrode are considered. Concentration profiles have been obtained, and reversibility criteria and methods for determining kinetic parameters of the charge transfer reaction are proposed. The theory has been experimentally verified with Fe(C2O4)3−3/Fe(C2O4)4−3, Tl+/Tl(Hg), Cr3+/Cr2+ and Zn2+/Zn(Hg) using a linear currrent scan and a current step. The kinetic parameters of the two last systems have been determined.

General analytical solution for a reversible i-t response to a triple potential step at an SMDE in the absence/presence of amalgamation

The theory corresponding to triple potential step chronoamperometry for a reversible charge transfer reaction in a static mercury drop electrode is presented. The equation for the current has been deduced assuming different diffusion coefficients for oxidized and reduced species and is valid when the reaction product is soluble in both the electrolytic solution and the electrode. The validity of equations proposed was verified by comparing theoretical i-t, double differential pulse and reverse differential pulse curves with those obtained experimentally for the system Cd2+Cd(Hg). It can be concluded that the double differential pulse and reverse differential pulse techniques are more sensitive to the presence or absence of amalgamation than double pulse techniques.

New methods for the application of an alternating current. Part II : Spherical electrodes

Abstract A theoretical study of the application of a pure alternating current to spherical electrodes (DME and SMDE) is presented. The conditions under which the transition time of oxidized species is never reached are discussed. The method proposed in this work permits characterization of the degree of reversibility of the process and the null current potential from the E - t and also the i - E curves. This method has been applied to several systems, in good agreement with the experimental results.

Cyclic voltammetry at constant sphericity

Cyclic voltammetry has been applied to the study of a simple charge transfer reaction at a spherical electrode such as the static mercury drop electrode. Theoretical and experimental curves have been obtained at constant sphericity both for a reversible and slow electrode process, taking into account amalgam formation. The use of constant sphericity lends great advantages since, on the one hand it allows us to discriminate between kinetic and curvature effects, and on the other reduces the interference from uncompensated IR drop. Methods for evaluating heterogeneous kinetic parameters are also proposed.