Juana Cabanes

Kojic Acid, a Cosmetic Skin Whitening Agent, is a Slow-binding Inhibitor of Catecholase Activity of Tyrosinase

Abstract— It was found that kojic acid, which is used in cosmetics for its excellent whitening effect, inhibits catecholase activity of tyrosinase in a non‐classical manner. A decrease in the initial velocity to a steady‐state inhibited velocity can be observed over a few minutes. This time‐dependence, which is unaltered by prior incubation of the enzyme with the inhibitor, is consistent with a first‐order transition. The kinetic data obtained correspond to those for a postulated mechanism that involves the rapid formation of an enzyme inhibitor complex that subsequently undergoes a relatively slow reversible reaction. Kinetic parameters characterizing this type of inhibition were evaluated by means of nonlinear regression of product accumulation curves.

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.

Characterization of catecholase and cresolase activities of monastrell grape polyphenol oxidase

Abstract The polyphenol oxidase of Monastrell grape was purified 126-fold by ammonium sulphate fractionation. The partially purified enzyme has both cresolase and catecholase activity. The latter activity has temperature and pH optima within the ranges 20–40° and 3.5–5.0, respectively. The apparent Km for 4-methyl catechol is 9 mM. Cresolase activity exhibits a lag period, which is modulated by different factors. An increase in enzyme concentration, temperature or pH (in a range 3.5–7.5) shortens the lag period. By contrast, an increase in the substrate concentration increases the lag period. The presence of o-diphenols in the reaction medium abolishes the lag period, by acting as co-substrates. The apparent Km towards p-cresol and the activation constant for o-diphenol (Kact) for cresolase activity are 0.5 mM and 1.6 μM, respectively.

Stabilization of the bioactive pigment of Opuntia fruits through maltodextrin encapsulation.

Betalains are water-soluble, nitrogen-containing pigments of growing interest in the food industry. They are present in most plants belonging to the order Caryophyllales, where they fulfill the role of anthocyanins, and are divided into two groups: violet betacyanins and yellow betaxanthins. They are bioactive molecules that account for health-promoting properties, recently described for cactus pears (Opuntia). In this work, the characteristic betalain of cactus pears, indicaxanthin, is obtained purely, and its stability is highly promoted by its encapsulation in a maltodextrin matrix. A suitable spray-drying procedure for encapsulation is described, and a bright yellow powder is obtained. The stability is analyzed under different conditions. In the absence of light, pure encapsulated pigment can be stored at 20 °C for months without appreciable loss of the bioactive substance and color variation. Furthermore, free radical scavenging and antioxidant properties of the pigment are studied under the ABTS(•+) radical and ferric reducing antioxidant power assays, in the presence and in the absence of maltodextrins. The stabilization of pure betalain pigments may boost the use of these bioactive and natural coloring molecules.

Chemical and enzymatic oxidation of 4-methylcatechol in the presence and absence of l-serine. Spectrophotometric determination of intermediates

A pathway for 4-methylcatechol oxidation by tyrosinase (monophenol, dihydroxy-L-phenylalanine:oxygen oxidoreductase, EC 1.14.18.1) in the presence of L-serine is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been performed. It has been possible to detect spectrophotometrically 4-methyl-o-benzoquinone as the first intermediate in this pathway after oxidizing 4-methylcatechol with epidermis tyrosinase or sodium periodate at pH 6.5. The steps for 4-methylcatechol transformation in the presence of L-serine could be: 4-methylcatechol----4-methyl-o-benzoquinone----5-methyl-4N-serine-catechol----5 - methyl-4N-serine-o-benzoquinone. Matrix analysis of the spectra obtained with a rapid-scan spectrophotometer verified that 4-methyl-o-benzoquinone was transformed into 5-methyl-4N-serine-o-benzoquinone at a constant ratio, the stoichiometry for this conversion being the equation: 2-(4-methyl-o-benzoquinone) + L-serine----4-methylcatechol + 5-methyl-4N-serine-o-benzoquinone.

L-mimosine, a slow-binding inhibitor of mushroom tyrosinase

Abstract It was found that L -mimosine is a slow-binding inhibitor of L -DOPA oxidation by mushroom tyrosinase. This inhibition is characterized by a prolonged transient phase. A mechanism is postulated according to the kinetic data.

A continuous spectrophotometric assay for phospholipase A2 activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).

Enzymatic oxidation by frog epidermis tyrosinase of 4-methylcatechol and p-cresol. Influence of L-serine

A study has been made of the kinetics of cresolase and catecholase activities of tyrosinase on the p-cresol/4-methyl-catechol pair in the presence of L-serine. For this, a spectrophotometer assay for both activities based on the spectrophotometric and stoichiometric characteristics of the chemical reactions in the evolution of 4-methyl-o-benzoquinone is proposed. The presence of L-serine in the reaction medium caused a modification in the lag period and the steady-state rate expressed during the cresolase activity of tyrosinase, but no modification was observed during the expression of catecholase activity. These results can be explained taking into account the complex mechanism proposed for tyrosinase which included the chemical steps involved in the process. Furthermore, a singular form of regulation of enzymatic activity by L-serine has been clarified, not by any direct interaction between the protein molecule and the nucleophile, but by modification of the chemical reactions involved in the mechanism of tyrosinase.

Evidence for a common regulation in the activation of a polyphenol oxidase by trypsin and sodium dodecyl sulfate.

Abstract Polyphenol oxidase (PPO) was extracted from beet root, in both soluble and membrane fractions, and in both cases the enzyme was in a latent state. PPO from the membrane fraction showed no diphenolase activity unless it was activated by trypsin or sodium dodecyl sulfate (SDS). The kinetics of the activation process of latent PPO by trypsin was studied and the specific rate constant of active PPO formation, k 3, showed a value of 0.03 s−1. The protease-activated form showed a pH optimum (6.5) and kinetic properties identical to those of the SDS-activated enzyme. Evidence is provided for the existence of a common peptide responsible for the regulation of the activity of the enzyme by both proteolysis and SDS detergent. Formation of the active proteolyzate was followed by spectroscopic measurements, Western blotting and partially denaturing SDS-PAGE.

Kinetic study of the suicide inactivation of latent polyphenoloxidase from iceberg lettuce (Lactuca sativa) induced by 4-tert-butylcatechol in the presence of SDS

In plants, polyphenoloxidase undergoes an irreversible inactivation during the oxidation of o-diphenol to o-quinones. In the present paper, using latent polyphenoloxidase from iceberg lettuce (Lactuca sativa), in the presence of an activating agent, SDS, the kinetic parameters that characterize the enzyme during its action on the suicide substrate 4-tert-butylcatechol have been determined. The effect of pH has also been considered. It was seen that the presence of SDS in the reaction medium changed the kinetic parameters of the enzyme during suicide inactivation, this phenomenon depended on the SDS concentration up to saturating concentrations. Variations were also observed in the kinetic parameters at pH values below 5 where SDS provoked inactivation of the enzyme. This differing kinetic behaviour during suicide inactivation in the presence of SDS may be caused by the conformational changes provoked by SDS in the enzyme in latent state. Thus, polyphenoloxidases showing suicide inactivation would present an enzymatic activity resulting from the balance between the activation process and suicide inactivation.