Francisco García-Carmona

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.

Characterization of the antiradical activity of betalains from Beta vulgaris L. roots

Betalains (betacyanins and betaxanthins), the main pigments of red beet (Beta vulgaris) roots, showed an antiradical effect when measured by the destruction of the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) free radical generated by the horse-radish peroxidase/hydrogen peroxide-mediated oxidation of ABTS. The antiradical activity of betacyanins was greater than that of the betaxanthins and increased with the pH of the reaction medium. The different antiradical properties shown by both types of betalain is discussed in light of the respective ease with which it is possible to withdraw one electron from their molecules and the stability of their corresponding radicals.

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.

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.

Biosynthesis of betalains: yellow and violet plant pigments

Betalains are the yellow and violet pigments that substitute anthocyanins in plants belonging to the order Caryophyllales. These pigments have attracted much attention because of their bioactivities, which range from an antioxidant capacity to the chemoprevention of cancer. However, the biosynthetic pathway of betalains remains under discussion; the main steps have been characterized in recent years, but multiple side reactions are possible. The key enzymes involved have only recently been described, providing clues about the regulation of betalain biosynthesis. In this review, we provide a comprehensive view of the biosynthetic scheme of betalains and discuss the different reactions that have been demonstrated experimentally or proposed in the literature.

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.

Evolution of ascorbic acid and peroxidase during industrial processing of broccoli

The ascorbic acid content and peroxidase activity of raw, canned and frozen (after blanching times of 60, 90, 120 and 150 s) broccoli florets and stems were determined. The ascorbic acid content represented 1.12 and 0.78 g kg−1 fresh weight in raw florets and stems respectively. After blanching (for different times) and freezing, the ascorbic acid content decreased to values of 0.55–0.56 g kg−1 fresh weight in florets and 0.35–0.36 g kg−1 fresh weight in stems. The industrial processing involved in canning decreased the ascorbic acid content to 0.18 g kg−1 fresh weight in florets. The peroxidase activity observed in the florets and stems of raw broccoli was 308.8 and 278.6 µmol min−1 per 100 g fresh weight respectively. The peroxidase activity remaining in frozen florets varied from 0.9 to 0.2%, while that in stems showed values of between 7.5 and 8.4%. These values for stems were within the range recommended for residual activity after blanching and freezing. The peroxidase activity of canned broccoli florets was practically undetectable. © 2000 Society of Chemical Industry

Structural implications on color, fluorescence, and antiradical activity in betalains

Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors on flowers and fruits of most plants of the order Caryophyllales. They are classified as betacyanins, exhibiting a violet coloration, and betaxanthins, which exhibit yellow coloration. Traditionally, betalains have been defined as condensation products of betalamic acid with different amines and amino acids, but the implication of the pigment structure for their properties has not been investigated. This paper explores different structural features of the betalains, revealing the clues for the switch from yellow to violet color, and the loss of fluorescence. A relevant series of 15 betalain-related compounds (both natural and novel semisynthetic ones) is obtained and characterized by chromatography, UV-vis spectrophotometry, fluorescence, and electrospray ionization mass spectroscopy. Antiradical properties of individual pure compounds in a broad pH range are studied under the ABTS•+ radical assay. Relevance of specific bonds is studied, and differences between betaxanthins and betacyanins are used to explore in depth the structure–antiradical activity relationships in betalains.

The effect of sodium dodecyl sulphate on polyphenol oxidase

Abstract Polyphenol oxidase (PPO) from Vicia faba is enzymically inactive in aqueous buffers at neutral pH, but is active at acidic pHs (pH 3–4). However, the presence of sodium dodecyl sulphate (SDS) in the reaction medium eliminates the activity at such acid pH and greatly increases the activity at neutral pH. This activation by SDS at various pHs is not dependent on the substrate. Cresolase and catecholase activities of the enzyme were activated by SDS at neutral pH. This activation is rapid and dependent on detergent concentration. The stability of the enzyme during relatively long treatments with SDS is also pH dependent. This relationship between proton and SDS concentrations is interpreted as a displacement of the sensitive p K s of the enzyme caused by the interaction with SDS molecules. This displacement depends on the binding of SDS to a specific centre, with a dissociation constant of 0.52 mM.

Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization

Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol oxidase activity. Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth. Significant levels of induction were only achieved when cholesterol emulsified with Tween 80. The presence of chloramphenicol at the time of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis. Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was quickly erased, while the activity of the particulate enzyme decreased sharply. The electrophoretic pattern on native Western blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa.