Francisco Javier García Carmona

Kinetic study on the suicide inactivation of tyrosinase induced by catechol

Tyrosinase has a suicide inactivation reaction when it acts on omicron-diphenols. In the present paper, this reaction has been studied using a transient phase approach. Explicit equations of product vs. time have been developed for the multisubstrate mechanism of tyrosinase, and the kinetic parameters which characterize the enzyme acting on the suicide substrate catechol have been determined. The effect of pH has also been considered.

Kinetic studies on the interaction of phosphatidylcholine liposomes with Triton X-100

Sonicated unilamellar and large multilamellar liposome suspensions have been treated with the non-ionic detergent Triton X-100, and the subsequent changes in turbidity have been studied as a function of time. Sonicated liposome suspensions exhibit an increase in turbidity that takes place in two stages, a fast, low-amplitude one is completed in less than 100 ms, and a slow large-amplitude one occurs in 20-40 s. The first increase in turbidity is associated to detergent incorporation into the bilayer, and the second one, to vesicle fusion. The fast stage may be detected at all detergent concentrations, while the slow one is only seen above the critical micellar concentration of Triton X-100. Both processes may be interpreted in terms of first-order kinetics. Studies of the variation of kexp with lipid and detergent concentration suggest a complex multi-step mechanism. In the case of multilamellar liposomes, a fast increase in turbidity is also seen after detergent addition, which is followed by a slow (20-60 s) decrease in turbidity and a very slow (up to 12 h) large scale decrease in turbidity. These processes do not conform to single-exponential patterns. The fast stage is also thought to reflect surfactant incorporation, while the decrease in turbidity is interpreted as bilayer solubilization starting with the outer bilayer (slow stage) and proceeding through the remaining ones (very slow stage).

A new spectrophotometric method for the determination of cresolase activity of epidermis tyrosinase

A sensitive, rapid, quantitative method for the determination of the activities of the bifunctional frog epidermis enzyme, tyrosinase (E.C. 1.14.18.1), has been developed. It is a spectrophotometric method using p-coumaric acid and caffeic acid as substrates at pH 7.0. Lineweaver-Burk plots yielded straight lines and the initial velocities for the reactions were proportional to enzyme concentrations. It is simpler, faster, and more economical than radiometric methods and, furthermore, permits continuous monitoring.

A kinetic study of the one-electron oxidation of Trolox C by the hydroperoxidase activity of lipoxygenase

The oxidation of Trolox C (a vitamin E analog) by the hydroperoxidase activity of lipoxygenase was studied. Trolox C was oxidized to its corresponding phenoxyl radical in the presence of hydrogen peroxide, evolving through a ketodiene intermediate to the Trolox C quinone. The H2O2/Trolox C quinone molar ratio was 1.0. The overall reaction followed an enzymatic-chemical second-order system and involved a substrate regeneration mechanism. From the equations derived from this mechanism, the dismutation constant of the Trolox C radical was evaluated by non-linear regression as 4 x 10(5) M(-1) x s(-1). The accumulation curve of Trolox C quinone was found to be linear, with no lag period, and dependent on enzyme concentration. No phenoxyl radical was detected when the reaction was carried out in the presence of ascorbate. This synergistic reaction between the Trolox C radical and ascorbate was quantitative and depended on the respective concentrations of enzyme, Trolox C and hydrogen peroxide. The results presented in this paper suggest that the diferences observed in the kinetic behaviour of monophenols (one-electron donors) and diphenols (two-electron donors) stem from the fact that the latter evolve directly into ferric form without taking the slow pathway once the steady state is reached, whereas the monophenols are always forced take the slow way, even in the steady state. This peroxidative oxidation of a vitamin E analog by the hydroperoxidase activity of lipoxygenase together with the oxidation produced by dioxygenase activity suggests that lipoxygenase might be a key enzyme in destroying the lipophilic antioxidant barrier against the reactive oxygen species in membranes.

Characterization of a novel N-acetylneuraminate lyase from Staphylococcus carnosus TM300 and its application to N-acetylneuraminic acid production.

The possibility of incorporating N-acetylneuraminic acid (Neu5Ac) in infant formulas and other functional foods has opened up the need to synthesize N-acetylneuraminic acid using N-acetylneuraminate lyases (NALs) by reversible aldol condensation of pyruvate and N-acetyl-d-mannosamine. Until now, NALs have been cloned from pathogenic microorganisms; however, this Article describes the expression and characterization of an N-acetylneuraminate lyase from the Staphylococcus carnosus TM300, a GRAS microorganism used in fermented meat. ScNAL showed a high level of expression in E. coli (403 mg L(-1) culture). This, combined with its simple two-step purification procedure, the highest recovery described to date (86%), its kinetic parameters, which are in the same order of magnitude as best reported NALs, and its optimum pH and temperature, make ScNAL a promising and cheap biocatalyst. To confirm its biotechnological potential, the Neu5Ac was synthesized in 3 h in simple industrial working conditions with a high degree of conversion (94%).

Chromatographic analysis of lipoxygenase products

Abstract Lipoxygenases (LOXs) are non-heme iron-containing enzymes, widely distributed both in the plant and animal kingdom. They catalyse the regio- and stereospecific dioxygenation of polyunsaturated fatty acids (PUFAs) which contain a (1Z,4Z)-pentadiene system. Although, most LOXs catalyze the formation of one particular regioisomer, it has become apparent that several LOXs exhibit a dual or even multiple positional specificity. Hydroperoxides, the primary products of LOX, are short-lived and are transformed into various families of metabolites. In plants, hydroperoxides are metabolized via several secondary pathways to form bioactive compounds such as jasmonates. In animals, the oxidation of arachidonic acid (AA) by LOX is the source of highly active bioregulators such as leukotrienes and lipoxins. The ability of LOX products to initiate the synthesis of different signaling molecules is determined by the positional and stereospecific nature of the hydroperoxides produced. Thus, the complete characterization of LOX products is essential for establishing the physiological role of this enzyme. Different methods to determine the positional specificity of LOX products have been proposed. In this review, we will describe the different chromatographic methods (RP-, SP- and CP-HPLC, LC/MS and GC/MS) reported to date for analyzing the regio- and stereospecificity of the primary reaction products of LOX.

Effect of 7-Degree Rearfoot Varus and Valgus Wedging on Rearfoot Kinematics and Kinetics During the Stance Phase of Walking

BACKGROUND The scientific evidence behind the mechanical function of foot orthoses is still controversial. Research studies that have investigated the kinematic effect of foot orthoses on the lower extremity have shown variable results, with orthoses causing either no significant change or a small significant change in foot kinematics. METHODS The right limbs of 12 healthy asymptomatic individuals were studied in three walking conditions: barefoot, with a 7 degrees rearfoot varus wedge, and with a 7 degrees rearfoot valgus wedge. Kinematic and kinetic variables measured were the foot progression angle, the peak internal tibial rotation angle, and net ankle inversion moments during the stance phase in the three conditions. RESULTS There were statistically significant differences in the foot progression angle between the barefoot and varus wedge conditions and between the varus and valgus wedge conditions. There were no significant changes in peak internal tibial rotation among the three conditions tested. However, rearfoot varus wedges significantly reduced net ankle inversion moments compared with barefoot and rearfoot valgus wedges. CONCLUSIONS These results support the idea that foot orthoses work by methods other than by changing kinematic parameters. The present study supports the concept that foot orthoses work primarily by altering kinetics, with their effects on kinematics being secondary.

A proposed subungual exostosis clinical classification and treatment plan.

Subungual exostosis is a slow-growing, benign outgrowth of normal bone under the nail that affects the nail unit. The most common location in the foot is the dorsal surface of the distal phalanx of the big toe. Clinically, it can appear in combination with a variety of nail disorders, masking the underlying bone condition, which is frequently unrecognized or misdiagnosed. A new classification system for these lesions is proposed on the basis of the clinical signs and symptoms present during examination and the associated disorders of the nail plate. Also, a therapeutic algorithm that describes surgical approaches to the different presentations of this disorder is presented. (J Am Podiatr Med Assoc 99(6): 519-524, 2009)

Kinetic study of the transient phase of a chemical reaction system coupled to an enzymatically catalyzed step. Application to the oxidation of epinine by tyrosinase

The present work deals with epinine oxidation by mushroom tyrosinase and sodium metaperiodate. Intermediates produced within short reaction times were characterized by repetitive scanning spectrophotometry and the stoichiometry of the appearance of the respective aminochrome was established. The oxidation pathway from epinine to aminochrome had the following steps: epinine----o-quinone-H+----o-quinone----leukoaminochrome----aminoc hrome. The stoichiometry for the conversion of o-quinone-H+ into the aminochrome of epinine followed the equation: 2 o-quinone-H+----epinine+aminochrome. A transient phase kinetic study has been developed for the system of chemical reactions coupled to an enzymatically catalyzed step, these taking place when epinine is oxidized by mushroom tyrosinase. Rate constants for the implied chemical steps at different temperature and pH values were calculated from analysis of the progress curves of aminochrome accumulation with time. The thermodynamic activation parameters of the chemical steps were also calculated.

Molecular characterization of a new N-acetylneuraminate synthase (NeuB1) from Idiomarina loihiensis.

N-Acetylneuraminate lyase synthase (NeuB; E.C. 2.5.1.56) is a key enzyme in pathogenic microorganisms for producing N-acetylneuraminic acid through the irreversible condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). However, nothing is known about this enzyme in non-pathogenic bacteria. This paper describes, for the first time, one of the two putative N-acetylneuraminate synthases from the halophilic non-pathogenic gamma-proteobacterium Idiomarina loihiensis NeuB1 (IlNeuB1). The obtained 95-kDa dimeric enzyme showed maximal activity at pH 7.0 and 40°C and was more stable at pH 8.0 (8 h half-life) than the previously described NeuB. Its catalytic efficiency towards ManNAc and PEP was 10- and 40-fold higher, respectively, than that determined for Campylobacter jejuni NeuB, but only half that found for Neisseria meningitidis NeuB towards PEP. The phylogenetic and structural analyses of NeuB enzymes revealed the new domain architecture 4 has no cystathionine-β-synthase domain (cystathionine-β-synthetase domain), unlike domain architecture 3. In addition, 10 conserved blocks (I-X) were found, and surprisingly, this study showed that the arginine essential for catalysis that is present in antifreeze-like domain (block X) was not fully conserved in NeuB, but is replaced by a serine in a long sequence (>700 residues) NeuB, such as that existing in domain architectures 3 and 4.