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Biomedical subjects

M G Roig

Publications and source records attributed to M G Roig.

At least 19 recordsLinked to original sources
PubMed2001

Thermally induced conformational changes in horseradish peroxidase.

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme N -->k D where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Calorimetry↗
PubMed1997

Rheological studies on the solubilization by the surfactant SDS of complexes between three acidic polysaccharides and an organic base chloride.

Developments in the solubilization of complexes between the cationic polymer, poly(hexamethylenebiguanidinium chloride) (PHMBH+Cl-) and acidic polysaccharides are reported. It was discovered that the anionic detergent sodium dodecyl sulphate (SDS) is an excellent solubilizer for these complexes, enabling several multi-component systems to be studied. SDS itself was shown to interact with PHMBH+Cl- to form a highly viscous solution. Maximum viscosity was obtained with a SDS:PHMBH+Cl- molar ratio of 15.6. SDS:PHMBH+Cl- at this ratio served as a good solubilizer for the acidic polysaccharides (sodium alginate, sodium carboxymethyl cellulose, and xanthan), forming highly viscous fluids. The effect of temperature on the viscosity of these solutions was also examined.

Alkalies↗
PubMed1995

Comparative kinetic study of lipases A and B from Candida rugosa in the hydrolysis of lipid p-nitrophenyl esters in mixed micelles with Triton X-100.

(1) Lipases A and B from Candida rugosa catalyzing the hydrolysis of esters in micellar media have been characterized kinetically by studies on substrate specificity, rate equation forms and modeling of enzyme mechanisms. (2) The study on specificity revealed that both lipases are non-specific esterases with similar activity against lipid p-nitrophenyl esters micellized with Triton X-100. The slight difference was that lipase A has its maximum activity centered in the caprylate while that of lipase B is in the laurate. (3) Kinetic studies for both lipases were carried out with p-nitrophenyl laurate under three experimental conditions: (I) the molar fraction of substrate is fixed and the bulk concentration of substrate and Triton X-100 are varied; (II) the bulk concentration of substrate is held constant and the molar fraction of substrate and bulk concentration of Triton X-100 are varied; and (III) the bulk concentration of Triton X-100 is held constant but the bulk concentration of substrate and molar fraction of substrate are varied. (4) In case I, a similar Michaelis-Menten behaviour was observed with both lipases; the curve fitting gave kappcat/Kappm values of 3.0.10(5) and 5.6.10(5) s-1 M-1 for lipases A and B respectively. In case II, for both lipases the relationship between rate and the molar fraction of substrate required a fitting equation of 2:2 degree polynomial quotient. In case III, both lipases showed non-Michaelian behaviour with concave-up curves in the Eadie-Hofstee plot, a minimum degree of 2:2 in substrate concentration being detected for the rate equation. (5) The above results are interpreted in terms of the hypothesis that the mechanism of both lipases must include at least two different inputs for the molecule of substrate which would explain the quadratic terms observed in the rate equation.

Candida↗
PubMed1995

High-alkaline protease from Bacillus PB92 entrapped in calcium alginate gel. Physicochemical and microscopic studies.

High-alkaline protease (HAP) has been entrapped in Manugel DMB (an alginate gel) and assayed with two sizes and types of substrates: neutral protein casein and synthetic chromogenic tripeptide substrate, Z-Gly-Pro-Cit-PNA. Increasing the concentration of calcium chloride used for capsule formation decreased the measured enzyme activity with both substrates. Capsules were found to be stable in water for long periods of time, but they dissolved in both phosphate and carbonate-bicarbonate buffers. The pH vs activity profiles of encapsulated enzyme showed pH optima between 10 and 11 with both substrates. The calcium alginate matrix surrounding the enzyme was quite effective in stabilizing the enzyme at 20-25 degrees C and even more so at 4 degrees C. Enzyme stability at 50 degrees C was quite impressive, some enzyme activity being evident even after remaining for 1 wk at this temperature in water. Increasing concentrations of sodium dodecyl sulfate (SDS) were also found to inhibit the protease progressively, whereas a polyhexamethylene biguanidium chloride (PHMBH+Cl-) and SDS:PHMBH+Cl- combination showed the opposite effect. Optical microscopy, especially polarized light microscopy, provided a sensitive physical means of ascertaining some of the structural properties (sphericity, disorganization or organization, distinct layer enveloping the capsules, intensity of the maltese cross) of the capsules with and without enzyme before and after different chemical treatments and the presence or absence of the substrate.

Alginates↗
PubMed1995

Perspectives for biophysicochemical modifications of enzymes.

This article reviews the strategies and successes of modifying enzymes by means of biophysicochemical transformations. By judicious choice of methods, it has been possible to modify enzymes through physical interactions, chemical reactions and/or mutagenesis to alter a very wide range of properties ranging from stability and solubility on the one hand to catalytic activity and selectivity on the other.

Antibody Formation↗
PubMed1995

A model study on rate of degradation of L-ascorbic acid during processing using home-produced juice concentrates.

The rate of degradation of L-ascorbic acid in solution has been investigated under varying conditions, such as temperature, level of dissolved oxygen, pH, amino acids, sugars and processing conditions. Changes in pH between pH 1.5 and 7.0 accelerate L-ascorbic acid degradation. The most important factor that determines its stability is storage temperature, on which the rate of degradation of L-ascorbic acid is directly dependent. Similarly, the deleterious effect of variables such as oxygen and pH are influenced by temperature. Therefore, low temperature storage is imperative in order to regard L-ascorbic acid delay. A definite role of amino acids in L-ascorbic acid degradation has not been identified.

Amino Acids↗
PubMed1994

Immobilization of carbohydrases on epoxide-, isocyanate-, acid chloride-, and carboxylic acid-activated plastic supports.

Methods for the covalent immobilization of alpha-amylase and glucoamylase on to epoxide-, isocyanate-, acid chloride-, and carboxylic acid-activated plastic supports have been optimized. The effects of the following parameters have been examined: coupling time, pH, and temperature; availability of the free essential reacting groups on the supports; porosity of the supports; and enzyme loading. Properties of the immobilized enzymes, such as coupled activity and operational stability were assessed.

Acids↗
PubMed1993

Principles of biotechnological treatment of industrial wastes.

This review includes current information on biodegradation processes of pollutants, digestor biocenosis and bioadditives, sludge production, measurement of pollution, and advances regarding biotechnological treatment of a series of specific industrial effluents.

Biodegradation, Environmental↗
PubMed1993

Alpha-amylase immobilized on plastic supports: stabilities, pH and temperature profiles and kinetic parameters.

The covalent immobilization of alpha-amylase on new isocyanate, acid chloride and carboxylic acid--activated plastic supports shows the viability of such supports for immobilizing enzymes, especially those reacting with 1,6-diaminohexane and glutaraldehyde for producing side arms. The operational stability of immobilized alpha-amylase could be extended by crosslinking the enzyme or by extending the support's side arm (substrate concentration has no effect). Inactive immobilized alpha-amylase were unfolded and then refolded at elevated temperature, these supports were found to be essential in increasing the stability of the enzyme during refolding. The pH curves for the immobilized enzyme were in general found not to be shifted from the soluble enzyme's pH optimum, although one isocyanate plastic support derivative shifted the pH activity profile of alpha-amylase to a higher range by 1.5 pH units, probably due to reaction between the enzyme and the free anhydride groups existing on the support's surface. In all cases, the immobilized enzyme's temperature activity profiles were shifted to a lower temperature range when compared to the soluble enzyme. The immobilized alpha-amylase Michaelis constants increased and the the maximum rates and specific activities decreased when compared to the soluble enzyme kinetic parameters.

Bacillus subtilis↗
PubMed1992

A reversed-phase HPLC method for measurement of 5-hydroxymethyl furfuraldehyde and furfuraldehyde in processed juices.

An HPLC method using a reversed-phase macroreticular PLRP-S column and phosphate buffer as eluent is described for the analysis of L-ascorbic acid degradation products, 5-hydroxymethyl furfuraldehyde and furfuraldehyde, in processed fruit juices. Measurement of the levels of 5-HMF and furfuraldehyde in citrus juices against time showed the presence of 5-HMF (0.45 mg l-1) even at zero time. An assessment on the effect of the additives on the formation of 5-HMF of reconstituted single-strength orange juice showed virtually the same results for all the samples stored at 4 degrees C and 20 degrees C, irrespective of the additive. For citrus juice samples which had been subjected to accelerated degradation, those that showed the highest decomposition of L-ascorbic acid, produced the highest level of 5-HMF. The presence of furfuraldehyde in any of the samples was not detected, probably due to the fact that furfuraldehyde was formed in such small amounts which are below the minimum detectability limit of the method (0.050 mg l-1).

Acetates↗
PubMed1991

Physico-chemical characterization of the main factors affecting the mode of action of liver alcohol dehydrogenase immobilized on nylon tubing.

Alcohol dehydrogenase (ADH) from horse liver (EC 1.1.1.1), cross-linked through the bifunctional reactive glutaraldehyde, onto nylon tubing was immobilized (35 micrograms cm-2 internal surface of nylon tubing). ADH inactivation kinetics of the immobilized enzyme are of first order (t1/2 = 84.3 h, k = 8.2 x 10(-3) h-1 at 5 degrees C; t1/2 = 2.6 h, k = 0.26 h-1 at 50 degrees C). The activity versus pH profile points to a smaller effect of pH on the activity of the enzyme, which is the case of ADH in solution, explicable on the basis of limitations to proton diffusion towards/from the support. A limiting effect to free external diffusion of the substrate (products) towards/from the support was observed; this effect seems to determine the effective kinetic behaviour of immobilized ADH.

Alcohol Dehydrogenase↗
PubMed1991

In vitro interaction between psychotropic drugs and alcohol dehydrogenase activity.

A series of CNS-stimulating and -depressant drugs have been studied for their in vitro interaction with horse liver alcohol dehydrogenase (ADH) activity. The depressant drugs studied included barbital, phenobarbital, thiopental, nitrazepam, chlorpromazine, sulpiride, clomethiazole, Li2CO3, diazepam, phenytoin, ethosuximide, morphine, and codeine. The stimulant drugs were theophylline, caffeine, amphetamine, imipramine, chlorimipramine, amitriptyline, and tranylcypromine. The results were as follows. First, ADH activity was inhibited by the action of chlorpromazine, tranylcypromine, imipramine, chlorimipramine, amitriptyline, sulpiride, amphetamine, codeine, ethosuximide, morphine, clomethiazole, nitrazepam, Li2CO3, theophylline, and phenobarbital, in descending order of inhibitory effect. Second, inhibition followed by activation of ADH activity was observed for imipramine and chlorimipramine. Third, activation of ADH activity was observed for phenytoin. Finally, the following drugs were not seen to exert any effect on ADH activity: barbital, thiopental, diazepam, and caffeine.

Alcohol Dehydrogenase↗
PubMed1991

Kinetic aspects of human placental alkaline phosphatase enzyme membrane.

The crosslinking of alkaline phosphatase of human placenta with human serum albumin has been optimized. During the physico-chemical characterization of this immobilized biocatalyst, special attention was paid to attributes such as the irreversibility of the enzyme support bonding, the stability of the catalytic activity, and the effects of pH and temperature on this activity. Regarding stability, patterns of denaturation are proposed, to account for inactivation curves over time and under storage/operation conditions. These patterns, in some cases, indicate the existence of different populations of immobilized enzyme molecules, with a different degree of sensitivity to denaturation. The activity vs pH profiles are clearly modified by the immobilization process. This is because the pH of the free homogeneous solution, measurable with a pH-meter, differs from the real pH of the immediate microenvironment of the immobilized enzyme molecules due to the effects of proton accumulation in the microenvironment (in the reaction catalysed by alkaline phosphatase, protons are produced), to limitations to the free diffusion of H+ and to the possible partition effects of H+ due to polar interactions with residues or molecules of the enzyme membrane. In the experimental working conditions, the apparent optimum temperatures are centered at 40 degrees C, inactivation (thermal denaturation) occurring above this temperature. In the temperature range 10-40 degrees C, the kinetic control over the overall activity of the immobilized enzyme was observed, causing the Arrhenius profiles to be linear.

Alkaline Phosphatase↗
PubMed1990

Liver alcohol dehydrogenase immobilized on polyvinylidene difluoride.

A physical method for immobilization of liver alcohol dehydrogenase (ADH) by hydrophobic adsorption onto a supporting membrane of polyvinylidene difluoride (PVDF) was performed. Simultaneously, a physicochemical characterization of the immobilized enzyme regarding its kinetic behaviour was performed. The activity/pH profile observed points to an effect of pH on activity that is completely different from the case of ADH in solution. The disturbance in the typical bell-shaped profile owing to the fact that the enzyme was immobilized is explained on the basis of a potent limitation to the diffusion of the protons in the support. The findings of the present work also reveal the existence of an effect that limits free external diffusion of the substrate towards and/or the product from the support; this effect seems to be the determinant of the overall rate of the enzymatic reaction and is thus of great importance in the effective kinetic behaviour (v([S])) of immobilized ADH, whose kinetic behaviour is complex (non-Michaelian), as may be seen from the lack of linearity observed in the corresponding double reciprocal and Eadie-Hofstee plots. By non-linear regression numerical analysis of the v([S]) data and application of the F-test for model discrimination, the minimum rate equation necessary to describe the intrinsic kinetic behaviour of PVDF-immobilized ADH proved to be one of the polynomial quotient type of degree 2:2 (in substrate concentration).

Alcohol Dehydrogenase↗
PubMed1987

Conditions when statistical tests for model discrimination have high power. Some examples from pharmacokinetics, ligand binding, transient and steady-state enzyme kinetics.

The situation where data pairs xi, yi are actually generated by a true model, f(delta, x), but erroneously fitted by a deficient model, g(phi, x), is explored. A function, Q(delta), is described which is the average squared distance between f(delta, x) and the best-fit false model, g(phi, x). For a range of x covering 5-95% 'saturation' of f(delta, x), Q(delta) is calculated numerically for sums of exponentials, binding functions and rational functions. In each case, the region of delta when the second model in the series can be reliably differentiated from the first by statistical tests is described.

Enzymes↗
PubMed1982

Kinetic studies of the transphosphorylation reactions catalyzed by alkaline phosphatase from E. coli: hydrolysis of p-nitrophenyl phosphate and o-carboxyphenyl phosphate in presence of Tris.

1. Transphosphorylation of p-nitrophenyl phosphate and o-carboxyphenyl phosphate to Tris, has been studied at alkaline and acid pH. 2. The rate of release for all reactions products was Tris-dependent for both substrates, with a slight maximum for phenol at alkaline pH. These dependences have been analyzed from a mechanistic standpoint. 3. Individual constants of rate of a simple transphosphorylation mechanism have been determined. 4. At high Tris concentration (greater than 1.0 M) a slight competitive inhibition has been observed. 5. Inhibition in NH4+-NH3Cl buffer has been found at alkaline pH but not at acid pH. It would therefore seem that the non-protonated NH2 group of Tris is responsible for inhibition. 6. The results suggest the formation of complexes between Tris and the enzyme. Other possible alternatives are also analyzed.

Alkaline Phosphatase↗