J. M. Cachaza

Thermodynamics of the binding of salicylate to human serum albumin: evidence of non‐competition with imidazole

Abstract— The thermodynamic characteristics of the binding of salicylate to human serum albumin have been studied using a technique based on the variation of the quantum yield of fluorescence of salicylate when it binds to the protein. The binding constants, number of sites and the values of ΔG°, ΔH° and Δ° were determined. The results are consistent with a model that proposes two equal and independent types of binding site with a predominantly ionic interaction and an important hydrophobic contribution in one of the sites. The technique was also used to demonstrate that imidazole and salicylate (that can be found simultaneously in plasma following administration of imidazole‐2‐hydroxybenzoate) do not compete for the same binding sites on the protein.

Evaluation of the Main Factors Determining the Mode of Action of Immobilized Enzymes

Biotechnology and Applied Biology Section Evaluation of the Main Factors Determining the Mode of Action of Immobilized Enzymes* MANUEL G ROIG, FELIPE BELLO ESTEVEZ, FERNANDO GONZALEZ VELASCO and JUAN M CACHAZA Departamento de Fisicoquimica Aplicada y T~cnicas Instrumen tales Facuhad de Farmacia Universidad de Salamanca Salamanca, Spain Introduction Most enzymes, except for those of the digestive tract, exist in vivo associated with highly organized cellular material. Even the so-called soluble glycolytic enzymes are in fact immobilized either physically or chemically within the cytoplasmic matrix, i lowevcr, much further research is necessary to ehtcidatc the kinetics of the action of cnzymcs bound to solid supports, both natural and synthetic. Thc kinctic bchaviottr of an immobilized enzyme may differ from that of the frcc enzyme in several ways: (a) the interaction between the immobilized enzyme and the suhstratc takes place in

Biotechnology and applied biology section applications of immobilized enzymes

Recently developed techniques for immobilizing and modifying enzymes without destroying their activity can considerably increase their usefulness since it is possible to use and re-use them because of their increased stability and also preventing contamination of the product in the industrial processes involved. These immobilizing tech- niques facilitate analytical applications such as enzyme electrodes and medical applications such as the treatment of inborn errors of metabolism. The design of a plant in which heterogeneous biological catalysts are used requires thorough investigation of the process-engineering aspects of the supported enzyme system to be used. Up to the present, this kind of study has received little attention. A thorough evaluation of the mode of action and efficiency of the various kinds of enzymatic reactors (batch, stirred tank, packed bed, fluidized bed) should be carried out. The design and analysis of flow reactors of immobilized enzymes has been reviewed. The aim of an enzymatic reactor is to allow enzyme and substrate to come into contact for a sufficient period of time for reaction to take place; enzyme and product may then easily be separated. The systematics followed for the design of enzymatic reactors is the same as that employed for any chemical reactor.2 4 Both Atkinson ~ and Vieth et al I deal specifi- cally with immobilized enzyme reactors. The choice of one particular kind of reactor (Fig 1) depends on the characteristics of the process, both in the preliminary stages before the enzymatic reaction and in the later phases after it. Venkatasubramanian 6 has made a fairly detailed study of the parameters to be taken into consideration in the design of reactors for the enzymatic production of jellies with a high fructose content. Since this article is only a review, interested readers should consult the literature referring to the design and operation of reactors, such as enzyme kinetics 7 9 and mass trans- fer.] o-]3

Impact of drought on the ecological and chemical status of surface water and on the content of arsenic and fluoride pollutants of groundwater in the province of Salamanca (Western Spain)

The impact of drought on the ecological and chemical status of surface and groundwaters of the River Tormes (River Duero basin, northwestern Iberian Peninsula) was studied to evaluate the evolution of the quality of the river during its passage through the city of Salamanca (Spain). The Water Quality Index (WQI) of the river revealed that the drought period of 2005 did not significantly affect water chemical quality. However, during the study period differences were found in surface water ecological quality, using phytoplankton quality as AN indicator. These differences may be accentuated as a result of regulation of the River Tormes by the Santa Teresa reservoir. Arsenic and fluoride concentrations were measured in water wells, finding higher arsenic concentrations after the drought period and no correlation between the arsenic and fluoride contents. The results are useful for an overall understanding of potential impact of climate change on the ecological and chemical status of water in regional systems.

Human placental alkaline phosphatase covalently immobilized on a cross-flow microfiltration polyvinylidene difluoride membrane. Part I. Physicochemical effectors

Abstract Human placental alkaline phosphatase has been chemically immobilized on a hydrophilic cross-flow microfiltration membrane made of polyvinylidene difluoride (PVDF) chemically labelled with 1-carbonylimidazole groups. Physicochemical characterization of this immobilized biocatalyst focused especially on attributes such as the immobilization isotherms (Langmuir type) of the enzyme to the support, the stability of the catalytic activity, the effects of pH and temperature on this activity and the existence of limitations of external diffusion for H+, substrate and/or products. Regarding enzyme stability and its dependence on different experimental conditions, patterns of hysteresis or memory are proposed.

Solubility of diazepam and prazepam in aqueous non-ionic surfactants

The solubility of diazepam and prazepam in aqueous polyoxyethylen‐10‐dodecanol, polyoxyethylen‐23‐dodecanol and polyoxyethylen‐20‐hexadecanol, has been determined at 25.0°C. Diazepam seems to achieve a higher micellar penetration than prazepam, in spite of an expected smaller hydrophobic character. Thermodynamic interpretation of the micellar solutions is carried out using the regular solutions approach. A surfactant‐independent relation between solubilities of both drugs has been derived.

Human placental alkaline phosphatase covalently immobilised on a cross-flow microfiltration polyvinylidene difluoride membrane Part II. Activity vs. [substrate] kinetic behaviour

Abstract Human placental alkaline phosphatase was chemically immobilized on a hydrophilic cross-flow microfiltration membrane made of polyvinylidene difluoride (PVDF) chemically labelled with 1-carbonylimidazole groups and characterized physicochemically (in Part I, J. Mol. Catal . (Vol. 93, 85–104)) The intrinsic kinetic behaviour (rate vs. substrate concentration) in the absence of diffusional constraints was analysed graphically and numerically, by non-linear regression and use of the F statistical test for model discrimination, postulating a minimum rational rate equation of 2:2 degree in substrate concentration. According to the results found, a mechanism for the catalytic kinetic action has been postulated.

Kinetic Behavior of Native Alkaline Phosphatase from Human Placenta

The kinetic behavior of human placental alkaline phosphatase, which catalyses the hydrolysis of p-nitrophenyl and of o-carboxyphenyl phosphates, was studied by means of graphical and non-linear regression statistical fitting analysis of data of rate versus substrate concentration. Non linear Lineweaver-Burk and Eadie-Hofstee plots and rational functions of degree 2:2 (F-test assessing the goodness of fit) show non-Michaelian kinetic behavior. In the same way, the behavior of the enzyme was also non-Michaelian in the simultaneous presence of these two substrates.

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.