Roque Bru

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.

Phase Separation of Biomolecules in Polyoxyethylene Glycol Nonionic Detergents

The advantage of aqueous two-phase systems based on polyoxyethylene detergents over other liquid-liquid two-phase systems lies in their capacity to fractionate membrane proteins simply by heating the solution over a biocompatible range of temperatures (20 to 37 degrees C). This permits the peripheral membrane proteins to be effectively separated from the integral membrane proteins, which remain in the detergent-rich phase due to the interaction of their hydrophobic domains with detergent micelles. Since the first reports of this special characteristic of polyoxyethylene glycol detergents in 1981, numerous reports have consolidated this procedure as a fundamental technique in membrane biochemistry and molecular biology. As examples of their use in these two fields, this review summarizes the studies carried out on the topology, diversity, and anomalous behavior of transmembrane proteins on the distribution of glycosyl-phosphatidylinositol-anchored membrane proteins, and on a mechanism to describe the pH-induced translocation of viruses, bacterial endotoxins, and soluble cytoplasmic proteins related to membrane fusion. In addition, the phase separation capacity of these polyoxyethylene glycol detergents has been used to develop quick fractionation methods with high recoveries, on both a micro- and macroscale, and to speed up or increase the efficiency of bioanalytical assays.

Partial purification of a thylakoid-bound enzyme using temperature-induced phase partitioning.

Triton X-114 was used to partially purify broad bean polyphenol oxidase, a thylakoid membrane-bound enzyme, in latent form, free of phenolic compounds and chlorophylls, with a high recovery rate. The activation of the latent enzyme by detergents or trypsin was 10 times higher than that obtained when the enzyme was purified by other methods used in plant biochemistry, such as acetone powders and ammonium sulfate fractionation. The kinetic parameters of the latent and activated enzyme are also given.

Triton X-114 phase partitioning in plant protein purification

Abstract A brief overview is given of how Triton X-114 can be used not only to solubilize plant membranes but also as an excellent reagent in a bulk fractionation method to purify enzymes compared with the classical drastic methods using acetone powder or ammonium sulphate fractionation. Triton X-114 removes the tenacious phenols and chlorophylls on centrifugation. There is no need to use insoluble synthetic resins or organic solvents as Triton X-114 is so mild that the enzymes can be extracted in their natural form without activating them. The methods developed with Triton X-114 are easily reproducible and sufficiently cheap to be used in large-scale purification procedures. The classical topological use of Triton X-114 in plant membranes is also discussed.

Kinetic properties of polyphenoloxidase in organic solvents A study in Brij 96-cyclohexane reverse micelles

Polyphenoloxidase entrapped in Brij 96‐cyclohexame reverse micelles showed both cresolase activity towards monophenols and catecholase activity towards diphenols. The kinetic parameters, such as apparent K m, temperature effect and pH profile, were determined for catecholase activity, the findings being very close to those found in aqueous systems. The enzyme activity was dependent on the molecular ratio of water to surfactant (ωo), obtaining a maximum activity at ωo=10. The stability of the system with time increased with the water content until ωo=10, and then decreased. An inverse relation was found between activity and water volume fraction (θ) when ωo was held constant. In this situation V max was constant while K m varied proportionally with θ.

Triton X-114-aided purification of latent tyrosinase.

Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The phenols were reduced to 8% of the original content, avoiding pre- and post-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH and to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetically characterized, latent enzyme showed both monophenolase and diphenolase activities, the latter activity displaying an unexpected lag period before reaching the steady-state rate. This behaviour is characteristic of a hysteretic enzyme, and has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhibitor.

Aggregation of polyunsaturated fatty acids in the presence of cyclodextrins

Abstract The aggregation behaviour of the polyunsaturated fatty acids (PUFA) linoleic acid and arachidonic acid was studied in the presence of cyclodextrins (CDs). The influence of CD concentration of CMC of PUFA suggests that two CD molecules bind sequentially to one molecule of PUFA. Two equilibrium constants, K 1 representing the interaction of the first CD molecule, and K 2 , the interaction of the second, were determined by non-linear regression of the PUFA CMC versus CD concentration data to an expression deduced from the reaction scheme in the equilibrium. The effect of pH and the structure of the CD on the equilibrium constants was studied. It is postulated that the first CD molecule interacts with the carboxyl group of PUFA through hydrogen bonding when the fatty acid is protonated, while the second CD molecule binds to the hydrocarbon chain of the PUFA through hydrophobic interaction. The formation of hydrogen bonds was principally affected by the inner diameter of the CD, while the hydrophobic interactions were very strongly affected by the polarity of the CD group coating the inner channel. The relevance of the results for the development of enzyme assays involving fatty acids is discussed.

Kinetic characteristics of the enzymatic conversion in presence of cyclodextrins: study of the oxidation of polyunsaturated fatty acids by lipoxygenase.

The capability of cyclodextrins (CDs) to greatly enhance the solubility in water of poorly water-soluble substances makes them an ideal alternative system for studying the expression of enzyme activity with such substrates in aqueous solution. In order to evaluate the behaviour of the enzymes in presence of CDs a study of the lipoxygenase (LOX)-catalyzed oxidation of polyunsaturated fatty acids (PUFA) as model reaction has been carried out. This was done by using LOX from two different sources (soybean and potato tuber), at two pH values (6.3 and 9.0), with two substrates (linoleic acid and arachidonic acid) and three types of CD (beta-CD, methyl-beta-CD and monoglucosyl-beta-CD). PUFA have been shown to form inclusion complexes of 1:2 stoichiometry which are in equilibrium with free PUFA and free CD, thus complexation is governed by two equilibrium constants, K1 and K2 (J.M. López-Nicolás et al., Biochem. J. 308 (1995) 151-154; R. Bru et al., Colloids Surf. 97 (1995) 263-269). For the oxidation of PUFA by LOX in the presence of beta-CD we propose a model in which free PUFA is the only effective substrate, thus the oxidation of the complexed substrate requires the previous dissociation of the complex. The equilibrium constants of complex formation are determined by both a physico-chemical and an enzymatic method. In spite of giving quite similar results, the second was proven to be more accurate so it was employed in further studies. CD was shown to slow down the reaction rate of LOX, specifically due to the increase of Km, Vmax remaining unchanged. That apparent inhibition is due to removal of effective (free) substrate in the form of inclusion complexes. This sequestered substrate can, however, be converted since it is in equilibrium with the free. The feasibility of realizing a CD-mediated accurate control over the conversion rate is demonstrated in the experiment called cyclodextrin assay in which the concentration of the free substrate is calculated by using the equilibrium constants of complex formation and setting the initial concentrations of total substrate and total CD. From the observation of the reaction progress curves in the conditions of the CD assay, we have studied some characteristic parameters of the oxidation of PUFA by LOX in this new medium, such as enzymatic activity, duration of linear product accumulation and the lag phase.

Characterization of cholesterol oxidase activity in AOT-isooctane reverse micelles and its dependence on micelle size

SummaryCharacterization of cholesterol oxidase in AOT reverse micelles was performed. pH and temperature profiles show that the entrapment of the enzyme does not change its characteristics appreciably. The enzyme tends to behave as it does in water when micelle size increases and does not maximum rate at some intermediate micelle size. Km was 55–60 fold that in water

Trypsin-SBTI interaction in reverse micelles A slow intermicellar exchange-dependent binding

Solubilisate exchange between reverse micelles must take place before any reaction inside reverse micelles occurs if the reactants are confined to the aqueous micellar core. When the interacting species are 2 small molecules or one small molecule and one macromolecule, it has been shown that the exchange is faster than the typical turnover of an enzymatic reaction. The study of the interaction between 2 macromolecules (trypsin and soybean trypsin inhibitor) in reverse micelles carried out in this work reveals that the exchange between these macromolecule‐containing reverse micelles slows down by a thousand times and the limiting‐step in the exchange, the fusion, by 10* times. Both reverse micellar size (ωo = [water]/ [surfactant]) and temperature affected the rate of the fusion process. A hypothesis for the proposed active role of macromolecules in the exchange process is also given.