Lorenzo Braco

Improving lipase activity in solvent-free media by interfacial activation-based molecular bioimprinting

Abstract Nine lipases of mammalian, fungal and bacterial origin and two different model reactions, direct esterification and transesterification (alcoholysis), have been used to probe the potential in solvent-free media of the recently reported strategy of interfacial activation-based molecular (bio)imprinting (IAMI) [Mingarro et al., Proc. Natl. Acad. Sci. USA , 92 (1995) 3308]. The results demonstrate that the imprinting treatment permits nonaqueous rate accelerations which are lipase-dependent and span in some cases up to higher than two orders of magnitude. For several lipases, the method allows conversion yields after short reaction times (in either of the model reactions assayed) which are remarkably higher than those obtained with either the corresponding powdered commercial sample, the pH-optimized control lipase or even the lipase ‘protected’ by conventional means such as the use of lyoprotectants (sugars, polyols) or inorganic salts. Therefore, IAMI is proposed as a straightforward, convenient approach to improve lipase performance in more practical solvent-free-based applications.

Structural characterisation of the natural membrane-bound state of melittin: a fluorescence study of a dansylated analogue.

The binding of a dansylated analogue of melittin (DNC-melittin) to natural membranes is described. The cytolytic peptide from honey bee venom melittin was enzymatically labelled in its glutamine-25 with the fluorescent probe monodansylcadaverine using guinea pig liver transglutaminase. The labelled peptide was characterised functionally in cytolytic assays, and spectroscopically by circular dichroism and fluorescence. The behaviour of DNC-melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. We used resonance energy transfer to measure the state of aggregation of melittin on the membrane plane in synthetic and natural lipid bilayers. When bound to erythrocyte ghost membranes, the extent of energy transfer was found to be equivalent to when bound to small unilamellar vesicles of phosphatidylcholine. Our results correlate best with a proposed model in which the initial interaction between melittin and the red blood cells could be merely electrostatic and the peptide remains in a low alpha-helical conformation. The next step would be a peptide stabilisation in the membrane in a monomeric alpha-helical conformation that would imply the collapse of the membrane structure and liberation of the cell contents.

Time-dependent monomerization of gramicidin A, enhanced by phosphatidylcholine in non-polar solvents : A high-performance liquid chromatographic and spectrofluorometric study

Abstract The usefulness of size-exclusion high-performance liquid chromatography for the study of gramicidin A dimer—monomer conformational equilibrium in non

Biocatalysis and biorecognition in nonaqueous media. Some perspectives in analytical biochemistry

Biocatalysis and, to a lesser extent, biorecognition in non-aqueous media (including organic solvents as well as supercritical fluids and gases) constitute at present an exciting research area which has already demonstrated its biotechnological potential in numerous, varied applications. Less attention, however, has been paid to its analytical possibilities, even though many advantages have been postulated and a wide range of poorly water-soluble analytes are present in samples (or waste materials) from food and drink, petrochemical, pharmaceutical, military and other industries. The main approaches, developed in recent years to exploit the use of enzymes, antibodies or antibody mimics in water-restricted environments for analytical purposes, as well as possible future directions are briefly discussed.

New high-performance liquid chromatography-based methodology for monitoring the conformational transitions of self-associating hydrophobic peptides, incorporated into liposomes

A new high-performance size-exclusion chromatographic strategy is reported for the analysis of the hydrophobic self-associating peptide gramicidin A, incorporated into artificial phospholipid vesicles (liposomes). The method is based on the direct injection of a few microlitres of the gramicidin A-containing liposome suspension into the column, which is eluted with a non-polar solvent, such as tetrahydrofuran. The type and amount of information which can be derived from this methodology have been evaluated. Using this chromatographic approach, a correlation has been unambiguously shown to exist between the organization of the peptide in the vesicles and a number of variables involved in the method of preparation of liposomes. Finally, a gramicidin A conformational transition has been monitored in the phospholipid vesicles which proved to be dependent on the class of phospholipid present in the liposome.

Solution properties of polyelectrolytes : IV. Use of a new hydrophilic size-exclusion chromatographic packing for the separation of anionic and cationic polyions

Abstract The chromatographic behaviour of sodium polystyrenesulphonate in Ultra-hydrogel aqueous size-exclusion chromatographic packings was analysed under different experimental conditions. Three types of mobile phases (salt-free water, sodium nitrate solution and buffers of different pH) were investigated in order to characterize the elution mechanisms and to optimize the separation. Several parameters, such as sample concentration, injection volume, eluent pH and ionic strength were considered. Finally, a “universal” calibration graph was obtained under simple, mild optimized conditions of the mobile phase (0.2 M acetate buffer, pH 4.0), which is congruent for both polyanions and polycations and also for uncharged polymers.

Dimer-monomer conformational equilibrium of gramicidin A in 1-alkanols as studied by h.p.l.c. and fluorescence spectroscopy

Abstract High performance size-exclusion chromatograph (HPSEC) has been successfully used to characterized the dimer-monomer conformational equilibrium of gramicidin A (GA) in a series of 1-alkanols, from methanol to 1-pentanol. The chromatographic methodology proposed has allowed a rapid and accurate determination of kinetic and thermodynamic constants in the different alcohols assayed. The validity of this chromatographic approach has been tested by comparing the values of the kinetic and thermodynamic constants obtained with those reported in the literature deduced from spectroscopic techniques. Taking advantage of the chromatographic results, the differential fluorescent features of the monomer relative to the dimer have been investigated in terms of the quantum yields ratio of the individual species, as a function of solvent polarity. Finally, the possibility of applying this HPSEC approach to the study of other auto-associating polypeptides and of GA incorporated in liposomes is also considered.

HPLC Characterization of the Gramicidin A Dimer-Monomer Conformational Equilibrium in Ethanol and Study of the Effect of Calcium Ion

Abstract The usefulness of size-exclusion high-performance liquid chromatography for the study of gramicidin A (GA) dimer-monomer conformational equilibrium in polar organic solvents is demonstrated for the first time. The monome-rization process of GA in ethanol has been analyzed using an Ultrastyragel 1000 A column isocratically equilibrated with tetrahydrofuran, which has allowed the determination of kinetic and thermodynamic constants. Furthermore, the kinetics of interaction of Ca2+ with GA in ethanol has been followed using this methodology, and the binding mechanism has been investigated in terms of the poly-peptide dimer-monomer conformational equilibrium.

Solution properties of polyelectrolytes: VII. Non-ideal mechanisms in size-exclusion chromatography of synthetic polyions, peptides and proteins

Abstract Chromatographic data for sodium polystyrene sulphonate were obtained on both silica- and polymer-based size-exclusion supports using mobile phases of various pH and ionic strength. Deviations of the elution volume were observed towards both lower and higher values relative to the reference calibration graph obtained with uncharged standards. An empirical correlation is proposed in order to account for all the secondary effects observed. The general applicability of this correlation was further tested for chromatographic data obtained for a series of peptides and proteins on a silica-based support under very different eluent conditions. Deviations from ideal elution behaviour such as ion-exclusion and hydrophobic effects were analysed in the light of this approach.

High-performance liquid chromatographic separation of modified and native melittin following transglutaminase-mediated derivatization with a dansyl fluorescent probe

The 26-amino acid linear, amphiphilic peptide melittin was enzymatically modified with the fluorescent probe monodansylcadaverine using guinea pig liver transglutaminase and a fluorescent derivative of stoichiometry 1:1 was obtained. Reversed-phase and size-exclusion high-performance liquid chromatographic modes were tested in order to resolve the labelled peptide and native species. The influence of several operational variables was analysed and the elution conditions were optimized so that a satisfactory resolution could be achieved in both instances in a rapid, easy manner. Both chromatographic modes offer the possibility of accurate monitoring of the time course of the enzyme-mediated conversion and more interestingly, can be applied to the semi-preparative purification of the labelled peptide.