0000000000004979
AUTHOR
Lorenzo Braco
Trapping of Different Lipase Conformers in Water-Restricted Environments
Based on a recently reported strategy to rationally activate lipolytic enzymes for use in nonaqueous media [Mingarro, I., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3308-3312], we compared the behavior in water-restricted environments of activated vs nonactivated forms of different lipases toward their natural substrates, triacylglycerols. To this end, nine lipases from varied origins (mammalian, fungal, and bacterial) were assayed using simple acidolyses as nonaqueous model reactions. The experimental results for several (though not all) lipases, discussed in the light of current structural and functional information, were collectively consistent with a model where, depending on the "…
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) whic…
Characterization of gramicidin A in an inverted micellar environment. A combined high-performance liquid chromatographic and spectroscopic study
We have investigated the conformational adaptability of gramicidin A incorporated into reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water, a so far unexplored "host" membrane-mimetic model system for this peptide. A high-performance liquid chromatographic strategy previously developed for the study of gramicidin in phospholipid vesicles and normal micelles [Bañó et al. (1989) FEBS Lett. 250, 67; Bañó et al. (1991) Biochemistry 30, 886] has been successfully extended to this system. The method has permitted the separation of peptide conformational species, namely, double-stranded dimers and monomers, and an accurate quantitation of their proportion in the invert…
Time-dependent monomerization of gramicidin A, enhanced by phosphatidylcholine in non-polar solvents
Abstract The usefulness of size-exclusion high-performance liquid chromatography for the study of gramicidin A dimer—monomer conformational equilibrium in non
Solution properties of polyelectrolytes
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 a…
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…
Structural characterisation of the natural membrane-bound state of melittin: a fluorescence study of a dansylated analogue
Abstract 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 bo…
HPLC in the characterisation of conformational species of linear gramicidins
Abstract High-performance size-exclusion chromatography (HPSEC) has proved to be a highly simplified and rapid procedure to investigate the conformational behaviour of gramicidin A inserted in different model membrane systems (lipid dispersions, liposomes, micelles) based on the separation of double-stranded dimers and monomers present in the lipid assemblies. The HPSEC approach has been extended to the characterisation of acylated analogs of gramicidin (palmitoyl- and oleoyl-derivatives) and a series of peptide analogs where tryptophan residues were chemically modified or replaced by less polar residues (phenylalanine or naphtylalanine) lacking H-bonding ability. The Chromatographic result…
Direct HPLC Monitoring of Lipase Activity in Reverse Micellar Media
Given the profusion of biotechnological applications of the nonaqueous use of lipases, we have evaluated the possibilities of exploiting the inherent advantages of high performance liquid chromatography (HPLC) for a simple, rapid assay of lipase activity in reverse micellar media, as a convenient alternative to previously reported spectroscopic methods, using both a model system and esterification reaction, and different commercial lipases. The results obtained after a screening for optimized chromatographic conditions in the reverse-phase mode indicate that a satisfactory resolution of the reaction components can be obtained following a straightforward protocol, which permits an accurate, …
Lipase-enhanced activity in flavour ester reactions by trapping enzyme conformers in the presence of interfaces
In order to improve the lipase-catalyzed synthesis of flavour esters, we have used the reported strategy of interfacial activation-based molecular (bio)imprinting [Mingarro et al. 1995. Proc. Natl. Acad. Sci. U.S.A. 92: 3308], later called trapping in the presence of amphiphile interfaces (TPI) [Mingarro et al. 1996. Biochemistry 35: 9935]. Five lipases of fungal and mammalian origin typically used for esterification process have been explored to improve production by TPI treatment. A marked enhancement of enzymatic activity has been observed in all TPI-treated lipases assayed and the activation factor obtained was up to 90-fold. The dependence on chain length of acyl donors in the esterifi…
Solution properties of polyelectrolytes. III. Effect of sodium polystyrene sulfonate concentration on viscometric and size exclusion chromatographic behavior at different ionic strengths
The effect of sodium polystyrene sulfonate concentration on the shape of its molecules in aqueous solution at different NaNO3 ionic strengths has been analyzed by viscometry and size-exclusion chromatography (s.e.c). An equation has been developed which predicts the intrinsic viscosity, [η] p.cp.cs, at finite concentration of both polyion (cp) and electrolyte (cs). The experimental results obtained by both techniques can be accounted for in terms of the theory. Several factors involved in the elution mechanism have been considered and the variation of the slopes of s.e.c. calibration curves with cp and cs has been discussed in terms of polyion conformation changes.
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 w…
Interfacial activation-based molecular bioimprinting of lipolytic enzymes
Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the presen…
A Study of Lipid-Lipid and Lipid-Polypeptide Interactions by High Performance Liquid Chromatography
Abstract Ternary systems containing phosphatidylcholine-cholesterol, phosphatidylcholine-gramicidin A or cholesterol-gramicidin A in tetrahydrofuran have been examined by high performance liquid chromatography. Preferential solvation of cholesterol and especially gramicidin A by phosphatidylcholine is observed. These results are interpreted in terms of hydrophobic interactions between membrane components.
Conformational transitions of gramicidin A in phospholipid model membranes. A high-performance liquid chromatography assessment.
We have investigated the conformation of gramicidin A reconstituted in different phospholipid environments, small unilamellar vesicles, extensive bilayers, and micelles by exploiting a recently proposed experimental approach based on high-performance liquid chromatography [Bano et al. (1988) J. Chromatogr. 458, 105; Bano et al. (1989) FEBS Lett. 250, 67]. The method allows the separation of conformational species of the peptide namely, antiparallel double-stranded (APDS) dimers and β 6.3 -helical monomers, and quantitation of their proportions in the lipid environment. Various experimental parameters (e.g., nature of organic solvent, time of incubation in organic solvent, lipid-to-peptide m…
Characterization of Acylating and Deacylating Activities of an Extracellular Phospholipase A2 in a Water-Restricted Environment
The behavior of porcine pancreatic phospholipase A2 (ppPLA2) in monophasic low-water media has been explored, for the first time, in a systematic manner. It has been investigated how a number of variables can modulate both acylating and deacylating activities of the enzyme, and several interesting, unexpected results are presented. Among the most relevant, when placing ppPLA2 in the water-restricted environment, are the following: (i) it displays a remarkable alteration of its specificity toward the substrate polar head relative to all-water medium; (ii) it is quite severely inhibited by lysophosphatidylcholine (LPC), which has important implications, particularly concerning its acylation a…
Solution properties of polyelectrolytes. II. Comparison of the effects of both polyion and eluent salt concentrations on the calibrations in aqueous exclusion chromatography
The elution behaviour of charged water-soluble polymers (sodium polystyrene sulphonate) in both salt-free and NaCl-containing aqueous mobile phases has been investigated by size-exclusion chromatography. A comparative analysis has been established between the results as a function of polyion and salt concentrations, which demonstrates that the chromatographic behaviour is similar in both cases. The variation of the slopes of the different concentration-dependent calibration curves is discussed in terms of polyelectrolyte conformational changes. Finally, a semi-empirical model has been developed to predict the dependence of the elution volume in either polyion or salt concentration, and its …
Phosphatidylcholine-Gramicidin a Interaction Study in Non-Aqueous Solvent with a new HPLC-SEC Column
Abstract A new HPLC-SEC column, Ultrastyragel 500 A, has been assayed in order to check the separation between gramicidin A and phosphatidylcholine in tetrahydrofuran. The good resolution of both compounds has allowed the quantitation of lipid-polypeptide interaction in terms of the binding ratio parameter, BR. A BR value of 3.6 moles of phosphatidylcholine per mole of gramicidin A has been obtained for a 0.1 % (w/v) phosphatidylcholine solution as eluent. The suitability of this support for studies on lipid-polypeptide interactions in non-aqueous solvents is demonstrated.
High-performance liquid chromatographic separation of modified and native melittin following transglutaminase-mediated derivatization with a dansyl fluorescent probe.
Abstract 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-m…
Solution properties of polyelectrolytes. I. Exclusion chromatography of sodium polystyrene sulphonate in salt-free water as eluent
The effect of concentration of sodium polystyrene sulphonate on the elution volume in exclusion chromatography has been investigated using salt-free water as eluent. A non-linear increase in the elution volume has been observed with increasing injected sample concentration. Concentration-dependent calibrations have been obtained which approach the universal calibration for uncharged polymers (dextrans) as polyelectrolyte concentration increases. The separation mechanisms are interpreted in terms of electrostatic and hydrodynamic interactions. A semi-empirical model is proposed to predict concentration effects of linear polyions in exclusion chromatography and good agreement has been found b…
HPLC demonstration that an all Trp--Phe replacement in gramicidin A results in a conformational rearrangement from beta-helical monomer to double-stranded dimer in model membranes.
We have taken advantage of our previously reported high performance liquid chromatographic (HPLC) strategy to investigate the conformational behavior of the optically reversed gramicidin M (gM-), an analog of gramicidin A with all tryptophans replaced by phenylalanines, in different model membranes. It is quantitatively demonstrated for the first time that once inserted in the lipid environment, gM- (unlike the native peptide) undergoes a conformational transition from beta-helical monomers to thermodynamically stable double-stranded dimers. This transition is faster the higher the incubation temperature and can be neatly observed in both small unilamellar phospholipid vesicles and lysophos…