Carlos P. Sotomayor

Laurdan properties in glycosphingolipid-phospholipid mixtures: a comparative fluorescence and calorimetric study

Laurdan (6-dodecanoyl-2-dimethylamine-naphthalene) is a fluorescent membrane probe of recent characterization. It was shown that this probe discriminates between phase transitions, phase fluctuations and the coexistence of phase domains in phospholipid multilamellar aggregates. We measured the excitation and emission generalized polarization (GP(ex) and GP(em)) of Laurdan in aggregates of complex glycosphingolipids in their pure form and in mixtures with dipalmitoylphosphatidylcholine (DPPC). Our results show that Laurdan detects the broad main phase transition temperature of the neutral ceramide-tetrasaccharide Gg(4)Cer (asialo-G(M1)) and shows a value of GP(ex) in between that of DPPC and that of ganglioside G(M1). In contrast, Laurdan was unable to detect the thermotropic phase transition of G(M1). The probe also appears to be unable to detect phase coexistence in both types of pure glycolipid aggregates. Deconvolution of the excess heat capacity vs. temperature curves of pure Gg(4)Cer and DPPC/Gg(4)Cer mixtures indicates that the thermograms are composed by different transition components. For these cases, Laurdan detects only the high cooperativity component of the transition of the mixture. The peculiar behaviour of Laurdan in aggregates containing complex glycosphingolipids may result from the inherent topological features of the interface that are conferred by the bulky and highly hydrated polar head group of these lipids.

INTRAMOLECULAR TRYPTOPHAN HEME ENERGY TRANSFER IN HORSERADISH PEROXIDASE

Abstract— Chain folding of horseradish peroxidase allocates its sole tryptophanyl residue at a distance of 18 A from the active site heme group as determined by electronic energy transfer. This finding confirms that the phosphorescence spectrum observed in the peroxidase catalyzed oxidation of isobutyraldehydc is due to the excited triplet state acetone produced.

A Pyranine based Procedure for Evaluation of the Total Antioxidant Potential (TRAP) of Polyphenols: A Comparison with closely related Methodologies

A novel procedure for the evaluation of total reactive antioxidant potentials (TRAP) is described. The method is based on the measurement of the bleaching of pyranine by peroxyl radicals. The addition of the antioxidants produces a clear induction time whose magnitude is directly related to the antioxidant concentration. A comparison of the values obtained with those reported employing closely related methodologies shows that the results are significantly affected by the substrate employed to monitor the steady state free radical concentration. Possible sources of this dependence are discussed.

Membrane lipid diffusion and band 3 protein changes in human erythrocytes due to acute hypobaric hypoxia

Because it has been reported that hypoxia in rats may promote lipid peroxidation and other free radical reactions that could modify membrane lipids and proteins, the effect of acute hypobaric hypoxia on human erythrocyte membranes was investigated. 12-(1-Pyrene)dodecanoic acid fluorescent probe was used to assess short-range lateral diffusion status in the membrane bilayer. Membrane protein modification was detected by SDS-PAGE. Healthy young men were exposed for 20 min to the hypobaric hypoxia, simulating an altitude of 4,500 m. Under this condition, erythrocyte membrane lipids reached a state of higher lateral diffusivity with respect to normobaric conditions and membrane band 3 protein was modified, becoming more susceptible to membrane-bound proteinases. These observations suggest that acute hypobaric hypoxia may promote an oxidative stress condition in the erythrocyte membrane.

Effect of polyunsaturated fatty acid deficiency on dipole relaxation in the membrane interface of rat liver microsomes

The influence of a fat-free diet on the lipid dynamics of rat liver microsomes and liposomes of microsomal lipids was studied by using different fluorescence methods. Lifetime distribution and rotational diffusion of probes with different localization in the lipid bilayer were measured using multifrequency fluorometry. Lateral mobility was studied by measuring excimer formation of pyrenedodecanoic acid. Dipolar relaxation in the interfacial region was studied using 2-dimethyl-amino-6-lauroylnaphthalene (Laurdan). In spite of large changes in the fatty acid composition of microsomal lipids, polyunsaturated fatty acid deficiency showed no effect on the lifetime distribution and rotational mobility of 1,6-diphenyl-1,3,5-hexatriene (DPH). l-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 2- 7- and 12-(9-anthroiloxy)stearic acids. The treatment did not change the lateral diffusion of pyrenedodecanoic acid, either. However, generalized polarization of Laurdan fluorescence was higher in polyunsaturated fatty acid deficient microsomes as compared to the polyunsaturated fatty acid sufficient ones. This effect was also observed in liposomes of the total microsomal lipids, indicating that the changes in fatty acid composition resulting from polyunsaturated fatty acid deficiency produced a small but significant decrease in the rate of dipolar relaxation in the region of the lipid polar groups of the bilayer. The absence of lipid gel phase domains in rat liver microsomes was also indicated by Laurdan fluorescence features.

Main phase transition depression by incorporation of alkanols in DPPC vesicles in the gel state: Influence of the solute topology

Diphenylhexatriene is employed to sense the changes elicited in dipalmitoylphosphatidylcholine large unilamellar vesicles by the incorporation of several alkanols. From the dependence of the observed anisotropy values upon the surfactant concentration, pseudo partition constants of alkanols are obtained under conditions in which the vesicles are intermediate between the gel and liquid crystalline states. These data, together with previous data obtained at lower alkanol concentrations, allow an evaluation of the dependence of the alkanol incorporation on the solute concentration. The effect of the solute topology is investigated by employing linear and highly branched alkanols.

Dimethyl sulfoxide as chemical and biological probe: Conformational effect on peroxidase systems

Abstract The variation of the spectra and its reactivity towards 2-methylpropanal, indole-3-acetic acid and malonaldehyde of solutions of horseradish peroxidase in dimethyl sulfoxide-water mixtures has been studied. A broad pattern of changes was observed in the CD spectra of peroxidase, especially in the 400 nm region. These variations influenced strongly the excited triplet acetone emission from the 2-methylpropanal system which is generated in the active site of the enzyme protected from external quenching. This means that presumably the active site is more uncovered in the presence of dimethyl sulfoxide than the native form. Energy transfer parameters indicate that in fact there is a conformational effect produced by dimethyl sulfoxide in the horseradish peroxide active site. Dimethyl sulfoxide appears to be an important conformational probe in biochemistry.

STRUCTURAL EFFECTS OF VERAPAMIL ON CELL MEMBRANES AND MOLECULAR MODELS

ABSTRACT Verapamil is one of the frequently prescribed calcium channel blockers used in the treatment of hypertension and angina pectoris . Results of evaluations of the therapy have led to reports of toxic effects. This study presents several evidences that verapamil affects human cells. Scanning electron microscopy observations of intact human erythrocytes indicated that they underwent morphological alterations as increasing verapamil concentrations starting from 5 μM changed their discoid normal shape, and finally to hemolysis. Fluorescence spectroscopy on isolated unsealed human erythrocyte membranes confirmed these outcomes. In fact, the assays showed that verapamil induced a significant increase of the anisotropy parameters and a moderate one of the generalized polarization, indicative of enhanced order at the acyl chain and polar head regions of the erythrocyte membrane lipid bilayer. X-ray diffraction experiments on dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine bilayers, classes of the major phospholipids present in both outer and inner sides of the erythrocyte membrane, respectively showed that verapamil perturbed the polar head and acyl chain regions of both lipid bilayers. These interactions were found to be stronger with DMPC bilayers. On the other hand, human SH-SY5Y neuroblastoma cells incubated with verapamil suffered a sharp decrease of cell viability.

Effect of gramicidin addition upon the physicochemical properties of dipalmitoyl phosphatidyl choline large unilamellar vesicles

Abstract In this work, we report the results bearing on the effect of gramicidin incorporation upon the dynamic and structural properties of dipalmitoyl phosphatidyl choline (DPPC) large unilamellar vesicle (LUVs). Results were obtained at different depths in the bilayer, both in the bulk of the bilayer and the vicinity of the added pentadecapeptide. This analysis was performed employing a series of extrinsic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 6-dodecanoyl-2-dimethyl aminenaphthalene (Laurdan), pyrenedodecanoic acid, pyrene butyric acid, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (Py–C10–PC) and 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (bis-Py–C10–PC)). Fluorescence measurements were carried out after direct excitation of the probe or after its excitation by resonant energy transfer (RET) from the intrinsic tryptophan groups of the polypeptide. The last procedure allows an evaluation of the lipidic annulus properties in the vicinity of the incorporated polypeptide. In the gel state, gramicidin incorporation increases the order near the vesicle interface, as sensed by TMA-DPH. This increase is observed, above the phase transition temperature, over all the bilayer. This effect is probably related to a decrease in the lateral diffusion of the lipids, as evidenced by the reduced formation of intermolecular excimers following Py–C10–PC excitation. Gramicidin incorporation also increases the penetration and/or mobility of water molecules located near the bilayer interface. In the gel phase, these changes are more relevant in the central part of the bilayer. When the LUVs are in the liquid crystalline state, the effect is observed through all the bilayer. In this state, the polypeptide incorporation also increases the diffusion/concentration of oxygen, irrespective of the probe localization. Resonance energy transfer between Trp residues and Laurdan, Py–C10–PC or bis-Py–C10–PC dyes was employed to evaluate the properties of the lipidic annulus surrounding the polypeptide. In the gel phase, gramicidin incorporation produces an increase in the penetration and/or dynamics of water molecules, while in the liquid crystalline state it leads to a decrease of the acyl chain mobility in the deepest parts of the bilayer.

Transbilayer asymmetry of pyrene mobility in human spherocytic red cell membranes

The diffusion-dependent formation of pyrene excimers (excited dimers) was studied in normal and spherocytic red cell membranes. Pyrene emission was alternatively quenched in either bilayer half by non radiative energy transfer to haemoglobin. Pyrene excimer to monomer fluorescence intensity ratio, I/I, was 0.35 +/- 0.03 (S.E.) in washed red blood cells obtained from normal donors (n = 8) and 0.45 + 0.03 (n = 13) in the corresponding isolated, haemoglobin-free resealed membranes (P less than 0.02). In the spherocytic condition the respective values were 0.28 +/- 0.01 (n = 9) and 0.53 +/- 0.03 (n = 9), P less than 0.001. In contrast to the decrease of I/I in red cells as compared to isolated membranes, being 22% in normal cells and 47% in spherocytic ones, haemoglobin added to the exofacial side of isolated membranes, respectively, reduced I/I by 18% and 5%. In normal red cell membranes, pyrene mobility appears to be higher in the inner monolayer than in the outer one. In spherocytic membranes our results indicate an enhanced transmembrane asymmetry in lipid monolayer fluidity, probably due to a defect of the membrane protein skeleton organization.