Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

Protein and DNA adsorption on dehydroxylated and aluminated silochromes]

A macroporous silica - silochrom - used for immobilization and purification of biopolymers was modified by dehydroxylation and alumination. The adsorption capacity of the modified silochroms for proteins and DNA is higher than that of the initial sorbent. The biopolymers adsorbed on aluminated silochrom are strongly attached to its surface.     

Chromatic fluorescence transitions in bean chloroplasts]

Chromatic fluorescence transients of isolated bean chloroplasts were investigated. In the presence of methylviologen we observed a decrease in the chloroplasts fluorescence under the influence of light which mainly excited the photosystem I. The effects of preliminary illumination with the white light and Mg2+ ions concentration on the fluorescence of chloroplasts were also studied.     

Vitamin B6 fluorescence in cells]

The band of cell fluorescence with the maximum of 395-400 nm is registered. This band is exposed on the phone of the tryptophane by wavelength excitation lambdaex=250-260 nm, and in pure scape by lambdaex=310-326 nm. Pyridoxin - substrate of vitamin B6 has identical parameters of spectra excitation and emission of neutral (pH 7) and acid (pH 2) solutions. After temperature damage of cells the intensity of this band increases.     

Kinetics of corneal fluorescence in experimental keratitis]

It was shown by the method of scanning photometry suggested by the authors that rabbits with experimentally-induced keratitis displayed a three-phasic reaction of the area of the corneal burn: that of the enhanced fluorescein sorption at the reactive phase: that of the loss of the sorption capacity in the dystrophic stage, and that of the secondary absorption intensification during the regenerative stage. The mentioned phasic changes can be used for the diagnosis and objective assessment of the clinical course of keratitis.     

Measuring the adsorption of Fatty acids to phospholipid vesicles by multiple fluorescence probes

Fatty acids (FA) are important nutrients that the body uses to regulate the storage and use of energy resources. The predominant mechanism by which long-chain fatty acids enter cells is still debated widely as it is unclear whether long-chain fatty acids require protein transporters to catalyze their transmembrane movement. We use stopped-flow fluorescence (millisecond time resolution) with three fluorescent probes to monitor different aspects of FA binding to phospholipid vesicles. In addition to acrylodan-labeled fatty acid binding protein, a probe that detects unbound FA in equilibrium with the lipid bilayer, and cis-parinaric acid, which detects the insertion of the FA acyl chain into the membrane, we introduce fluorescein-labeled phosphatidylethanolamine as a new probe to measure the binding of FA anions to the outer membrane leaflet. We combined these three approaches with measurement of intravesicular pH to show very fast FA binding and translocation in the same experiment. We validated quantitative predictions of our flip-flop model by measuring the number of H⁺ delivered across the membrane by a single dose of FA with the probe 6-methoxy-N-(3-sulfopropyl) quinolinium. These studies provide a framework and basis for evaluation of the potential roles of proteins in binding and transport of FA in biological membranes.     

Dynamic characteristics of the fluorescence of human peripheral blood neutrophilic polymorphonuclear leukocytes and lymphocytes intravitally fluorochrome-stained with acridine orange]

Neutrophil segmentonuclear leukocytes and lymphocytes of the human peripheral blood vitally stained with Acridine Orange (AO) in concentrations of 250 and 330 mcg/ml show different fluorescence dynamics. The number of neutrophil segmentonuclear granulocytes with green fluorescence of nuclei decreases, whereas the number of cells with red fluorescence of nuclei increases. As a criterion of this process, time T1/2 is taken during which the number of green-fluorescent cells decreases twofold. With AO concentrations of 250 and 330 mcg/ml, T1/2 is equal to 40 or 5 minutes, resp. The nuclei of lymphocytes within a 60 minutes observation show green fluorescence. This effect is likely to be due to structural-functional peculiarities of neutrophil segmentonuclear granulocytes and lymphocytes.     

Immunoaffinity purification and fluorescence studies of human adenosine deaminase

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.     

Quinacrine fluorescence of variant and abnormal human Y chromosomes

Quinacrine fluorescence has been used to examine variant and abnormal human Y chromosomes, at interphase and mitosis. The length difference in variant Y chromosomes has been shown to involve the highly fluorescent segment only. Eight abnormalities of the Y chromosome have been positively identified, two isochromosomes of the long arms of the Y, five pericentric inversions, and a dicentric Y chromosome with two long arms. Contributory evidence for two further abnormalities, a ring Y and a dicentric with two short arms, is also given.     

A novel method of analysis of ligand-polynucleotide adsorption isotherms]

The adsorption of ethidium bromide on the synthetic double-stranded polyribonucleotide poly(G)poly(C) was investigated by adsorption spectroscopy. The adsorption isotherms were analyzed in the framework of the Crothers-Gursky model. It was shown that adsorption parameters (e.g., binding constant) may also be determined from the analysis of dispersion of some bound molecules. In some cases this method may be more convenient.     

The fluorescence quenching resolved spectra and red-edge excitation fluorescence measurements of human alpha 1-proteinase inhibitor

The human alpha 1-proteinase inhibitor (alpha 1-PI) and its reactive site modified form (alpha 1-PI*) have been examined using the fluorescence quenching resolved spectra method. The red-edge excitation measurements were applied for the study of structural differences between these forms. The crystallographic data of alpha 1-PI* structure have shown that its polypeptide chain includes only two tryptophan residues. The fluorescence quenching data have indicated that the conversion of the intact inhibitor molecule into its nicked form is accompanied by changes in the tryptophan environments. The red-edge excitation measurements have proved that the dipolar relaxation process around the Trp-194 residue is much bigger in alpha 1-PI* form than in the nicked one.     

Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics.

The total internal reflection/fluorescence photobleaching recovery (TIR/FPR) technique (Thompson et al. 1981. Biophys. J. 33:435) is used to study adsorbed bovine serum albumin dynamics at a quartz glass/aqueous buffer interface. Adsorbed fluorescent labeled protein is bleached by a brief flash of the evanescent wave of a focused totally internally reflected laser beam. The rates of adsorption/desorption and surface diffusion determine the subsequent fluorescence recovery. The protein surface concentration is low enough to be proportional to the observed fluorescence and high enough to insure that the observed recovery rates arise mainly from adsorbed rather than bulk protein dynamics. The photobleaching recovery curves for rhodamine-labeled bovine serum albumin reveal both an irreversibly bound state and a multiplicity of reversibly bound states. The relative amount of reversible to irreversible adsorption increases with increasing bulk protein concentration. Since the adsorbed protein concentration appears to be too high to pack into a homogeneous surface monolayer, the wide range of desorption rates possibly results from multiple layers of protein on the surface. Comparison of the fluorescence recovery curves obtained with various focused laser beam widths suggests that some of the reversibly bound bovine serum albumin molecules can surface diffuse. Aside from their relevance to the surface chemistry of blood, these results demonstrate the feasibility of the TIR/FPR technique for measuring molecular dynamics on solid surfaces.     

Synchronous scan fluorescence spectroscopy of proteins and human eye lenses

Scanning both the excitation and emission monochromators synchronously while recording the fluorescence spectrum results in a considerable decrease in the apparent band width and shift in the peak position. We demonstrate the potential of this approach in the studies on proteins and their interactions as well as fluorophores in condensed media. We have chosen crystallins, eye lens proteins and human lenses. Synchronous scan spectra of alpha-, beta- and gamma-crystallins are clearly distinguishable and appear to provide specific signatures. The spectrum of the mixed solution could be simulated by the linear combination of components indicating that these proteins might not have any specific interaction in the dilute solutions. Synchronous spectra of the human lenses, both normal and cataractous, show several distinguishable features.     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.