Fluorescence analysis study of the lability of biomembranes and their components. III. Kinetics of complex formation between bovine serum albumin molecules and higher saturated fatty acids]

Kinetic peculiarities of the sorption of natural limited fatty acids on the molecules of bovine serum albumine (BSA) were studied by investigating fluorescent parameters of ionic (1-anilinonaphtalin-8-sulphonate-ANS) and neutral (N-phenyl-1-naphtylamine-PNA) probes. The following regularities were found: 1. The parameters which characterize the microsurroundings of both probes (quantum yield of fluorescence, the binding constant) did not change significantly during the sorption of the fattyn acids (laurinic, palmitinic and methyl ether of the stearinic acid). An exponential character of BSA fluorescent titration with fatty acids points to a competitive character of the relationship dye -- fatty acid for the binding sites in hydrophobic sacks of BSA. 2. The study of the character of the effect of solution ionic strength on the sorption of fatty acids showed that along with hydrophobic interactions the electrostatic interaction between carboxyl residues of fatty acids and charged protein groups also significantly contributed to this process. 3. Temperature relationship of AMS and PNA fluorescence intensity in the complex BSA -- laurinic acid correlates well with temperature relationship obtained from a pure protein system.     

Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.     

Microscopic studies of fatty acid vesicles

Microscopic vesicles enclosed by membranes formed entirely of oleic or linoleic acids (ufasomes) were studied by the freeze-etching and birefringence techniques. The results suggest the presence of one or more membranes around the particles, in which the fatty acid chains lie perpendicular to the surface. Comparison with results obtained with phospholipid liposomes shows that both types of particles are basically similar, although ufasomes have a less regular structure.     

Use of the fluorescent probe method to study the interaction of barbiturates with biological membranes]

The binding of the negatively charged fluorescence dye ANS and neutral dye NPN2 with lipid and erythrocyte membranes in the presence of barbiturates was studied. It was found that barbiturates decreased the amount of binding sites of ANS and NPN2 with membranes did not affect the quantum yield and the dissociation of the membrane-dye complex. It was shown that all barbiturates investigated were bound with the membranes in a neutral form.     

Molecular basis of chromosome banding

Silver and mercury ions are known to react with the bases of nucleic acids in solution. At low cation/base ratios Ag+ has an affinity for GC pairs in DNA, whereas Hg++ is preferentially bound to AT-rich nucleic acids. We have used fluorometry to measure the effect of these cations on the fluorescence intensity of preformed complexes of acranil and DNA in solution. The results are: 1) Ag+ enhances the fluorescence intensity presumably by affecting the dye intercalated in the vicinity of GC-pairs. 2) The addition of Hg++ leads to a quenching of the fluorescence intensity of the complex at low ion/base ratios, suggesting an effect on the dye molecules bound to AT pairs. At high GC-content of the nucleic acid, slight enhancement of the fluorescence intensity occurs with Hg++. 3) With both metals there is a correlation between base content of DNA and effect on the intensity of fluorescence indicating base specificity of the dye-polymer interaction.     

Fluorescence and binding properties of phenazine derivatives in complexes with polynucleotides of various base compositions and secondary structures

The interactions of two phenazine derivatives, one with a neutral chromophore (glycoside) and the other with a cationic one (quaternary salt), with various synthetic single- and double-stranded polynucleotides and natural DNA were studied by fluorescence techniques, conducting measurements of steady-state fluorescence intensity and polarization degree as well as fluorescence lifetime. These dyes show fluorescence quenching upon intercalation into the GC sequences of the double-stranded nucleic acids and an increase in fluorescence emission and lifetime upon incorporation into the AT and AU sequences. GC base pairs in continuous deoxynucleotide sequences were found to be preferred as binding sites for both phenazines, in contrast to AT base pairs. On the contrary, the continuous ribonucleotide GC sequence binds the phenazines more weakly than does the AU sequence. With regard to the interaction of the phenazines with single-stranded polynucleotides, a stacking interaction of the dye chromophores with the nucleic bases was observed. In that case the guanine residue quenches the cationic phenazine fluorescence, while the stacking interaction with the other bases results in an increase in the fluorescence quantum yield. Unlike the cationic dye, the fluorescence of the neutral phenazine was quenched by both purine bases.     

Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye

Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25 equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5–8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.     

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.     

FIT probes: peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.     

Absorption and fluorescence spectra of ring-substituted indole-3-acetic acids

The absorption and fluorescence spectra of indole-3-acetic acid (1), a plant growth regulator (auxin) and experimental cancer therapeutic, 29 ring-substituted derivatives and the 7-aza analogue (1H-pyrrolo[2,3b]pyridine-3-acetic acid) are compared. Two to four absorbance maxima in the 260-310-nm range are interpreted as overlapping vibronic lines of the 1La<--1A and 1Lb<--1A transitions. Two further maxima in the 200-230-nm region are assigned to the 1Ba<--1A and 1Bb<--1A transitions. 4- and 7-Fluoroindole-3-acetic acid exhibit blue shifts with respect to 1, most other derivatives show red shifts. All indole-3-acetic acids studied, with the exception of chloro-, bromo- and 4- or 7-fluoro-derivatives, fluoresce at 345-370 nm when excited at 275-280 nm. 7-Azaindole-3-acetic acid emits at 411 nm. The fluorescence quantum yield of 6-fluoroindole-3-acetic acid significantly exceeds that of 1 (0.3); the other derivatives have lower quantum yields. The plant-growth promoting activity of the ring-substituted indole-3-acetic acids studied correlates with the position of the 1Bb<--1A transition band.     

The interactions between the fluorescent dye thiazole orange and DNA

The interaction of the fluorescent dye thiazole orange (TO) with nucleic acids is characterized. It is found that TO binds with highest affinity to double-stranded (ds) DNA [log(K) ≈ 5.5 at 100 mM salt], about 5–10 times weaker to single-stranded polypurines, and further 10–1000 times weaker to single-stranded polypyrimidines. TO binds as a monomer to dsDNAs and poly(dA), both as a monomer and as a dimer to poly(dG) and mainly as a dimer to poly(dC) and poly(dT). The fluorescence quantum yield of TO free in solution is about 2 · 10⁻⁴, and it increases to about 0.1 when bound to dsDNA or to poly(dA), and to about 0.4 when bound to poly(dG). Estimated quantum yields of TO bound to poly(dC) and poly(dT) are about 0.06 and 0.01, respectively. The quantum yield of bound TO depends on temperature and decreases about threefold between 5 and 50°C. © 1998 John Wiley & Sons, Inc. Biopoly 46: 39–51, 1998     

Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry

The fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein-labeled alpha-glucosylated serum albumin to 3LL cells at 4 degrees C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37 degrees C in the presence of a fluorescein-labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4 degrees C in the presence of monensin, a proton/sodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4 degrees C, the high cell-associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.     

Detection of hybrid formation between peptide nucleic acids and DNA by fluorescence polarization in the presence of polylysine.

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9-13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.     

Characterization of the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II), ethidium bromide and nucleic acids.

The purpose of this study was to characterize the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II) (cis-DDP) and nucleic acids, in the presence of the intercalating compound ethidium bromide (EtBr). In these ternary complexes, some EtBr is tightly bound to the nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or thin layer chromatography at basic pH. These ternary complexes are formed with double stranded but not with single stranded nucleic acids. They are not formed if cis-DDP is replaced by transdiamminedichloroplatinum(II). The amount of tightly bound EtBr depends upon the sequence of the nucleic acid, being larger with poly (dG-dC).poly(dG-dC) than with poly(dG).poly(dC). Spectroscopic results support the hypothesis that the tight binding of the dye is due to the formation of a bidentate adduct (guanine-EtBr)cis-platin. The visible spectrum of the ternary complexes is blue-shifted as compared to that of EtBr intercalated between the base pairs of unplatinated DNA and it depends upon the conformation of the ternary complex. The fluorescence quantum yield of the ternary complexes is lower than that of free EtBr in water. Tightly bound EtBr stabilizes strongly the B form versus the Z form of the ternary complex poly(dG-dC)-Pt-EtBr and slows down the transition from the B form towards the Z form. The sequence specificity of cis-DDP binding to a DNA restriction fragment in the absence or presence of EtBr is mapped by means of the 3'----5' exonuclease activity of T4 DNA polymerase. In the absence of the dye, all the d(GpG) sites and all the d(ApG) sites but one in the sequence d(TpGpApGpC) are platinated. The d(GpA) sites are not platinated. In the presence of EtBr, some new sites are detected. These results might help to explain the synergism for drugs used in combination with cis-DDP and in the design of new chemotherapeutic agents.     

Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols.     

Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 1. Equilibrium studies.

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica are described. One class of saturable ethidium sites was blocked by alpha-bungarotoxin and therefore reflects direct binding to the receptor (Kd approximately 3 micrometers; stoichiometry--one ethidium site per two alpha-bungarotoxin sites). The second class of sites was nonsaturable and unaffected by alpha-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the dissociation constant and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anaesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in flourescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium appears not to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.     

Lipopeptidophosphoglycan from Trypanosoma cruzi. Amide and ester-linked fatty acids.

Lipopeptidophosphoglycan, extracted from whole cells of epimastigote forms of Trypanosoma cruzi, has now been shown to contain 12.6% of fatty acids in addition to the previously identified content of neutral sugars (60%), glucosamine (0.8%), peptide (9.5%) and acid-hydrolyzable phosphate (2%). The main fatty acids are palmitic (6.9%) and lignoceric (4.6%) acids. Stearic (0.55%), oleic (0.15%) and myristic (0.18%) acids were also found. One third of the fatty acids are bound in the lipopeptidophosphoglycan as esters (14 mmol%) and two thirds as amides (28 mmol%). Lignoceric acid was found to be bound only as amide. Two ninhydrin-positive compounds, obtained by chloroform extraction of a total acid hydrolysate of the lipopeptidophosphoglycan, were tentatively identified as sphingosine bases.     

Fluorescent molecular rotors: a new class of probes for tubulin structure and assembly

9-(Dicyanovinyl)julolidine (DCVJ) is a fluorescent dye whose intramolecular rotational relaxation is solvent dependent. Since its quantum yield increases with decreasing free volume, this molecule has been very useful in monitoring synthetic polymer reactions and measuring local microviscosity changes in phospholipid bilayers [Loutfy, R. O. (1986) Pure Appl. Chem. 58, 1239-1248; Kung, C. E., & Reed, J. K. (1986) Biochemistry 25, 6114-6121]. We have used DCVJ to follow the polymerization of tubulin, a protein that can assemble into a variety of polymorphic microstructures. DCVJ binding to free tubulin is accompanied by an increase in quantum yield, indicating that DCVJ has become partially immobilized. At 4 degrees C, DCVJ binds to a single population of high-affinity hydrophobic sites (Kd = 1.12 +/- 0.26 microM) with a stoichiometry that is protein concentration dependent. n, the number of moles of DCVJ bound per mole of alpha beta dimer, approaches 1 at concentrations less than or equal to 0.5 mg/mL but decreases to a lower limit of approximately 0.3 at concentrations greater than or equal to 2.0 mg/mL. The quantum yield also increases with increasing protein concentration. This trend is unaltered by the presence of microtubule-associated proteins. These results are analyzed in terms of a concentration-dependent oligomerization of tubulin at 4 degrees C. When tubulin is polymerized at 37 degrees C to microtubules or to sheets in the presence of Zn2+, the fluorescence intensity of DCVJ increases although the magnitude of this increase differs significantly. We are able to use the distinct fluorescent and binding characteristics of the bound dye to distinguish between these two polymorphs on a molecular level.     

Fluorescent membrane probes incorporating dipyrrometheneboron difluoride fluorophores.

The spectroscopic properties of a new series of fatty acid analogs in which a dipyrrometheneboron difluoride fluorophore forms a segment of the acyl methylene chain are presented and their characteristics as fluorescent membrane probes are examined. When incorporated as a low mole fraction component in model phospholipid membranes, the probes retain the principal characteristics of the parent fluorophore: green fluorescence emission with high quantum yield, extensive spectral overlap, and low environmental sensitivity. The fluorescence quantum yield is typically two to three times that of comparable membrane probes based on the nitrobenzoxadiazole fluorophore. The spectral overlap results in a calculated F?rster energy transfer radius (Ro) of about 57 A. Consequently, increasing fluorescence depolarization and quenching are observed as the mole fraction of the probe species incorporated in the membrane is increased. Low environmental sensitivity is manifested by retention of high quantum yield emission in aqueous dispersions of fatty acids. Partition coefficient data derived from fluorescence anisotropy measurements and iodide quenching experiments indicate that in the presence of fluid phase phospholipid bilayers the aqueous fraction of fatty acid is very small. Fluorescence intensity and anisotropy responses to phospholipid phase transitions are examined and found to be indicative of nonrandom fluorophore distribution in the gel phase. It is concluded that the spectroscopic properties of the fatty acid probes and their phospholipid derivatives are particularly suited to applications in fluorescence imaging of cellular lipid distribution and membrane level studies of lateral lipid segregation.     

Endogenous fatty acids are covalently and noncovalently bound to interphotoreceptor retinoid-binding protein in the monkey retina

Interphotoreceptor retinoid-binding protein (IRBP) purified from monkey interphotoreceptor matrix contains relatively high concentrations of endogenous fatty acids, 6.51 mol/mol of protein. Sixty-five percent of the total fatty acid bound to IRBP was found to be noncovalently attached, with the remainder covalently bound. The fatty acids are not residual components of phospholipids or neutral lipids, as judged by microchemical methods. The major fatty acids bound to IRBP are: palmitic (35%), stearic (21%), palmitoleic (7%), oleic (29%), linoleic (6%) and docosahexaenoic acids (2%). These fatty acids account for about 90% of the total fatty acid bound to interphotoreceptor matrix proteins extracted with organic solvents. Thus, IRBP may function as an intercellular fatty acid carrier and may depend on the covalently bound fatty acids for anchoring in the outer leaflet of cell membranes.