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At least 289 records · Page 16Linked to original sources
PubMed1991

Changes of fluorescence lifetime and rotational correlation time of bovine serum albumin labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride in guanidine and thermal denaturations.

The nanosecond fluorescence depolarization method was applied to measure the fluorescence lifetime (tau) and the rotational correlation time (phi) of bovine serum albumin (BSA) labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-Cl). Changes of tau and phi of dansyl BSA in the guanidine denaturation and in the thermal denaturation were examined. In parallel, the secondary structural change of dansyl BSA was followed by circular dichroism measurements. The magnitude of tau was almost unchanged between 1 and 2M guanidine, where the secondary structure of the protein was predominantly disrupted; whereas that of phi began to increase before the disruption of secondary structure in the guanidine denaturation. In the thermal denaturation, in contrast, changes of both tau and phi occurred in a temperature range where the secondary structure was predominantly disrupted. The volume of equivalent sphere (Ve) and the axial ratio (rho) for the BSA were 3.6-3.8 x 10(-19) cm3 and 3.6 at 2 M guanidine as against 2.1 x 10(-19) cm3 and 2.2 in the absence of guanidine (25 degrees C), respectively. The magnitudes of Ve and rho were 4.9 x 10(-19) cm3 and 4.5 at 65 degrees C, respectively. Although the secondary structural change of dansyl BSA was irreversible in the thermal denaturation, Ve and rho were reversible.

Dansyl Compounds↗
PubMed1990

Fluorescence lifetime and rotational correlation time of bovine serum albumin-sodium dodecyl sulfate complex labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride: effect of disulfide bridges in the protein on these fluorescence parameters.

A fluorescent dye, 1-dimethylaminonaphthalene-5-sulfonyl chloride, was used to label bovine serum albumin (BSA), intact and disulfide bridges-cleaved. The fluorescence lifetime and fluorescence anisotropy of the adducts in sodium dodecyl sulfate (SDS) solutions were studied by the nanosecond fluorescence depolarization method. The volume of equivalent sphere (Ve) was calculated to be 2.1 x 10(-19) cm3 for BSA with the intact disulfide bridges from the rotational correlation time. The value of Ve was 4.4 x 10(-19) cm3 for the disulfide bridges-cleaved BSA. With an increase in SDS concentration, the rotational correlation time of the intact BSA became longer, while that of the disulfide bridges-cleaved BSA became shorter. This suggests that upon the binding of SDS, the total volume of the intact BSA increases while the expanded state of the protein, caused by the cleavage of the disulfide bridges, becomes compact.

Circular Dichroism↗
PubMed1992

Structural differences in active site-labeled thrombin complexes with hirudin isoinhibitors.

Hirudin, a 65 amino acid polypeptide from the medicinal leech, is the most potent thrombin inhibitor known to date. Recently, recombinant forms have been reported, which are as effective as the isolated forms. The studies presented here demonstrate sensitive spectroscopic methods for distinguishing binding of two recombinant hirudins, HV1 and HV2-Lys 47, with active site-labeled human alpha-, epsilon- and zeta-thrombins. Specifically, fluorosulfonylphenyl nitroxide spin labels, dansyl fluoride and p-nitrophenylanthranilate, were employed as active site-directed covalent reporter groups. In general, the nitroxide immobilization was greater for spin-labeled thrombin complexes with HV2-Lys 47 vs. HV1. The two fluorophore moieties, dansyl and anthraniloyl, were also sensitive to differences in HV1 and HV2-Lys 47 binding, including interactions with loop 145-150 of the thrombin structure where the epsilon- and zeta-thrombin cleavages exist. Speculation over sequence differences between the two isoinhibitors centers on residues 24 and 47, both of which involve either a loss or gain of charge on the side chain.

Amino Acid Sequence↗
PubMed1988

A screening method for the simultaneous determination of putrescine, cadaverine, histamine, spermidine and spermine in fish by means of high pressure liquid chromatography of their 5-dimethylaminonaphthalene-1-sulphonyl derivatives.

A simple and rapid method is described for the extraction, derivatization and subsequent determination by means of high pressure liquid chromatography of putrescine, cadaverine, histamine, spermidine and spermine as their 5-dimethylaminonaphthalene-1-sulphonyl derivatives. The amines are extracted from the fish material by an aqueous trichloroacetic acid solution and derivatized by means of 5-dimethylaminonaphthalene-1-sulphonylchloride (dansyl chloride). The reaction mixture is then injected on a RP-8 column using gradient elution to separate the amine derivatives. Use of the method results in a considerable saving with respect to both time and costs.

Animals↗
PubMed1986

[Determination of piperazine in hen eggs with HPLC following oral administration].

After oral administration of piperazine citrate to hens, this antihelmintic agent appeared unchanged in the eggs. After a therapeutic dose of ca. 0.9 g piperazine citrate per hen, a maximum level of 1.5 mg piperazine/kg egg was found two days after the application. The elimination half-life was 29 h and piperazine was found in the eggs up to 17 days after administration (limit of detection: 1 microgram/kg). Piperazine determination was performed by HPLC of the dansylderivative after clean-up by TLC.

Administration, Oral↗
PubMed1983

Endogenous ligand(s) decrease drug--protein binding in uremic sera: a fluorescence probe study.

Human serum albumin (HSA) was isolated and purified (greater than 97% purity) from normal sera, from sera of patients with severe chronic renal insufficiency and from sera to which a strongly protein bound acidic drug--clofibrinic acid--was added as a model ligand. The binding properties were evaluated using dansylglycine as a fluorescent probe. Data were analyzed according to Scatchard, the binding constants were calculated by least square approximation. The binding of dansylglycine to HSA from uremic sera was substantially decreased, reflected mainly by a lower product n1 . K1, as was the binding of dansylglycine to HSA from model sera containing clofibrinic acid. The binding was restored to almost normal when HSA was treated with charcoal. It is concluded that the impaired binding of many mostly acidic drugs to HSA in uremia is due to the presence of endogenous ligands. In addition a minor contribution by changes in HSA structure cannot be excluded.

Adult↗
PubMed1987

Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments.

The direct binding of Ca2+ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnCDANZ) was measured (25 degrees C) at three Mg2+ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca2+ to high- and low-affinity sites of troponin. The binding of Ca2+ to the high-affinity sites had a greater sensitivity to Mg2+ (KMg = 1 x 10(4)M-1) than the low-affinity sites (KMg = 1.2 x 10(3)M-1). The fluorescence change of thin filaments reconstituted with TnCDANZ was titrated with Ca2+ in the same solutions used for binding assays. The Ca2+-dependent fluorescence change had nearly the same sensitivity to Mg2+ (KMg = 9.4 x 10(2)M-1) as did Ca2+ binding to the low-affinity sites. The Ca2+ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca2+ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca2+ binding to the low-affinity sites of isolated TnCDANZ was measured (4 degrees C). From these results the binding of Ca2+ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca2+ affinity.

Actin Cytoskeleton↗
PubMed1990

Surface dielectric constant, surface hydrophobicity and membrane fusion.

Membrane fusion induced by ions and its associated membrane property, surface dielectric constant, were studied with the use of acidic and neutral phospholipid vesicles. The fusion of vesicles was monitored by utilizing two fluorescence fusion assays: fluorescence content mixing method and fluorescence labelled membrane component dilution method. For the surface dielectric constant measurements, a fluorescence method was used which detected the environmental effect on the membrane surface upon the addition of various fusogenic cations. Also, the effects of poly-(ethylene glycol) on both fusion and surface dielectric properties were examined. It was found that the extent of fusion correlated well with the degree of lowering in the dielectric constant of the surface membrane, which corresponds to the increase in hydrophobicity of the membrane surface. This agrees with the previously obtained experimental results that the increase in interfacial tension of the membrane, which also corresponds to the increase in surface hydrophobicity, correlates with the extent of membrane fusion.

Calcium↗
PubMed1987

Phospholipase C and the physical states of polar head groups of lipids.

Activity of phospholipase C from Clostridium perfringens on liposomes made from sn-3-phosphatidylcholine, dimyristoyl (DMPC), dipalmitoyl (DPPC) or distearoyl (DSPC) was measured at various temperatures and was correlated with their gel/liquid-crystalline phase transitions (Tc:23, 41.5, 52 degrees C for DMPC, DPPC, DSPC, respectively). In all cases, the activity of phospholipase C was high in the gel phases of the substrates and was almost zero in their liquid-crystalline phases. Fluorescence depolarization measurements of N-dansyl-sn-3-phosphatidylethanolamine (DPE) and 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the liposomes showed that both the head group and the alkyl chains of the lipids were immobilized in the gel phases but were highly mobile in the liquid-crystalline phase. These results indicate that the rotational mobility of lipids (both of the head groups and the alkyl chains) was not a major factor in the phospholipase C reaction. It is inferred that some electrostatic and/or hydrophobic interactions might play important roles in regulation of the phospholipase C activity.

Dansyl Compounds↗
PubMed1985

Local dielectric properties around polar region of lipid bilayer membranes.

Local dielectric constant was evaluated from the Stokes shifts of fluorescence spectra of L-alpha-dansylphosphatidylethanolamine (DPE) incorporated into liposomes made of synthetic phosphatidylcholine (dipalmitoyl or distearoyl) or bovine brain phosphatidylserine. The evaluation was established as follows. First, the Stokes shift of DPE was assured to follow Mataga-Lippert's equation and was a function of the dielectric constant and the refractive index in some standard organic solvents. Second, the change of the refractive index did not contribute much to the change in the Stokes shift. Third, the time resolved fluorescence depolarization of DPE in liposomes showed that the cone wobbling diffusion was rapid relative to the fluorescence lifetime and therefore that the dielectric relaxation did not affect the evaluation of the constant in the polar region of membranes. We then investigated the characteristics of the local dielectric constant in the polar region of the lipid bilayer and found that the dielectric constant varies between 4 and 34 depending upon calcium binding and also gel/liquid-crystal phase transition. Such large changes of the local dielectric constant were further correlated with the dynamic structure of lipid bilayer membranes measured by conventional fluorescence depolarization techniques. The large changes of dielectric constant around the polar region suggest that electrostatic interactions at this region can be altered 10-fold by such ionic or thermotropic factors and therefore that local dielectric properties can play crucial roles in membrane functions.

Dansyl Compounds↗
PubMed1984

Potential-dependent phase partitioning of fluorescent hydrophobic ions in phospholipid vesicles.

Fluorescent, dansyl derivatives of triphenylalkylphosphonium ions have been synthesized and exhibit fluorescence intensities in small sonicated phospholipid vesicles that are dependent upon transmembrane potentials. The voltage-dependent fluorescence changes are a result of changes in quantum yield that accompany a voltage-dependent phase partitioning of the probe. This phase partitioning is easily quantitated by calibrating the intensities of totally membrane-associated and aqueous probe. The voltage-dependence is well accounted for by a simple thermodynamic model and allows an estimation of potentials from fluorescence intensities in small vesicle systems.

Dansyl Compounds↗
PubMed1984

Membrane structure and the tenuously maintained resistance to staining with N epsilon-dansyl-L-lysine shown by many cells.

The ability to resist staining by N epsilon-dansyl-L-lysine is tenuously maintained in the majority of live nucleated cells taken from tissues concerned with immune function. Resistance is lost under a variety of nonphysiological conditions known to, or likely to, cause protein denaturation or aggregation. In contrast to that of dansyl-gamma-aminobutyrate, the fluorescence intensity of N epsilon-dansyl-L-lysine is only weakly enhanced by native proteins. This is further reduced on denaturation or aggregation of the proteins. It is unlikely, therefore, that cellular uptake of, and staining by, N epsilon-dansyl-L-lysine is a direct consequence of membrane protein denaturation/aggregation but may result from a decrease in protein-phospholipid interactions leading to formation of phospholipid domains. Previous work has indicated that such features are stained by N epsilon-dansyl-L-lysine (Humphries, G.M.K., Lovejoy, J.P., 1983, Biophys. J. 42:307-310; Humphries, G.M.K., Lovejoy, J.R., 1983, Biochem. Biophys. Res. Commun. 111:768-774). Although it appears likely that passage through a dansyl-lysine-staining state is a common, if not universal, prelude to cell death (as monitored by uptake of trypan blue), not all cells that lose resistance to dansyl-lysine staining are moribund. Resistance to staining is also lost by macrophages on binding to solid substrates and multivalent ligands. The possible physiological significance of this is discussed.

Animals↗
PubMed1984

Direct observation of enzyme substrate complexes by stopped-flow fluorescence: mathematical analyses.

The fluorescence changes which occur upon the interaction of enzyme and substrate under stopped-flow conditions can provide a sensitive means to directly observe ES complexes. The interconversion of the intermediates during catalysis causes changes in fluorescence, signaling directly their existence, and allowing their quantitation. We have studied extensively an approach which measures radiationless energy transfer (RET) between enzyme tryptophanyl residues and a fluorescent peptide or ester substrate. Our studies of a number of proteolytic enzymes have validated the approach, which is sensitive and applicable to a variety of enzymes under a wide range of experimental conditions, including subzero temperatures. Direct excitation of fluorescent substrates can also be used to observe ES complex formation and breakdown and is complementary to the RET approach. Here we review both the RET and direct excitation kinetic approaches, with particular emphasis on the mathematical foundations we have developed which are critical to the successful interpretation of these or any other spectroscopic approach which yields a signal that is unique to the ES complex.

Carboxypeptidases↗