Biomedical subjects

D Toptygin

Publications and source records attributed to D Toptygin.

8 recordsLinked to original sources

PubMed1998

A nanosecond fluorescence study of the simultaneous influx of Ca2+ and Cd2+ into liposomes.

Nanosecond fluorescence decay characteristics of the calcium-binding probe Quin2 and two of its cation complexes were examined by time-resolved fluorescence spectroscopy. Binding of Ca2+ and Cd2+ resulted in fluorescence lifetime enhancements as compared to that of free Quin2 ('tau' = 0.9 ns). The Quin2-Ca2+ complex displays a monoexponential decay of tau = 7.4 ns, while the cadmium complex gives an average decay time of ca. 4 ns. Lifetime measurements made on heterogeneous cationic solutions demonstrate that decay times for individual complexes can be retrieved. Time-resolved measurements were used to monitor the kinetics of ionomycin-mediated calcium and cadmium transport across artificial membranes. Fluorescence decays, collected on the time-scale of second, were sufficient to measure individual ion fluxes or those of mixtures into liposomes. The combination of steady-state and time-resolved fluorescence techniques offers the unique advantage of simultaneously detecting other cations in the presence of calcium.

Aminoquinolines↗

PubMed1998

Fluorescence study of the multiple binding equilibria of the galactose repressor.

A fluorescence assay has been developed to study the multiple linked equilibria which function in regulation of the Escherichia coli galactose operon. Fluorescein 5-isothiocyanate was attached to Amino-Modifier C6dT at different positions in an oligonucleotide containing the sequence for the OE site of the galactose operon. These fluorescently labeled oligonucleotides were used to study OEDNA-GalR-d-galactose interactions. The data were analyzed and fit to various models including the classical competitive binding model as well as models involving the formation of a ternary DNA-repressor-inducer complex. Examination of the reduced chi-square of the various fits and comparison of fitted parameters with those obtained in independent experiments were used to distinguish different models. Since the ternary complex is likely to exist under physiological conditions, our results suggest that obligatory dissociation of GalR from DNA may not be required for induction of the gal operon. Rather, induction may involve the formation of a ternary complex of OEDNA with GalR and D-galactose with a different conformation than the GalR-OEDNA binary repressor complex.

Amino Acid Sequence↗

PubMed1998

The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II and a solution-based assay for the binding of a biantennary glycopeptide.

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299-303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (lambda[ex] = 295 nm, lambda[em] = 350 nm) decay is complex and can be described by four decay times with the following values: tau1 = 7.4 nsec, alpha1 = 0.22; tau2 = 2.9 nsec, alpha2 = 0.25: tau3 = 1.0 nsec, alpha3 = 0.34, tau4 = 0.2 nsec, alpha4 = 0.18. The addition of a biantennary glycopeptide (carbohydrate sequence [see text]) to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8 x 10(5) M(-1). The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.

Carbohydrate Sequence↗

PubMed1996

Steady-state and time-resolved fluorescence measurements for studying molecular interactions: interaction of a calcium-binding probe with proteins.

The binding of 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)-methyl] 6-methoxy-8-bis[carboxymethyl] aminoquinoline, the fluorescent calcium probe Quin2, to serum albumin and several other proteins has been investigated. Changes in fluorescence emission spectra and fluorescence anisotropy revealed interactions between Quin2 and several proteins including human serum albumin, bovine serum albumin, aldolase, phosphoglucose isomerase, glyceraldehyde-3-phosphate dehydrogenase, and alkaline phosphatase. Protein-probe interactions were inhibited by the presence of calcium. Binding was also measured by resonance energy transfer and gel permeation chromatography. Equilibrium binding constants for Quin2 were quantitated by the application of the recently-developed "SPECTRABIND' program to spectroscopic data (D. Toptygin and L. Brand, Anal. Biochem., 224 (1995) 330-338). Binding of Quin2 to human serum albumin is discussed in terms of the published X-ray crystal structure of human serum albumin (X.M. He and D.C. Carter, Nature, 358 (1992) 209-215).

Aminoquinolines↗

PubMed1995

Analysis of equilibrium binding data obtained by linear-response spectroscopic techniques.

A new computational approach for the analysis of equilibrium binding data obtained by spectroscopic methods is described. The approach is aimed at systems involving multiple interactions, where the changes in the spectroscopic signal cannot be attributed to a single interaction. A family of absorption or fluorescence spectra or decay curves obtained in a course of multiple titrations can be decomposed into basic components in an infinite variety of different ways. Model equilibrium equations are used as constraints to determine the basic components associated with the actual chemical species and the concentrations of these species. No assumption is made regarding the parametric form of the component spectra or decays. Binding constants are evaluated simultaneously with the resolved spectra and/or decays in a nonlinear least-square procedure. A key benefit of the method presented is the reduction in the number of fitting parameters. The conditions necessary and sufficient for the existence of a unique solution are considered. The application of the method is demonstrated with experimental data involving titrations of horse liver alcohol dehydrogenase with NADH and isobutyramide.

Alcohol Dehydrogenase↗

PubMed1993

Dynamic fluorescence measurements of two-state systems: applications to calcium-chelating probes.

A fluorescence technique for characterizing ligand binding is evaluated. This technique uses the combination of steady-state and time-resolved methods to recover molar concentrations and overcomes errors inherent in the use of either method alone. The technique is applicable to time-resolved measurements made either with time-domain or frequency-domain instrumentation. A straightforward single-frequency phase/modulation approach is presented to determine whether an experimental system can be described by a two-state system. The approach is based on a nonlinear transformation of the phase/modulation data that results in a linear model function. Here, the theory is applied to the fluorescent calcium-binding probes Quin-2 and Calcium Green, but is relevant to studies involving other interacting systems. The technique described is used to assess the fraction of bound ligand (Ca2+) and binding constants for these probes.

Aminoquinolines↗

PubMed1993

Fluorescence decay of DPH in lipid membranes: influence of the external refractive index.

The radiative decay rate of a fluorescent probe in an optically thin layer is known to depend on the orientation of the probe and on the refractive indices inside and outside the layer (W. Lukosz, Phys. Rev. B 22 (1980) 3030). Fluorescent probes in phospholipid bilayer membranes approximate such a system. The natural lifetime is expected to vary with the refractive index of the medium surrounding the bilayer. The lifetime variation with the refractive index depends on the orientation of the fluorescent probe. This can be used to retrieve the second-rank orientational order parameter, . The fluorescence decay of all-trans 1,6-diphenyl-1,3,5-hexatriene in L-alpha-dipalmitoyl-phosphatidylcholine large unilamellar vesicles (LUVs) was measured at a temperature well below that of the phase transition. The refractive index of the medium was varied by addition of glycerol or sucrose. The observed change of decay time with the refractive index followed the theoretical prediction. The value of the order parameter, , recovered is significantly lower than that obtained from fluorescence polarization data. Possible reasons for this disagreement are discussed.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1992

Time-resolved intrinsic fluorescence of Enzyme I. The monomer/dimer transition.

Enzyme I of the bacterial phosphotransferase system can exist in a monomer/dimer equilibrium which may have functional significance. Each monomer contains two tryptophan residues. It is demonstrated that the decay of both the monomer and the dimer can be described by a biexponential. The decay times depend on the temperature and at 6 degrees C the decay times are tau 1 = 0.4 ns and tau 2 = 3.2 ns for the monomer and tau 3 = 3.2 ns and tau 4 = 7.2 ns for the dimer form of the enzyme. The changes in the fluorescence decay parameters can be utilized to measure the equilibrium constant for the monomer/dimer transition.

Escherichia coli↗