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V V Khramtsov

Publications and source records attributed to V V Khramtsov.

At least 37 records · Page 2Linked to original sources
PubMed1992

The application of pH-sensitive spin labels to studies of surface potential and polarity of phospholipid membranes and proteins.

The effects of pH titration on the EPR spectra of imidazolidine nitroxides located at the surface of mixed bilayers composed of dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylcholine (DMPC), and at the surface of the protein, human serum albumin (HSA), have been investigated. It is found that the shift in pKa of the amino group of the imidazolidine radical from its value of 4.6 in water depends both on the interfacial polarity (delta pKapol) and on the electrostatic surface potential (delta pKael) when it is positioned at the bilayer/water interface by an anchoring hydrocarbon tail. The polarity shift is determined to be: delta pKapol = -1.3 units at the surface of DMPC bilayers at 17 degrees C, corresponding to an effective interfacial dielectric constant of epsilon approximately 37, and depends on the temperature with a coefficient of d delta pKapol/dT approximately -0.01 per degree. The electrostatic shift at the surface of DMPG bilayers is delta pKael = +1.6 units in 0.1 M KCl, which corresponds to an electrostatic surface potential of -95 mV. This electrostatic shift depends strongly both on ionic strength and on the fraction of charged lipid in the DMPC/DMPG mixtures, in a manner that agrees with the predictions of electrostatic double-layer theory. It is found that the shift in pKa of an imidazolidine radical covalently bound at the surface of HSA is determined mainly by the surface electrostatics (delta pKapol approximately 0) and corresponds to an electrostatic potential of +33 mV in 0.01 M KCl at a pH below the isoelectric point of the protein.

Dimyristoylphosphatidylcholine↗
PubMed1991

Measurement of the local translational diffusion rates of proteins by saturation transfer EPR spectroscopy.

The concentration dependence of the normalized integral of the saturation-transfer EPR spectrum of human serum albumin spin-labelled on amino groups is found to be sensitive to viscosity and to temperature in aqueous solution and in 60% glycerol. The dependence on protein concentration is consistent with theoretical predictions for a diffusion-controlled Heisenberg spin-exchange interaction and the experimentally derived bimolecular collision rate constants are in reasonable agreement with those calculated theoretically for a diffusion-controlled process. The method is therefore applicable to the determination of translational diffusion coefficients of proteins in solution and, because of the nature of the saturation transfer EPR method, should be ideally suited to the study of local translational diffusion of proteins in membranes.

Diffusion↗
PubMed1991

Monitoring of enzymatic activity in situ by EPR.

A novel approach using an EPR method for the determination of activity of enzymes, whose substrates or products are low molecular weight compounds containing SH-groups, is proposed. The approach is based on thiol-disulfide exchange of stable nitroxyl biradical containing a sulfur-sulfur bond with low molecular weight thiol. The method is applied to determine activity of acetylcholinesterase in a larvae bollworm (from the formation of thiocholine from acetylthiocholine). The method is characterized by high sensitivity (up to 2 x 10(-12)m thiocholine) and makes possible measurements in optically unclear (scattering and coloured) media and determination of enzymatic activity (ca. 1 min) directly in the homogenate obtained from the heads of individual larvae. Thus, the method can be recommended for the fast monitoring of many enzymatic systems (including glutathione-dependent) directly in biological tissues of warm-blooded animals and insects.

Acetylcholinesterase↗
PubMed1991

[Quantitative determination and reversible modification of sulfhydryl groups in low and high molecular weight compounds using a biradical spin marker].

A new method for quantitation of sulfhydryl groups of low and high molecular weight compounds is proposed. The method is based on the use of a biradical spin label carrying a disulfide bond, RS-SR, where R is the imidazoline radical. It was found that this biradical is involved in the reaction of thiol-disulfide exchange with thiols; the EPR spectra of the original biradical and monoradical products differ essentially. This circumstance made it possible to determine the bimolecular rate constant for the biradical interaction with cysteamine, cysteine, glutathione and human serum albumin. The method was used for measuring glutathione and cysteine levels in murine and rat blood and for assaying the insect acetylcholine esterase activity and reversible inhibition of NADPH-cytochrome P-450 reductase. The method is marked for a high sensitivity (10(-6)-10(-7) M) towards sulfhydryl groups and allows the determination of thiol groups in coloured and nontransparent solutions.

Acetylcholine↗
PubMed1990

[Macromolecular spin pH-probes on the basis of human serum albumin].

Human serum albumin has been chemically modified by two different spin pH-sensitive labels of the imidazoline series containing in their structure alkylating and carboxyl groups, respectively. The ESR spectra of spin-labeled proteins are sensitive to pH of the medium. The pK values of spin-labeled proteins measured by the ESR method are: pKI = 3.2 +/- 0.1; pKII = 4.75 +/- 0.1. The resulting macromolecular spin pH probes may be used for measuring the local values of pH by the ESR technique within the pH range of 1.8-6.2.

Electron Spin Resonance Spectroscopy↗
PubMed1989

Quantitative determination of SH groups in low- and high-molecular-weight compounds by an electron spin resonance method.

To quantitatively determine SH groups in high- and low-molecular-weight compounds, a disulfide biradical (RS-SR), where R is imidazoline residue, has been used. The biradical is shown to participate in a thiol-disulfide exchange reaction with compounds containing SH groups. In this case the ESR spectra of the biradical RS-SR and the resulting monoradical R-SH are different. The reaction of the biradical with cysteine, glutathione, and human serum albumin has been studied using the ESR method and the rate constants kf of this reaction have been calculated. Studies of the pH dependence of kf indicate that the thiol-disulfide exchange occurs by reaction with mercaptidione. Protein human serum albumin and hemoglobin have been modified by RS-SR. It has been shown that the treatment of modified proteins with reduced glutathione leads to removal of the radical from the protein; such modifications are thus reversible. The method proposed has been used to quantitatively determine the SH groups of cysteine and glutathione in mouse and rat blood. The method is shown to coincide within experimental error with the determination of glutathione and cysteine by titration with p-chloromercuribenzoate or reaction with Ellman's reagent. This method allows detection of 10(-6)-10(-7) M SH compounds even in colored and highly absorbing samples. The kinetics of the SH group modification can also be determined, leading to deduction about accessibility of the SH group in protein.

Animals↗
PubMed1989

ESR study of proton transport across phospholipid vesicle membranes.

A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently bound to glutathione. This modified glutathione is pH sensitive at pH 1.5-4.5 and does not penetrate across phospholipid membranes. Using ESR this probe was applied to register the kinetics of pH variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH approximately 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCl, HClO4, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media.

Biological Transport↗