Rotational correlation times of IgG and its fragments spin-labeled at carbohydrate or protein moieties. Spatially fixed position of the Fc carbohydrate.
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Animals↗
Biomedical subjects
I V Dudich
Publications and source records attributed to I V Dudich.
[Study of the structural properties of recombinant gamma-interferon by circular dichroism and differential scanning microcalorimetry].
Recombinant human gamma-interferon is dimeric in solution at pH 7-4 as revealed by analytical gel-filtration. It was shown by circular dichroism that decreasing pH to 5.0 does not affect the secondary and tertiary structures of gamma-interferon macromolecule. It was established that heat denaturation process of gamma-interferon obeys the two-state transition model and can be described as the first-order reversible reaction. Temperature dependence of the denaturation-renaturation rate constants was shown to be consistent with the Arrhenius law. The equilibrium value of the denaturation temperature was found. Effective enthalpy of denaturation was determined both by thermodynamic and kinetic approaches. The data obtained showed that in the pH range 7-4 the dimeric IFN-gamma structure may be considered as a single cooperative thermodynamic domain. Thus, it may be concluded that gamma-interferon dimerization is necessary for the existence of the corresponding tertiary structure of the macromolecule.
Calorimetry, Differential Scanning↗
[Study of the structural properties of recombinant leukocyte interferon A by means of fluorescence polarization, circular dichroism, and differential microcalorimetry].
Human leukocyte interferon-A1 (IFN-alpha A) structure in solution was investigated by fluorescence polarization, circular dichroism and scanning microcalorimetry techniques. Using gel-filtration it was established that at neutral pH values and at concentration not exceeding 0.3 mg/ml IFN-alpha A has a dimeric configuration in solution. At pH below 5, IFN-alpha A exists as a monomer. Using circular dichroism technique the IFN-alpha A molecule was shown to preserve a native structure upon decreasing pH to 3.5. The rotational correlation time of IFN-alpha A molecule in dimeric and monomeric form was measured using fluorescence of DNS, conjugated with the protein, and fluorescence of tryptophan residues. Our data indicate that the shape of IFN-alpha A molecule may be approximated by the rigid ellipsoid of revolution with the axis ratio = 4:1. The intramolecular melting of IFN-alpha A was studied by scanning microcalorimetry and circular dichroism in the acidic pH range. Thermodynamic analysis reveals two independent cooperative transitions. These transitions can be explained by assuming that the IFN-alpha A molecule consists of two structural domains.
Calorimetry, Differential Scanning↗
[Polarity of the environment as a factor determining DNA conformation].
Polarity of double and ternary water-nonelectrolyte systems at the component ratio, corresponding to a half-transition point of DNA from B to A form was evaluated from ESR spectra of a spin-probe. In all cases examined the isotropic super-fine splitting constant (aN) is the same with an accuracy of 0.05 gauss. Small differences in aN are well correlated with the concentration of the groups which are able to form hydrogen bonds with the nitroxide fragment of the radical. Thus, media polarity is a factor which determines the A--B equilibrium of DNA in solution.
Chemical Phenomena↗
[Macromolecule rotative correlation time measurement by ESR for covalently bound spin label].
The dependence from temperature and viscosity of the shifts of the internal and external wide extremums in the ESR spectra of spin labelled bovine serum albumin has been studied. 2,2,6,6-tetramethylpiperidine-NI-oxyl-4-iodacetamide was used as a spin label. The obtained dependences was shown to be a consequence of the label participation in two types of rotations: an anisotropic fast rotation with tau less than 10(-9) sec relatively to a macromolecule, and the isotropic one with tau greater than 10(-8) sec due to rotation of the macromolecule itself. These conclusions were done on the basis of a model for complex rotation of the spin label. Comparison of theoretical and experimental data makes it possible to determined the correlation time for the protein moiety, to evaluate quantitatively the polarity of surroundings of the iminoxyl and to introduce a numerical parameter for the degree of mobility of the spin label relatively to protein molecule.
Electron Spin Resonance Spectroscopy↗
[Measurements of the time of rotational correlation of bovine serum albumin molecule using iodine and mercury-containing spin-labels].
New spin-labels based on iodine and hydrargirum containing imidazolids were approbated on the bovine serum albumin (BSA) molecule. It is shown that all hydrargirum labels are binded to the external SH-group of BSA practically immediately in comparison with earlier known spin-labels based on piperidine with maleimide and iodacetamide groups, requiring some minutes of hours, correspondingly. Rotational correlation times and the character of relative mobility of the spin-label were measured. Values of the rotational correlation times of the protein molecule obtained by hydrargirum containing labels were found in the range of correlation times typical for the BSA molecule and represent at pH 7.0 a rigid stretch ellipsoid of rotation. The result obtained revealed that the relative reorientation nature of spin-labels is essentially different and is taken into account in different values of order parametres S according to model--fast anisotropic rotator on slow isotropic rotator. ESR spectra based on theoretical calculations by means of computer are given.
Computer Simulation↗
[Study of binding of spin probes of the carboline and benzocarboline series to bovine serum albumin].
The dependence of the external and internal wide hyperfine extreme shifts of the ESR spectra on temperature and viscosity for spin-probes in solution of BSA was studied. Seven homologous spin-probes of carboline and benzocarboline derivatives were used. The obtained dependences are a consequence of the involvement of the spin-probe in two types of rotation: an anisotropic fast reorientation with tau > 10(-9) s with respect to a macromolecule and the isotropic one with tau > 10(-8) s due to rotation of the macromolecule itself. It was shown, that extrapolation values of a separation between hyperfine extreme do not reflect the degree of the immediate spin-probe environment polarity, but are determined by the hyperfine tenzor partial averaging as a result of the rapid anisotropic reorientation of the spin-probe. All spin-probes used were shown to be bound by the BSA molecule in the near vicinity of the tryptophan residue. The rotation correlation time of the BSA molecule was determined to be equal to 40 ns.
Adsorption↗
[Interaction of ribulose-1,5-diphosphate carboxylase with ribulose-1,5-diphosphate and with the magnesium ion by the spin label method].
A selective modification of external SH-groups of riboluse 1,5-diphosphate carboxylase (EC 4.1.1.39) from spinach by a spin label on the basis of maleimide was performed. From the EPR spectra of spin labels of three types of SH-groups were classified. It is shown that part of spin labels pre-reduced with dithiothreitol are reoxidized by the enzyme after the removal of dithiothreitol from the reaction mixture. The reoxidation is connected with the fact that one of the spin labels is bound to the SH-group at the enzyme active site and can act as its substrate, which results in alteration of the electron structure of the greater than N--O fragment in the spin label upon interaction of the spin-labeled enzyme with the substrate and Mg2+.
Carboxy-Lyases↗
[Preparation and properties of hybrid RNAase S', formed by S-protein and S-S-bound conjugates of (Nle-13,Cys-20)S-peptide with 3'-thiopropyl derivatives d(T)15].
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Catalysis↗