Biomedical subjects
Publications and source records attributed to A E Myshkin.
It has been shown that low concentrations of potassium chloride change almost similarly the rate constant of oxyhemoglobin autooxidation in tris-buffer and the rate constant of its oxidation by ferricyanide in the same buffer. A conclusion has been made on the principal possibility of using the latter constant as a test for oxyhemoglobin state in solution and, particularly, for its predisposition to autooxidation.
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The results are discussed of studies on oxyhemoglobin coagulation in neutral phosphate buffer and acidic acetate buffer at pH ranging from 5.85 to 4.90. Peculiarities are shown of the effect of strong complexon on the oxyhemoglobin-coagulum-mercuric acetate system in neutral tris-buffer. Coagulation characteristics are cited for polymeric oxyhemoglobin in presence of mercury ions.
An analytical review of studies on human oxyhemoglobin coagulation has been performed by the author jointly with V. S. Koniaeva and L. D. Bogdanova within a period from 1985 to 1990. It was shown that the oxyhemoglobin coagulation modified by mercurials proceeded without any essential alteration of native protein conformation. A hypothesis is discussed that the oxyhemoglobin coagulation results from the primary polyaggregation of dimer fragments and that hydrophobic sites which provide for dimer-to-dimer contacts in native tetrameric oxyhemoglobin, participate in this process.
It was shown that the mechanism regulating the oxyhemoglobin coagulation in presence of a mercury reagent in large amount differs from that in presence of the reagent in relatively small concentrations. The significance of a large class of ligands at mercury atom during the oxyhemoglobin coagulation was demonstrated. Several theoretical generalizations are drawn.
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The kinetics of human oxyhemoglobin coagulation in neutral phosphate buffer in the presence of mercury acetate at 20 degrees has been studied using turbidimetric methods. The addition of small amounts of concentrated Hg2+ solution leads to rapid local protein coagulation with subsequent dissolution of the formed coagulate. Coagulation can be inhibited by addition of Tris that binds to mercury ions. The pattern of oxyhemoglobin coagulation is determined by molar Hg2+/protein ration rather than by total Hg2+ concentration.
A significant difference was discovered in low temperature (77 K) gamma-radiolytic behavior of 20% aqueous solutions of human oxyhemoglobin and partly denaturated methemoglobin. In the latter case twice as high yield of the sum of free radicals and OH radicals was observed, as well as presence in the ESR spectrum of a narrow singlet line at g 2.00 (absent for irradiated solutions of oxyhemoglobin) ascribed to the stabilized electron.
ESR spectra of gamma-irradiated and frozen at 77 K human oxyhemoglobin and partially denaturated methemoglobin solutions were analysed. The quartet signal ascribed to the anion-radical of proximal histidine was shown to dominate in the spectra of both solutions. The spectra of methemoglobin solution irradiated with relatively small doses have an intensive singlet ascribed to the stabilized electron. The formation mechanism of free radicals is discussed.
The initial velocity of coagulation of human oxyhemoglobin in tris-HCl buffer measured by turbidimetric method, pH 7.2 in the presence of phenylmercuryacetate made it possible to estimate the amount of moles of this reagent stechiometrically binding with hemoglobin without coagulation of the latter. At 15-30 degrees C this amount is 30-34 mole per hemoglobin-tetramer. At temperature increase from 30 to 42.5 degrees C the amount of the reagent necessary for protein coagulation sharply decreases. A model is proposed assuming that oxyhemoglobin coagulation proceeds only during binding of the reagent with specific protein sites.
By the dynamics of human oxyhemoglobin coagulation in the presence of phenyl mercury acetate in tris-AcOH buffer, pH 7.2 the number of moles of PhHg+ stechiometrically bound with protein at different temperatures was estimated. Within the temperature range 15-30 degrees C this value is constant--32-34 mole per 1 mole of HBO2-tetramer. Within the range 30-40 degrees C it rises to approximately 40. Coagulation of oxyhemoglobin modified with PhHg+ cation is reversible in contrast to HBO2 coagulation modified with uncharged PhHgCl.
Oxidation kinetics-characterized state of human oxyhemoglobin bound with p-chloromercuribenzoic acid (PCMB) (above 2 mole per tetramer) is changed during incubation at 20 degrees C. This suggests transfer of PCMB molecules from primary occupied centres to the secondary ones having higher affinity to PCMB. The same effect is observed at increased temperature without incubation. By means of gel-chromatography the hypothesis that the change of oxyhemoglobin state is accompanied by its increased equilibrium dissociation into dimers is acknowledge.
When the series of 4-substituted derivatives of 2,2,6,6-tetramethyl piperidine-I-oxide (TMPO) interacts with human serum albumin (HSA) in 0.01 M Na-acetate buffer, pH 5.6 in differential spectra there appears the intensive maximum at 232-235 nm characteristic of protein denaturation. It is suggested that specifics of nitroxyl binding with HSA is mainly determined by the conformational state of TMPO (twist or chair), since corresponding 4-nitro piperidines having other conformation result in the appearance of the band at 232.5 nm with a much lower intensity. It is recommended to take into account possible significant structural changes of albumins when studied by the spin label method using TMPO or its derivatives.
The rate of human hemoglobin oxidation in the presence of various sulfhydryl reagents essentially depends on the degree of binding of different sulfhydryl groups of hemoglobin during the oxidation process. Kinetics of modified hemoglobin oxidation is biphasic, which allows to judge the effect of reagent-hemoglobin binding on the state of rapidly and slowly oxidated subunits separately.
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