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Robert Cassoly: Influence Statistics

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per ...

The time course of carbon monoxide binding to stripped valency hybrids (α ii β iii CN)2 and (α iii CNβ ii )2 was studied at pH 6.6 and T = 0.05. It exhibits a strong heterogeneous character. Two spectrophotometrically and kinetically distinct forms are apparent. At most wave lengths the fast component contributes about 60 to 80% of the total absorbance change and has a rate of binding similar to that of rapid hemoglobins. Correlation between the spectral properties of hybrids and their ...

Binding of the cytoplasmic fragment of band 3 protein to oxyhemoglobin in solution caused a spectral change in the absorbance of the hemoglobin beta chain at a ratio of one monomer of band 3 protein per alpha beta dimer of hemoglobin. This spectral change was reversed at higher ratios of cytoplasmic fragment to hemoglobin. The unusual dependence on protein concentration was interpreted as indicating the formation of higher aggregates of the complex between hemoglobin and the cytoplasmic ...

The association of the isolated cytoplasmic fragment of band 3 protein with human hemoglobin was studied by rate zonal centrifugation in sucrose density gradients, by quenching of fragment fluorescence by hemoglobin, and by flash photolysis of carbon monoxidebound hemoglobin as a function of fragment concentration. The centrifugation results showed that both proteins interact and that the interaction is abolished upon addition of glyceraldehyde-3-phosphate dehydrogenase. The fractions ...

The identification of the protein that exerts the function of Cl-/HCO3- exchange is still unresolved in cardiac tissue. We have addressed this issue by using a multiple technical approach. Western blotting analysis with an antibody raised against human erythroid whole band 3 protein, the so-called protein that mediates the Cl-/HCO3- exchange in erythrocytes, showed that adult cardiomyocytes expressed two proteins immunologically related to the erythroid band 3. These proteins migrated in ...

Spectrin extracted from human red blood cells has been spin labeled in its dimeric and tetrameric forms with five different nitroxide derivatives of increasing chain length between their maleimide binding group and their nitroxide reporter group. Three molecules of spin label are bound per spectrin dimer. Electron spin resonance spectra show the simultaneous presence of strongly and weakly immobilized spin labels. Their relative proportion depends on the label length and is suddenly ...

Hemoglobin binds to the cytoplasmic domain of band 3 protein (CDB3) at physiologic pH and ionic strength in an oxygen-linked fashion, with deoxyhemoglobin having the higher affinity. The evidence in the literature suggests functional communication between the hemoglobin-binding site on CDB3 and the anion transport sites within the membrane-bound domain of band 3. Since the hemoglobin-binding site is estimated to be over 200 A from the transport domain, the functional communication ...

The addition of inositolhexaphosphate to stripped nitrosyl hemoglobin provokes strong changes in the visible absorption spectrum of the protein. The stoichiometry of this reaction, determined by spectrophotometry, is one mole of IHP per tetramer. The equilibrium dissociation constant of IHP for HbNO, in 0·1 m-2,2-bis (hydroxymethyl)-2,2′,2″-nitrilotriethanol (pH 6·8) is 1·3×10−6 m and increases with ionic strength and pH. IHP has only a small effect on bis (N-maleimidomethyl) ether ...

Human hemoglobin has been labeled on cysteine 93(beta) with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and reassociated with erythrocyte membrane previously stripped of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase. The affinity of hemoglobin for the membrane is not affected by the presence of the label. Saturation transfer electron paramagnetic resonance measurements show that the diffusion rotational movements of hemoglobin are considerably ...

The preparation and some properties of valency hybrids derived from human hemoglobin are described. These derivatives (α23+ β22+ and α22+ β23+), containing both the chains of a given type (α or β) in the ferri state, are obtained through association of isolated partner chains, each in the desired valency state. The hybrids are stable and are tetramers in the normal pH range. The oxygenation behavior of the hybrids has been studied at different pH values; the two complementary valency ...

There does not appear to be any co-operativity manifest in the four combination rate constants for the binding of nitric oxide to deoxyhemoglobin. The time-course of the observed reaction is best fitted by statistically related rates, and the numerical relation between the rate constants for the binding of the fourth molecule of carbon monoxide and the fourth molecule of nitric oxide, which can be obtained independently, also argues for a statistical relation between the nitric oxide ...

The binding of various linear and branched chain alkylisocyanides to soybean leghemoglobin has been studied with respect to association and dissociation kinetics and the results compared with those obtained in parallel on sperm whale and horse heart myoglobins; the linear ligands used (methyl to n-heptyl) cover a greater distribution of chain lengths than hitherto used. The association rate constants are much higher for leghemoglobin than for myoglobin, while the dissociation rates are ...

Semihemoglobins α and β are derivatives of human hemoglobin which possess the usual chain composition (α2β2 or αβ) but which carry the heme prosthetic group on only one kind of chain (α or β), the complementary chain (β° or α°) being heme-free. In this paper, new results on two compounds of this type, namely, semiHbαD (αβ°) and semiHbβ (α°β) have been presented; existing data on other earlier preparations have also been reviewed. Methods for preparation of semiHbαD and semiHbβ have been ...

The effects of organic phosphates on the carbon monoxide binding and NMR spectra of the hemoglobin valency hybrids (αIIICN βII)2 and (αIIβIIICN)2, have been studied. The stripped deoxy hybrids show heterogeneity in kinetics with two spectrophotometrically distinct forms which are not in rapid equilibrium. At most wavelengths the fast component contributes about 60 to 80% of the total absorbance change and has a rate of binding similar to that of isolated chains or other rapid ...

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the ...