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Biomedical subjects

R E Benesch

Publications and source records attributed to R E Benesch.

At least 37 records · Page 2Linked to original sources
PubMed1982

Labeling of hemoglobin with pyridoxal phosphate.

The reaction of pyridoxal 5'-phosphate (PLP) with deoxyhemoglobin is confined to 2 residues in the beta chains, i.e. the alpha-amino group of valine 1 and the epsilon-amino group of lysine 82, both of which are located in the polyphosphate binding site. The major product is a hemoglobin in which only the two NH2-terminal amino groups are substituted (symmetric diPLPHb). It is formed by subunit rearrangement of monoPLPHb which is the initial product of the pyridoxylation under anaerobic conditions. TetraPLPHb, with substitutions at lysine 82 and valine 1 of both beta chains is found as a minor component. It results from subunit exchange of asymmetric diPLPHb consisting of one unmodified alpha beta dimer and one which is pyridoxylated at both sites. Anaerobic electrophoresis and oxygenation curves show that this reaction is readily reversed by mixing the tetrasubstituted derivative with unmodified hemoglobin.

Amino Acids↗
PubMed1981

Sickle cell hemoglobin fiber structure altered by alpha-chain mutation.

Hybrid hemoglobin molecules prepared with beta chains from hemoglobin S (beta 6 Glu leads to Val) and alpha chains from hemoglobin Sealy (alpha 47 Asp leads to His) form fibers with a novel structure. In contrast to the typical fibers of hemoglobin S with an average diameter of 22 nm and a solid cross section composed of 10 outer filaments surrounding a 4-filament core, the fibers of the alpha Sealy2 beta S2 hybrid are much larger, with a mean diameter of 32 nm and a unique double-hollow arrangement of filaments. Sealy--S fibers can be described by a model in which the two pairs of filaments most readily lost from fibers of hemoglobin S are missing to form the hollow regions, with an additional sheath of filaments added to form the overall larger structure.

Computers↗
PubMed1980

Solubilization of hemoglobin S by other hemoglobins.

The polymerization of mixtures of Hb S with hemoglobins A, A2, and F has been investigated by analysis of the proportions of S and non-S hemoglobin both in the supernate and in the pellet after centrifugation. In all cases the non-S hemoglobin was incorporated into the polymer even in the absence of hybrids in the order A > A2 > F. The solubility of Hb S is substantially increased by the other hemoglobins, especially by Hb F, which would account for its antisickling effect. It appears that the excluded volume effect of the other hemoglobin on Hb S is largely counterbalanced by the solubilizing effect arising from the interaction between the two hemoglobins in solution. The ability of hybrid hemoglobins to gel was demonstrated directly with tetramers in which alpha beta s dimers were covalently linked to alpha beta A, alpha delta A2, and alpha gamma F dimers.

Centrifugation↗
PubMed1979

alpha Chain mutations with opposite effects on the gelation of hemoglobin S.

The preparation of three hemoglobin tetramers containing the hemoglobin S mutation at beta 6 and an additional one at alpha 6, alpha 47, and alpha 75 is described. The effect of the substitutions in the alpha chains on polymerization was investigated by the equilibrium solubility of the gels as well as the abrupt change in oxygen affinity associated with the onset of gelation. Substitution of a histidine for aspartic acid at alpha 47 causes a marked inhibition of polymerization. This inhibition probably results from tetramers which carry the two substitutions on the same alpha beta dimer. By contrast, the introduction of a tyrosine at alpha 75 and an alanine at alpha 6 have the opposite effect and are the first examples of alpha chain mutations which potentiate the gelation of Hb S. The molecular mechanisms responsible for the effects of the mutations on the self-association of Hb S are discussed.

Gels↗
PubMed1977

Location and bond type of intermolecular contacts in the polymerisation of haemoglobin S.

The solubility of 14 hybrid haemoglobins composed of alpha chains with a single substitution and beta chains from HbS was compared with that of sickle haemoglobin. A substantial reduction in the insolubility of native deoxyhaemoglobin S results from surface mutations in certain regions of the alpha chain while changes in other areas have no effect. Also, the chemical nature of the substitution is decisive an points to the type of intermolecular bonding at several loci.

Hemoglobin, Sickle↗
PubMed1977

Reciprocal interaction of hemoglobin with oxygen and protons. The influence of allosteric polyanions.

The interaction of three inositol esters, inositol hexaphosphate (IHP), inositol pentaphosphate (IPP), and inositol hexasulfate (IHS), with hemoglobin has been investigated. The proton uptake method was used to obtain the six binding constants for deoxy- and oxyhemoglobin. These data combined with oxygen binding curves over a range of cofactor concentrations were used to test theoretical and empirical equations relating the affinity of hemoglobin for oxygen and allosteric effectors. The Bohr and Haldane coefficients in the presence of the inositol esters are unequal at low, but not at high, concentration of the cofactors. The maximum value reached by both parameters increases with the number of negative charges of the polyanion. 2,3-Diphosphoglycerate (DPG) differs sharply from the inositol esters since even at high concentrations of this cofactor, the Haldane coefficient remains elevated. This is a reflection of the negligible affinity of DPG for fully oxygenated hemoglobin.

Adult↗
PubMed1977

5'-deoxypyridoxal as a potential anti-sickling agent.

5'-Deoxypyridoxal, which reacts specifically with the terminal amino groups of the alpha chains of hemoglobin, increases the oxygen affinity of hemoglobin solutions as well as of dilute suspensions of normal and sickle red cells and whole blood. As a result, the proportion of deoxyhemoglobin (which is responsible for sickling) is decreased at venous oxygen tensions and this is reflected by a sharply reduced sickle cell count in this range of oxygen pressures.

Anemia, Sickle Cell↗
PubMed1976

The allosteric effect of inositol hexasulfate on oxygen binding by hemoglobin.

Myoinositol hexasulfate (IHS) is a powerful allosteric effector of oxygen binding by hemoglobin. It binds to deoxyhemoglobin at the same site as 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (IHP) with an affinity which is intermediate between that of the two phosphate esters. The binding constant calculated from the displacement of the oxygenation curve in the presence of low concentrations of IHS is 0.9X10(6) M. The value obtained directly from the number of protons bound as a function of IHS concentration is 1.0X10(6) M. The agreement between these two independent measurements provides an experimental verification of the empirical equation, relating the oxygen affinity to the binding constants, proposed previously (Benesch, R.E., Benesch, R., Renthal, R and Gratzer, W.B. (1971), Nature (London), New Biol. 2348 174).

Binding Sites↗
PubMed1976

alpha-Chain contacts in the polymerisation of sickle haemogloblin.

Five new double-mutant haemoglobins composed of betaS chains and alpha chains with different substitutions, which are located at the surface of the tetramer, have been prepared. Although all the hybrids are more soluble than deoxyhaemoglobin S, the individual differences between these molecules make it possible to evaluate several regions on the alpha chains for intermolecular contacts in the polymerisation of deoxyhaemoglobin S.

Hemoglobin, Sickle↗
PubMed1976

Reaction of haptoglobin with hemoglobin covalently cross-linked between the alpha beta dimers.

Hemoglobin tetramers which cannot split into alphabeta dimers, because they are covalently cross-linked between the beta chains across the polyphosphate binding site, form complexes with haptoglobin. The reaction is biphasic as measured by fluorescence quenching and peroxidase activity. A complex in which one of the alpha beta dimers of the cross-linked hemoglobin is bound to one of the sites in the divalent haptoglobin molecule, is formed reversibly during the initial fast phase. In the subsequent slower step, this product then either polymerizes, adds another cross-linked hemoglobin molecule or, in the presence of excess haptoglobin, combines with a second haptoglobin molecule. This latter complex, in which two haptoglobin molecules are bridged by a cross-linked hemoglobin tetramer, can still combine with normal alpha beta dimers at the vacant haptoglobin combining sites. In spite of the very low oxygen affinity of the cross-linked hemoglobin, combination with haptoglobin shifts if oxygen affinity to the very high value of the normal hemoglobin-haptoglobin complex.

Binding Sites↗