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Biomedical subjects

R Benesch

Publications and source records attributed to R Benesch.

At least 19 recordsLinked to original sources
PubMed2001

A comparison of liquid hydrocarbon calibration standards in piston cylinders and standard cylinders with eductor tubes.

Natural gas liquid standards consist of various components primarily in the C1-C6 range; they are available in three types of cylinder packages: piston cylinders and two other types of standard cylinders with eductor tubes. The three cylinder packages have different operation conditions and thus a sample may behave differently in each of the packages. The piston cylinder maintains the components in a single liquid phase at a constant pressure, while the standard cylinders maintain the components as a two-phase mixture. Typically, the components may distribute between the two phases in different concentrations depending on the various thermodynamic variables, such as temperature, pressure, and volume. This study will examine the integrity of the sample in the three cylinder packages during a controlled cylinder depletion. The results for the various cylinders will be compared with a theoretical model of the experiments.

Journal Article↗
PubMed1986

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers.

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.

Adenosine Triphosphate↗
PubMed1986

The interaction of folylpolyglutamates with deoxyhemoglobin. Identification of the binding site.

Previous studies have shown that pteroylheptaglutamate (PteGlu7) can form a 1:1 complex with deoxyhemoglobin. The solution and crystallographic studies reported in this paper delineate the nature of the PteGlu7 binding site. We find that the three structural elements of PteGlu7 (the pteridine moiety, the p-aminobenzoyl portion, and the glutamate groups) each contribute to the binding energy by interacting with residues in the central cavity between the beta subunits and with residues at the alpha 1 beta 1 interface. Identification of the 2,3-diphosphoglycerate (DPG) binding site as part of the PteGlu7 binding site was accomplished in two ways; first by the demonstration of reduced PteGlu7 binding to hemoglobin selectively modified by pyridoxylation at this site, and second by the finding that DPG and PteGlu7 bind to deoxyhemoglobin in a competitive manner. In addition, since analogs of PteGlu7 in which the pteridine moiety is modified display reduced binding, it can be concluded that the pteridine group also contributes significantly to the binding energy. The crystallographic studies are completely consistent with the results determined in solution. A difference electron density image at 4.3 A resolution shows that the pteridine and p-aminobenzoyl groups are nestled against an interior edge of the alpha 1 beta 1 interface with the pteridine ring interacting with Phe 36 alpha 1 and the p-aminobenzoyl group positioned against a portion of the H helix between residues Lys 132 beta 1 and Ala 135 beta 1. The difference density for the glutamate residues is less well resolved (for reasons described in the text), but it is clear that some of the carboxylate side chains must interact with residues at the DPG binding site.

Binding Sites↗
PubMed1985

The binding of folyl- and antifolylpolyglutamates to hemoglobin.

A binding method that detects only the strongest binding site for a ligand on a protein has been used to show that folates and folate analogs, conjugated with poly-gamma-glutamates, are bound to hemoglobin. When the concentration of hemoglobin is much larger than that of the polyglutamate, as is the case in the red cell, the fraction bound is a direct function of the hemoglobin concentration and is independent of the total polyglutamate concentration. Binding to deoxyhemoglobin tetramers is competitive with 2,3-diphosphoglycerate. In oxyhemoglobin the folyl and methotrexate polyglutamates are bound preferentially by free alpha beta dimers, but removal of the pteridine moiety leads to tetramer binding even in oxyhemoglobin. Changes in the length of the polyglutamate side chain and alterations of the pteridine structure such as reduction and/or methylation have a much larger effect on the constant for binding to deoxyhemoglobin tetramers than on that for oxyhemoglobin dimers. The implications of these results for the storage of pteroylpolyglutamates in the erythrocyte and their release from the red cell under the influence of the degree of oxygenation and variations in the 2,3-diphosphoglycerate level are discussed.

Folic Acid↗
PubMed1984

Changes in the Bohr effect due to pyridoxylation of the alpha-chain terminal amino groups of hemoglobin.

Deoxyhemoglobins substituted with pyridoxal 5'-phosphate or pyridoxal 5'-deoxymethylenephosphonate at the N-terminal amino groups of the alpha-chains were investigated by 31P-NMR spectroscopy. Titration curves of the 5'-side-chains show a substantial increase in acid strength in alpha-pyridoxylated deoxyhemoglobins when compared to the corresponding CO-liganded hemoglobins. These derivatives therefore contain a new oxygenation-linked acid group which opposes the normal Bohr effect. The loss in stabilization of the monoanion of the phosphate or phosphonate group derived from the three-dimensional structure can account for the lower pK of this ionization in deoxyhemoglobin as compared to CO-liganded hemoglobin. The reduction in the Bohr effect caused by modification of the alpha-chains with pyridoxal 5'-deoxymethyl-enephosphonate is quantitatively equal to the expected contribution of alpha-chain N-terminal amino groups.

Allosteric Regulation↗
PubMed1984

Enhanced oxygen unloading by an interdimerically crosslinked hemoglobin in an isolated perfused rabbit heart.

Coronary perfusion has shown that an intramolecularly crosslinked hemoglobin (Hb) with a very low affinity for O2 (Hb crosslinked covalently between the beta chains with 2-nor-2-formylpyridoxal 5'-phosphate, HbXL) has several advantages over ordinary Hb. As predicted from in vitro oxygenation curves, much more O2 was unloaded to the heart at three different heart rates, at two perfusion rates, and when the perfusate was equilibrated with 25% as well as 95% O2. In all cases, the improved O2 unloading occurred at higher tissue O2 pressures than with normal Hb. The greater O2 consumption with HbXL was accompanied by better mechanical performance because, after 90 min of perfusion, the HbXL-perfused hearts maintained two-thirds of their original contractility (dp/dt), while that of the Hb-perfused hearts had declined to one-fifth. A special advantage of HbXL is its ability to unload significant amounts of O2 even at low temperature (10 degrees C), in contrast to whole blood. This should make it useful for supporting aerobic metabolism during low-temperature cardioplegia in cardiac surgery and for organ preservation.

Animals↗
PubMed1983

Specific receptor sites for pyridoxal 5'-phosphate and pyridoxal 5'-deoxymethylenephosphonate at the alpha and beta NH2-terminal regions of hemoglobin.

hemoglobins have been prepared which are substituted with pyridoxal 5'-phosphate or pyridoxal 5'-deoxymethylenephosphonate at both NH2-terminal amino groups of either the alpha or the beta chains. 31P NMR titration curves of the 5' side chain show a substantial decrease in acid strength in hemoglobins pyridoxylated at the alpha chains and the reverse effect in the hemoglobins labeled in the beta chains. These changes can readily be explained by the interaction of the 5' side chains with surrounding residues which provide specific binding sites for the monoanion at the alpha chain termini and for the dianion in the case of the beta chains. Since the proportion of monoanion is at lest ten times greater for pyridoxal 5'-deoxymethylenephosphonate than for pyridoxal 5'-phosphate at neutral pH, the phosphonate reacts preferentially with the alpha chain NH2-terminal amino groups and the phosphate with the beta chain ones.

Amino Acid Sequence↗
PubMed1983

A pteroylpolyglutamate binds to tetramers in deoxyhemoglobin but to dimers in oxyhemoglobin.

The binding of a physiological concentration of pteroylhepta(glutamate) to oxy- and deoxyhemoglobin in large excess was measured by ultrafiltration. The variation of free to bound folate with hemoglobin concentration showed that a single molecule of the pteroylpolyglutamate is bound by deoxyhemoglobin tetramers and by alpha beta dimers in oxyhemoglobin. Although the binding sites are different, the affinity constants are the same and very similar to the 2,3-bisphosphoglycerate binding energy. Nevertheless, in view of the small proportion of dimers in oxyhemoglobin much more pteroylhepta(glutamate) is bound by deoxyhemoglobin over a wide range of hemoglobin concentrations. Because even 2% deoxyhemoglobin is enough to bind all of the erythrocyte folate as polyglutamate, the bulk of it will be bound at physiological oxygen pressures. Free folate could only be expected in fully oxygenated erythrocytes. Therefore, the reaction of pteroylpolyglutamates with hemoglobin represents an oxygenation-dependent storage mechanism that can account for the 40-fold excess of the vitamin in the erythrocyte over the amounts in the serum. Because methotrexate is also converted to polyglutamate derivatives in the erythrocyte, this drug is likely to be concentrated and stored there by the same mechanism.

Folic Acid↗
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↗