Reactivity of cyanate with valine-1 (alpha) of hemoglobin. A probe of conformational change and anion binding.
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The 3-fold increase in the carbamylation rate of Val-1 (alpha) of hemoglobin upon deoxygenation described earlier is now shown to be a sensitive probe of conformational change. Thus, whereas this residue in methemoglobin A is carbamylated at the same rate as in liganded hemoglobin, upon addition of inositol hexaphosphate its carbamylation rate is enhanced 30% as much as the total change in the rate between the CO and deoxy states. For CO-hemoglobin Kansas in the presence of the organic phosphate, the relative increase in the carbamylation rate of this residue is about 50%. These results indicate that methemoglobin A and hemoglobin Kansas in the presence of inositol hexaphosphate do not assume a conformation identical with deoxyhemoglobin but rather form either a mixture of R and T states or an intermediate conformation in the region around Val-1 (alpha). Studies on the mechanism for the rate enhancement in deoxyhemoglobin suggest that the cyanate anion binds to groups in the vicinity of Val-1 (alpha) prior to proton transfer and carbamylation of this NH2-terminal residue. Thus, specific removal with carboxypeptidase B of Arg-141 (alpha), which is close to Val-1 (alpha) in deoxyhemoglobin, abolishes the enhancement in carbamylation. Chloride, which has the same valency as cyanate, is a better competitive inhibitor of the carbamylation of deoxyhemoglobin (Ki = 50 mM) compared with liganded hemoglobin. Nitrate and iodide are also effective inhibitors of the carbamylation of Val-1 (alpha) of deoxyhemoglobin (Ki = 35 mM); inorganic phosphate, sulfate, and fluoride are poor competitive inhibitors. The change in pKa of Val-1 (alpha) upon deoxygenation may be due to its differential interaction with chloride. (+info)
2,3 diphosphoglycerate in Parkinson's disease.
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The red cell 2,3 DPG, the most important factor for oxygen delivery in the tissues, was found to be increased in Parkinsonism patients compared with controls. The aging process seems not to be a factor in the increased 2,3 DPG concentration. Other factors relevant to raised 2,3 DPG level such as physical activity, increased oxygen requirements, and metabolic changes are discussed. (+info)
Kinetic studies on the binding affinity of human hemoglobin for the 4th carbon monoxide molecule, L4.
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L4, the affinity of hemoglobin for the 4th CO molecule, has been determined for human adult hemoglobin (HbA) as a function of pH and the presence of organic phosphates by measuring the kinetic parameters for the reaction. l'4, the rate of combination of CO with the triliganded molecule, was measured by flash photolysis while l4, the rate of CO dissociation for the ligand-saturated molecule, was measured by ligand replacement. L4 is pH-dependent and affected by 2,3-diphosphoglycerate. Additionally, this pH dependence of the high affinity state is largely eliminated by carboxypeptidase A digestion. L4 for human fetal hemoglobin (HbF) in phosphate buffers was also determined and found to be pH-dependent. These results cannot be reconciled within the framework of the two-state allosteric model. Additional structures in the conformational equilibrium due to either intermediates in the T to R transition or two or more R states must exist. (+info)
Isolation and partial characterization of monophosphoglycerate mutase from human erythrocytes.
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Monophosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes as indicated by exclusion chromatography, polyacrylamide gel electrophoresis, and equilibrium centrifugation. Occasionally, the recommended purification procedure yields a small amount (3% or less) of a single extraneous protein which can be deleted from the enzyme preparation by employing an additional purification step. The native enzyme has a molecular weight of 54,000 to 56,000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of 28,600, indicating that the native macromolecule is a dimer composed of subunits of similar mass. Homogeneous monophosphoglycerate mutase is free of diphosphoglycerate mutase, enolase, and nonspecific phosphatase activities; however, the enzyme manifests intrinsic 2,3-diphospho-D-glycerate phosphatase activity as shown by thermal denaturation studies. The diphosphatase activity is stimulated by PPi and glycolate-2-P, but is inhibited by Cl-, HSO3-, and Pi. The pH optimum for both the diphosphatase and the mutase is 6.8. The Km for 2,3-diphospho-D-glycerate in the phosphatase reaction is 82 muM at 37 degrees and pH 7.2. The amino acid composition of homogeneous monophosphoglycerate mutase is given. (+info)
Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.
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Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1). (+info)
Dimethyl adipimidate: a new antisickling agent.
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A new approach to the prevention of sickling in vitro by use of the bifunctional crosslinking reagent, dimethyl adipimidate, is described. Prior treatment of sickle erythrocytes with dimethyl adipimidate will inhibit sickling in completely deoxygenated erythrocytes. Treated erythrocytes do not demonstrate the potassium loss and viscosity increase that usually accompany sickling. The oxygen affinity of hemoglobin in these cells is increased independently from changes in the concentration of 2,3-diphosphoglycerate. The hemoglobin obtained from treated erythrocytes contains a high-molecular-weight component as well as additional positively charged components. The relative degree to which chemical modification and/or crosslinking is an essential part of the antisickling properties of the material is not known. (+info)
Glycosylation of hemoglobin in vitro: affinity labeling of hemoglobin by glucose-6-phosphate.
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To determine the mechanism for the formation of hemoglobin A1c (Hb A1c) in vivo, we incubated human hemoglobin with glucose and metabolites of glucose. [14C]Glucose-6-phosphate (G6P) reacted readily with deoxyhemoglobin, and formed a covalent linkage. The reaction rate was considerably reduced in the presence of carbon monoxide or 2,3-diphosphoglycerate (2,3-DPG). Purified G6P hemoglobin had a lowered oxygen affinity and decreased reactivity with 2,3-DPG compared to Hb A. G6P behaved as a 2,3-DPG analog and reacted specifically at the NH2-terminal amino group of the beta chain. In contrast, the interaction of hemoglobin with glucose was much slower, and was unaffected by carbon monoxide or 2,3-DPG. Neither glucose-1-phosphate, fructose-6-phosphate, nor fructose-1,6-diphosphate formed a reaction product with hemoglobin. G6P behaves as an affinity label with the phosphate group forming electrostatic bonds at the 2,3-DPG binding site and the aldehvde group reacting with the NH2-terminal amino group of the beta chain. Thus, G6P hemoglobin may be an intermediate in the conversion of Hb A to Hb A1c. (+info)
Activation of the low oxygen affinity-inducing potential of the Asn108(beta)-->Lys mutation of Hb-Presbyterian on intramolecular alpha alpha-fumaryl cross-bridging.
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The Asn108 beta-->Lys mutation in hemoglobin (HbPresbyterian mutation) endows a low O(2) affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two alpha 99 lysine residues also induces a low O(2) affinity into HbA. We have now engineered an alpha alpha-fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O(2) affinity-inducing structural perturbations. Despite the presence of the additional epsilon-amino group of Lys108(beta) within the central cavity, the epsilon-amino group of Lys99(alpha alpha) of deoxy Hb-Presbyterian retained high selectivity for alpha alpha-fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The alpha alpha-fumaryl cross-linking of Hb-Presbyterian reduced its O(2) affinity much more significantly than that observed with HbA, indicating a synergy between the two low O(2) affinity-inducing structural perturbations. Apparently, the alpha alpha-fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O(2) affinity-inducing potential of Lys108(beta) that is generally activated in the presence of chloride. The synergy between the Asn108(beta)-->Lys mutation and the alpha alpha-fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, alpha alpha-fumaryl Hb-Presbyterian accessed a low O(2) affinity T-state that is accessed by HbA, alpha alpha-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the alpha alpha-fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99(alpha) and Lys108(beta) [presumably His103(alpha) and/or Glu101(beta)] to compensate for the extra positive charge of Lys108(beta). Molecular modeling studies identified two potential chloride binding sites per alpha beta dimer within the middle of the central cavity of alphaalpha-fumaryl HbA involving residues His103(alpha), Arg104(beta) and Asn108(beta). The affinity of these sites is increased in alpha alpha-fumaryl Hb-Presbyterian as a result of the Asn108(beta)-->Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the alpha alpha-fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the alpha alpha- fumaryl-Hb Presbyterian to access a low O(2) affinity T-state structure much more readily than either Hb-Presbyterian or alpha alpha-fumaryl HbA. (+info)