Reinhold Benesch

Equations for the spectrophotometric analysis of hemoglobin mixtures

Abstract The extinction coefficients for deoxyhemoglobin, oxyhemoglobin, and carbonmonoxyhemoglobin as well as those of ferrihemoglobin between pH 6.2 and 8.8 are given at a number of wavelengths. Equations are presented for the analysis of ternary mixtures of these hemoglobin derivatives.

Determination of oxygen equilibria with a versatile new tonometer

Abstract A spectrophotometric method is deseribed for determining oxygen equilibria of hemoglobin in concentrations ranging from 0.005 to 10%. A new tonometer and rotating assembly were devised to carry out these measurements. Equations are given for calculating the results in the various concentration ranges and for detecting impurities of methemoglobin. It is also shown how methemoglobin formation can be kept to a minimum in work with very dilute hemoglobin solutions.

The removal of organic phosphates from hemoglobin

Abstract It has been shown that pH is an important factor in the separation of 2,3-diphosphoglycerate (DPG) from hemoglobin. Hemolyzates of pH 7.5, but not below pH 7, can be freed of phosphate by gel filtration in 1 1 2 hr. It was also demonstrated that the mobility of DPG itself on Sephadex G-25 varies greatly with pH. The abnormally high mobility in neutral solution where the molecule is highly charged can be explained by electrostatic repulsion within the molecule, giving it an extended conformation.

The Mechanism of Interaction of Red Cell Organic Phosphates with Hemoglobin

Publisher Summary Oxygen release depends crucially both on the shape of the oxygen binding curve and on the overall oxygen affinity. This chapter overviews the interaction of organic phosphates with hemoglobin, D-2, 3-diphosphoglycera (DPG) and the Bohr effect, DPG and CO 2 transport, effect of DPG on abnormal hemoglobins, and organic phosphates and artificial hemoglobin hybrids. Human hemoglobin without organic phosphates binds oxygen far too tightly to release it under physiological conditions. The large intracellular concentration of DPG lowers the oxygen affinity into the useful range, where it is further modulated by organic phosphates, hydrogen ions, and CO 2 . The red cell DPG level is subject to metabolic control, including feedback mechanisms, which respond to oxygen requirement. On a molecular level, the basis for the lowering of the oxygen affinity by organic phosphates is their mole for mole interaction with a single site on deoxyhemoglobin, which is destroyed on ligand binding. The linkage of phosphate cofactors with ligand binding also adds a new dimension to studies on the mechanism of oxygen transport by hemoglobin and models for the mechanism of oxygenation must take account of the influence of these cofactors. The specific stabilization of the deoxy conformation by organic phosphates has provided a powerful tool for investigations on the conformational transitions, which are basic for the understanding of the mechanism of oxygenation.

Hemoglobin covalently bridged across the polyphosphate binding site

Abstract Hemoglobin has been cross-linked covalently by reaction with 2-nor-2-formylpyridoxal 5′-phosphate followed by reduction with sodium borohydride. It is shown that a bridge is formed across the polyphosphate binding site between the two β chains. The modified hemoglobin binds oxygen cooperatively with a greatly decreased affinity demonstrating that the cross-link stabilizes the deoxy conformation but does not prevent the conformational change associated with oxygenation.

Structure of human deoxyhemoglobin specifically modified with pyridoxal compounds

Abstract Previous studies (Benesch et al., 1972, 1973, 1975) have shown that a variety of specifically modified hemoglobins having a wide range of functional properties can be prepared by reacting human hemoglobin with different pyridoxal compounds and then reducing the products with sodium borohydride. In particular, the modified hemoglobins (α PLS ‡ ‡ Abbreviations used: PLP, pyridoxal 5′-phosphate; PLS, pyridoxal 5′-sulfate; NFPLP, 2-nor-2-formylpyridoxal 5′-phosphate; DPG, 2,3-diphosphoglycerate; HbXL, αβ-NFPLP-βα hemoglobin. ) 2 β 2 , α 2 (β PLP ) 2 and αβ-NFPLP-βα have been characterized, respectively, as the reduced products of the reactions of pyridoxal 5′-sulfate with the NH2-termini of the α chains, pyridoxal 5′-phosphate with the NH2-termini of the β chains, and 2-nor-2-formylpyridoxal 5′-phosphate (which contains two reactive aldehyde groups) with one amino group from each β chain to form a stable cross-link between the β chains. In this paper, we report the three-dimensional structures of the deoxy forms of these hemoglobins as determined by X-ray diffraction methods. We find the pyridoxal side chains of these hemoglobins to be oriented as follows. 1. (1) The PLS side chains of (αPLS)2β2 are positioned so that each sulfate group replaces a weakly bound inorganic anion to form salt bridges between the newly formed secondary amine of valine 1α and the guanidinium ion of arginine 141 on the opposite α chain. The pyridoxal ring interacts with residues of the H-helix on the same α chain to which it is attached. 2. (2) In α2(βPLP)2, the PLP phosphate groups are located very near the positions occupied by the phosphate groups of 2,3-diphosphoglycerate in the deoxyhemoglobin-DPG complex, and serve as permanently bound counterions for the basic side chains of histidine 143, histidine 2, and lysine 82 of both β subunits. Each pyridoxal ring displaces a firmly bound inorganic anion and interacts with residues of the EF corner on the same β chain to which it is attached. 3. (3) In the case of αβ-NFPLP-βα, we find that the cross-link is asymmetric with the 2′ and 4′ carbons of NFPLP joined to the amino nitrogens of lysine 82β1 and valine 1β2, respectively. The phosphate group replaces the firmly bound inorganic anion mentioned above, and the 3-oxygen of the pyridoxal ring interacts with the imidazole group of histidine 143β1. Relative to the structure of deoxyhemoglobin A, no significant changes in α chain tertiary structure are observed for (αPLS)2β2, only small changes in β chain tertiary structure occur in α2(βPLP)2, but exceptionally large perturbations to the structure of one β chain result from the cross-link in αβ-NFPLP-βα. The relationships between the structures of these hemoglobin derivatives and their functional properties are discussed.

Oxygen affinity as an index of hemoglobin S polymerization: A new micromethod☆

Abstract The decrease in oxygen affinity with increasing hemoglobin concentration, which occurs in solutions of pure hemoglobin S, can be used to determine the minimum concentration at which polymerization of the deoxy form takes place. On this basis a very sensitive method for measuring the minimum gelling concentration has been developed. The influence of temperature, pH, and other hemoglobins on the end points obtained by this method is described. In all cases excellent agreement with the minimum gelling concentration determined directly on larger samples was observed. The results of this investigation demonstrate that the decrease in oxygen affinity of red cells containing hemoglobin S with increasing intracorpuseular hemoglobin concentration [May, A., and Huehns, E. R. (1975) Brit. J. Haematol.30, 317] is a direct consequence of the gelling properties of hemoglobin S alone.

Some relations between structure and function in hemoglobin

The cardinal problem that presents itself when one surveys the impressive amount of data available for the structure of the hemoglobin molecule is, of course, how such a bewilderingly complex arrangement can account for the functions of this protein. The combined results of the X-ray crystallographers ( Perutz et al., 1960 ; Cullis, Muirhead, Perutz, Rossmann & North, 1962 ) and the organic chemists ( Braunitzer et al., 1961 ; Konigsberg, Guidotti & Hill, 1961 ; Braunitzer & Matsuda, 1961 ) have given us considerable information concerning the three-dimensional arrangements, not only of the hemes, but also of the polypeptide chains and, to an increasing extent, even the amino acid residues in the individual chains. The functional integrity of the haemoglobin molecule can be gauged from a number of criteria, all of which stem from its ability to combine reversibly with oxygen. However, in order to qualify as a physiologically useful oxygen carrier, a number of additional closely related properties must be within normal range. The most important of these are the oxygen affinity, the heme-heme interaction, the Bohr effect and the CO2 binding capacity.

Preparative isoelectric focusing of CO hemoglobins on polyacrylamide gels and conversion to their oxy forms

Abstract Isoelectric focusing on acrylamide gels has been used to prepare 100–200 mg amounts of modified hemoglobins in a state of high purity. A method for the quantitative elution of the protein from the gel is described. A new procedure for the complete conversion of carbonmonoxyhemoglobin derivatives to their oxy or deoxy forms which fully preserves their functional integrity has also been developed.

Spectra of Deoxygenated Hemoglobin in the Soret Region

Methemoglobin is easily formed during the deoxygenation of hemoglobin. It can be removed with methemoglobin reductase. The Soret spectrum of pure deoxyhemoglobin is reported. Comparison of these data with the published spectra shows that some of these are incorrect since they must represent mixtures of deoxy-and methemoglobin.