Structural and functional studies of hemoglobin Wayne: an elongated alpha-chain variant.

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated alpha-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity (P 1/2 = 0.60 and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect (-delta log P 1/2/pH (7 to 8) = 0.34 and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l' = 5.6 X 10(6) M-1 S-1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l' = 2.6 X 10(6) M-1 S-1), whereas Hb Wayne Asp showed heterogeneous binding (l' = 6.1 X 10(6) and 2.6 X 10(6) M-1 S-1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.

Structure of deoxyhemoglobin Cowtown [His HC3(146) beta----Leu]: origin of the alkaline Bohr effect and electrostatic interactions in hemoglobin.

Hemoglobin Cowtown [His HC3(146)-beta----Leu] exhibits high oxygen affinity and a halved alkaline Bohr effect. X-ray analysis shows the COOH-terminal leucine to be in equilibrium between two positions: one with the salt bridge between the terminal carboxyl and Lys C5(40)alpha intact and the leucyl side chain leaning against main chain atoms of helices F and FG and the other with the terminal salt bridge broken and the leucyl side chain touching Pro C2(37)alpha. Structural changes are confined to the immediate neighborhood of the COOH terminus, showing the halving of the alkaline Bohr effect to be due directly to the loss of the histidine, without significant contributions from changes in pK values of other ionizable groups due to structural changes elsewhere.

Functional properties of Hb Pasadena, alpha 2 beta 2 75(E 19) Leu replaced by Arg.

Functional properties of Hb Pasadena (beta 75[E19]Leu replaced by Arg) as well as correlations of its structure-function relationships with those of normal hemoglobin are presented. The homeotropic and heterotropic properties of the isolated variant were investigated by automatic recording oxygen equilibrium analysis. These studies indicate an increase of 02 affinity but normal cooperativity. The decrease of 02 affinity in response to organic phosphate (DPG and IHP) was found to be normal. However, the cooperativity of Hb Pasadena is less affected by IHP than is that of Hb A. The alkaline Bohr effect was found to be somewhat decreased, although the temperature effect is nearly normal. The autoxidation rate is increased by a factor of nearly 2 compared to normal. These observations are consistent with the conclusion that the beta (E19)75 leucyl hydrophobic residue of Hb A associates with beta (A8)11 Val, beta (A12)15 Trp and beta (H11)133 Val to form a hydrophobic cluster between the A, E and H helics which thereby stabilizes the heme of the beta chain in the deoxy ferrous state. The experimental results also suggest that the amino acid substitution in Hb Pasadena mainly disrupts the interior conformation of the beta subunit without affecting subunit interfaces. Thus it is predicted that Hb Pasadena should have a normal crystal structure in either the liganded (R) or unliganded (T) state. Its increased 02 affinity can be explained partially by a decrease of proton binding and partially by a shift of the R:T equilibrium towards the R conformation.

Hemoglobin variants with altered oxygen affinity.

To date 88 abnormal hemoglobins have been described with altered oxygen affinities. Of these, 60 variants have high affinity and 28 have low affinity. Twenty-six of these abnormal hemoglobins are chemically unstable. Twenty-seven of the high affinity hemoglobins are associated with erythrocytosis. An equal number are not associated with increased red cell mass. Anemia has been reported with 13 of the low affinity variants; however, the primary cause of the anemia of many may be the concurrent chemical instability. A strong correlaton can be shown for stable variants with altered oxygen affinity between the P50 value of the red cell and the hemoglobin concentration of the affected individual's blood. Hemoglobin variants with altered oxygen affinity can be classified into groups depending upon the amino acid change in specific structural sites. The sites include subunit interfaces, heme contacts, central cavity, DPG binding site, C-terminus, and other external and internal residues. Functional studies of six abnormal hemoglobins, four at the beta 101 residue in the central cavity near the alpha 1 beta 2 interfce, one near the DPG binding site and one at the C-terminus of the beta chain, indicate that the exact explanation of the structure-function relationship in hemoglobin depends upon the specific residue that is altered and the chemical properties of the substituted group. Comparative studies of multiple variants of the same position should provide insight into the role of critical residues in the binding of oxygen by hemoglobin.

Application of anaerobic ion-exchange chromatography to the separation of hemoglobins in R from T conformational states.

As an allosteric protein, hemoglobin can assume at least two different quatenary structures, a deoxy or T for tense conformation and an oxy or R for relaxed conformation (2,3) depending upon its state of ligation. Because the pK values of oxy and deoxy- hemoglobins are different, molecules in the R conformation should behave differently than molecules in the T conformation on ion exchange chromatography. Kilmartin et al. (4) designed an anaerobic cation exchange chromatographic procedure with which they separated the deoxy hemoglobin from oxy hemoglobin and other R conformation hemoglobin components like CO-hemoglobin and sulfohemoglobin. In this report we describe the further development of Kilmartin's procedure to separate two electrophoretically silent hemoglobin mutants, Hb Potomac [beta 101 Glu leads to Asp] (5) a high oxygen affinity variant, and Hb M-Milwaukee [beta 67 Val leads to Glu] (6,8) a low oxygen affinity mutant from Hb A. The modifications we have made include (a) applying the hemolysate at a partial pressure of oxygen determined from an examination of the oxygen equilibrium curve to produce a favorable ratio of T to R conformations of the two hemoglobins to be separated, (b) controlling the partial pressure of oxygen tension of the column effluent during the development of the chromatogram.

Further studies on the functional properties of hemoglobin M Hyde Park.

The oxygen binding properties of Hb M Hyde Park (92 beta, histidine leads to tyrosine) were reinvestigated directing special care to testing the wave length-dependence of the oxygen equilibrium curve and to stabilizing hemoglobin samples using a methemoglobin reductase system. There was no indication that the Hb M Hyde Park fraction separated on a DEAE Sephadex column contained an unknown hemoglobin derivative which appeared in earlier studies. Contrary to earlier observations, there was no significant wave length-dependence of the equilibrium curve of Hb M Hyde Park, verifying the spectrophotometric determination of oxygen saturation. The reductase system satisfactorily reduced the normal alpha chain met hemes without reducing the abnormal beta chain met hemes. The oxygen binding property of Hb M Hyde Park is characterized by 3 to 4 times higher oxygen affinity than that for normal hemoglobin, complete loss of cooperativity, and substantially preserved Bohr effect. These results are consistent in part but not entirely with those observed by earlier investigators. The oxygen affinity of Hb M Hyde Park is between the affinity of the oxy structure and the deoxy structure of normal hemoglobin. Oxygen equilibrium curve of red cell suspension and whole hemolysate containing Hb M Hyde Park were biphasic, indicating that Hb M Hyde Park also exhibited the high oxygen affinity in those samples.

Hemoglobin Cowtown (beta 146 HC3 His-Leu): a mutant with high oxygen affinity and erythrocytosis.

A new mutant, hemoglobin Cowtown, has been found in a white man and his father, both of whom have erythrocytosis. The father had previously been treated with 32P for polycythemia vera. The abnormal hemoglobin is not detectable on electrophoresis in alkaline buffers, but it resolves distinctively on electrophoresis in citrate agar, pH 6.0; similarly, the abnormal beta-globin chain does not separate from beta-A in urea 2-mercaptoethanol buffers of pH 8.9, but it moves anodically to beta-A at pH 6.0. Peptide chromatography and amino acid analysis of the beta chain reveal that the C-terminal histidine residue (beta 146) has been replaced by leucine. Like several other hemoglobins substituted at this residue, Hb Cowtown has a high oxygen affinity and a diminished Bohr effect.