The hemoglobins of Artemia salina. IV. A model for genetic control of hemoglobin 1, hemoglobin 2, and hemoglobin X

Two loci account for all genetic variation resulting in difference in electrophoretic mobility in three hemoglobins (Hb1, Hb2, and HbX) in the hemolymph of the brine shrimp. Four alleles and nine alleles have been studied. In shrimps of all genotypes and in electrophoresis in media with varying degrees of molecular sieving, Hb2 is approximately equidistant from Hb1 and HbX. A shrimp heterozygous at both loci has a three-banded Hb1, a four-banded Hb2, and a three-banded HbX. We conclude that Hb2 contains n -polypeptides and n -polypeptides. Hb1 contains 2n -polypeptides. HbX contains 2n -polypeptides. During electrophoresis, the three native hemoglobins undergo reversible dissociation to n subunits. Subunits with the same charge reassemble to migrate as molecules of the same size as the native molecules. Although there is no evidence for an additional polypeptide in the three hemoglobins, we cannot exclude such a possibility. If it exists, it is under three constraints: (1) it must be present in equal amounts in each of the three hemoglobins; (2) it must have the same molecular weight as the - and -polypeptides; and (3) it must be free of genetic variation (detectable by electrophoresis).Supported by National Institutes of Health Grant HE-11445.     

Oxidation-reduction reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park

The kinetics and equilibrium of the redox reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park using the iron (II) and iron (III) complexes of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (CDTA4-) as the reducing and oxidizing agents have been studied. With respect to the equilibrium it was found that hemoglobin M Iwate (where the beta chains were reduced) was more readily reduced than hemoglobin M Hyde Park (where the alpha chains are reduced). This difference was shown to be a result of a difference in the rate constant for reduction but not oxidation. The observed rate contants for the reduction of all three hemoglobins were shown to decrease with increasing pH. This was attributed to a decrease in the [T]/[R] ratio. The observed rate contants for the oxidation reaction were shown to increase with increasing pH. Accompanying this increase was a change in the kinetic profile for hemoglobin A from pseudo first order to one in which the rate increased as the extent of reaction increased. Inositol hexaphosphate had no effect on the rate of oxidation of deoxyhemoglobin A. This was a result of binding of FeCDTA2- or HCDTA3- to the protein. However, in the presence of inositol hexaphosphate, the reduction of methemoglobin A exhibited biphasic kinetics. This result was interpreted in terms of the production of a small amount of a conformation which was more readily reduced.     

Oxygen equilibria of hemoglobin A and hemoglobin S valency hybrids.

Oxygen equilibrium determinations with “unsymmetrical” MetHb/Hb hybrids derived from human hemoglobins A and S are reported. All four of the possible hybrids have higher oxygen affinity than the parent hemoglobins. The α₂^Metβ₂^S hybrid has a lower oxygen affinity than that of α₂^Metβ₂^S. However, both the β^Met hybrids have similar oxygen affinity. The Bohr value of α₂^Metβ₂^S is more negative than that of α₂^Metβ₂^A while the β^Met hybrids appear to have almost identical Bohr values. These findings favor the view that α and β chains in hemoglobin A have different conformations and indicate that hemoglobin S has a β-chain conformation different from that of β-chain of hemoglobin A. This difference is probably carried into the oxygenation properties of the α-chain in such a way as to be reflected only when the β chain is oxidized.     

The hemoglobins of Artemia salina. V. Genetic variation in hemoglobin 1, hemoglobin 2, and hemoglobin 3

Electrophoretic mobilities of three hemoglobins (Hb1, Hb2, and Hb3) were studied in 15 populations of brine shrimps. Genetic segregation data support the model that Hb2 contains n -polypeptides and n -polypeptides; Hb1 contains 2n -polypeptides. Hb3 contains neither - nor -polypeptides. There is no evidence of linkage of and loci with each other or with the locus (or loci) which governs Hb3 or with the nonhomologous portion of the sex chromosomes. Hemoglobins of different populations may be hybridized in vitro by incubation at high temperature. Reversible dissociation to subunits which contain only one ( or ) polypeptide occurs at 40 C (for Hb1) and at 50 C (for Hb2).Supported by Grant HD-11445 from the National Institutes of Health.     

A study of baboon hemoglobin.

Electrophoretic analysis of hemoglobin types of 409 baboons of various species, mostly from Senegal, corresponds with the findings of other authors. Baboon hemoglobin is homogeneous as a whole, but differs electrophoretically from that of other monkey species. However, a difference in the electrophoretic mobility of the nonhemoglobin fraction of Papio anubis and Papio cynocephalus suggests a possibly different amino-acid sequence. This information may be useful for the classification in doubtful cases.     

Blood pressure changes after intravenous administration of cell-free hemoglobin A and hemoglobin H in the rat.

Hemoglobin H (HbH) is a tetramer of four beta chains (present in erythrocytes of alpha thalassemia), whereas hemoglobin A is a tetramer of two alpha and two beta chains. Since HbH is known to bind four times more nitric oxide (a vasodilator) at its sulfhydryls compared to HbA, the present studies were conducted to see the effect of HbH and HbA on rat blood pressure. The acute administration (20-2000 nmol/kg) of both HbH and HbA produced a dose-dependent effect on blood pressure. The net change in mean arterial pressure was significantly higher with HbH compared to HbA. Partially nitrosylated (in which SH groups are occupied with NO) HbH retained the property of raising blood pressure to some extent while HbA lost this property. Completely nitrosylated (in which both heme and SH groups are occupied with NO) derivatives of both HbH and HbA reduced the blood pressure to the same extent. The preliminary studies with chronic administration of HbA and HbH resulted in nonsignificant increase in blood pressure. It is concluded that these findings may explain the earlier observations of increased risk of hypertension in individuals with alpha thalassemia.     

Hereditary persistence of fetal hemoglobin, beta thalassemia, and the hemoglobin delta-beta locus: further family data and genetic interpretations.

Three Negro kindreds with hereditary persistence of fetal hemoglobin (HPFH) alone and in combination with various other hemoglobin abnormalities including beta thalassemia are presented. Among 11 offspring of two women heterozygous for both HPFH and the delta chain mutation Hb B2, five inherited the HPFH gene and six inherited the Hb B2 gene. In another kindred, a man inferred to be heterozygous for both HPFH and Hb C had six children; three offsprivg obtained the Hb C gene and three the HPFH gene. Similarly, a woman heterozygous for both Hb S and HPFH transmitted the Hb S gene to one of her two children and the HPFH gene to the other. Thus among 19 offspring, no crossovers between the HPFH locus or the Hb delta-beta locus were observed. These and earlier data are compatible with deletion of the Hb beta and delta loci as the primary event to explain the genetic origin of HPFH. Genetic considerations indicate that the finding of a single person with a hematologically normal phenotype among offspring of heterozygotes for both the African type of HPFH and a Hb beta or Hb delta structural abnormality would invalidate the deletion model.     

The kinetics and equilibria of squirrel-fish hemoglobin. A Root effect hemoglobin complicated by large subunit heterogeneity.

The functional properties of squirrel-fish hemoglobin have been measured by studying ligand binding equilibria and kinetics. The results show that squirrel-fish hemoglobin has a Root effect with a corresponding stabilization of the low affinity state. The properties of this state are pH dependent even in the absence of cooperativity. The effect of ATP shifts the overall ligant affinity towards the low affinity state and is characteristic of the allosteric effect caused by organic phosphates. Under pH and ATP conditions favoring the low affinity conformational state, a 10-fold difference in the binding kinetics of carbon monoxide to the alpha and beta subunits is observed.     

Gelation of sickle hemoglobin. III. Nitrosyl hemoglobin.

Sickle cell nitrosyl hemoglobin was examined for gelation by an ultracentrifugal method previously described (Briehl &; Ewert, 1973) and by birefringence. In the presence of inositol hexaphosphate gelation which exhibited the endothermic temperature dependence seen in gels of deoxyhemoglobin S was observed by both techniques. In the absence of inositol hexaphosphate no gelation was observed, nor did nitrosyl hemoglobin A exhibit gelation. On the assumption that gelation is dependent on the deoxy or T (low ligand affinity) as opposed to the oxy or R (high ligand affinity) quaternary structure this supports the conclusion that nitrosyl hemoglobin S in inositol hexaphosphate assumes the T structure, in contrast to the other liganded ferrohemoglobin derivatives oxy and carbon monoxide hemoglobin. Assuming further that the quaternary structures and isomerizations are the same in hemoglobins A and S it can also be concluded that nitrosyl hemoglobin A in inositol hexaphosphate assumes the T state. Since no gelation was seen in stripped nitrosyl hemoglobin S, inositol hexaphosphate serves to effect an R to T switch in this derivative. Thus R-T isomerization in nitrosyl hemoglobin occurs without change in ligand binding at the sixth position of the heme group confirming the conclusion of Salhany (1974) and Salhany et al. (1974).Lowering of the pH toward 6 favors gelation of NO hemoglobin S as it does of deoxy and aquomethemoglobin S (Briehl &; Ewert, 1973,1974), consistent with a favoring of the T structure due to strengthening of the interchain salt bridges and the binding of inositol hexaphosphate and/or changes in site-to-site interactions on which gelation depends.     

Resonance Raman and EPR of nitrosyl human hemoglobin and chains, carp hemoglobin, and model compounds. Implications for the nitrosyl heme coordination state.

We report the joint resonance Raman (RR) and electron paramagnetic resonance (epr) study of five- and six-coordinate nitrosyl heme model compounds and of the titled nitrosyl hemoproteins. Both epr and RR spectra fall into two types which, in the models, correspond to five- and six-coordinate nitrosyl hemes. However, neither RR nor epr spectroscopy is highly sensitive to the nature of the bond between a nitrosyl heme and a coordinated nitrogenous base, nor do the results of one technique uniformly correlate with those of the other. It is not possible to use epr spectroscopy as a test for the coordination state of a nitrosyl heme. The position of the highest frequency (depolarized) RR band possibly provides such a test. Any breaking of the very weak bond between nitrosyl heme and proximal histidine in T state human HbNO is more a consequence of tertiary structural features unique to the human alphaNO chains than it is of properties of the T quaternary conformation.     

Cys-93-betabeta-succinimidophenyl polyethylene glycol 2000 hemoglobin A. Intramolecular cross-bridging of hemoglobin outside the central cavity

Bis(maleidophenyl)-PEG2000 (Bis-Mal-PEG2000), a new bifunctional protein cross-linker targeted to sulfhydryl groups, introduces intra-tetrameric cross-links into oxy-HbA in nearly quantitative yields. Structural as well as crystallographic analyses of the cross-linked species, Bis-Mal-PEG2000 HbA, identified Cys-93(beta) as the site of intramolecular cross-linking. The cross-bridging had only a limited influence on the O(2) affinity and cooperativity of HbA in 50 mM BisTris acetate, pH 7.4. However, the Bohr effect was reduced by approximately 60%. Bis-Mal-PEG2000 HbA retained sensitivity to the presence of allosteric effectors 2, 3-diphosphoglycerate, IHP, and chloride, albeit to a lesser degree compared with HbA. Crystallographic analysis revealed the overall structure of deoxy-Bis-Mal-PEG2000 HbA to be similar to deoxy-HbA but for the loss of the salt bridge between Asp-94(beta) and His-146(beta). The large influence of the cross-bridging on the alkaline Bohr effect of HbA is consistent with the loss of this salt bridge. Unlike the "central cavity cross-bridges" described previously, the cross-link introduced by Bis-Mal-PEG2000 into HbA is an "outside the central cavity cross-bridge." In view of its oxy-conformational specificity and limited influence on O(2) affinity, this new cross-linking strategy holds promise for the stabilization of new designer low O(2) affinity Hbs generated by recombinant DNA technology for applications as Hb based therapeutics.     

Interaction of butene with human hemoglobin A.

The binding of various alkanes by proteins was recognized years ago. We have studied the effect of butene (C4H8), a short-chain aliphatic hydrocarbon, on the functional properties of human adult hemoglobin. Under 1 atm pressure (100 kPa) butene decreased the affinity of hemoglobin (Hb) for oxygen (p50) by 45% without altering the cooperativity of ligand binding. This effect was independent of pH (from 7.0 to 8.0) and of ionic strength. The changes in the affinity of hemoglobin for oxygen were dependent upon the partial pressure of butene and evoked a saturating mechanism of the binding site(s). Mathematical simulation of the curve relating p50 to the concentration of dissolved butene allowed us to calculate the apparent association constants for one single binding site KHb = 10.4 mmol-1 and KHbO2 = 1.53 mmol-1 to Hb and HbO2 respectively. The larger binding of butene by Hb was confirmed by a 25% decrease in K1, the first association constant of oxygen to the tetrameric hemoglobin. It is concluded that butene is an allosteric effector of human Hb which acts most likely through hydrophobic interactions. It is postulated that the oxygen-linked binding site may be located at the alpha 1 beta 2 interface.