The initiation of fetal hemoglobin biosynthesis.

Biosynthesis of the alpha and beta chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the delta chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent gamma chain at the time of removal of its N-terminal methionine was estimated to be 40--60 from the relative yields of labeled tryptic peptides.     

The molecular basis of hemoglobin Grady.

DNA from individuals heterozygous for the extended alpha-chain variant Hb Grady were studied by gene counting and restriction enzyme analysis. Neither method indicated the presence of an extra (fifth) alpha gene, which argues that if this variant arose by unequal crossing over, the event most likely involved mispairing between alleles rather than between the separate alpha 1 and alpha 2 loci.     

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.     

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.     

Action of hemoglobin. The energy of interaction

The energy of interaction is developed for three of the models applied to the action of hemoglobin. Evaluation of an energy of interaction requires a model defining the unperturbed or statistical process from which the real system is derived. The shape of a ligand binding curve by itself does not yield an energy of interaction since a steepened ligand binding curve may result from either a negative or positive energy of interaction.     

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.     

The preferential adsorption of hemoglobin to polyethylene.

Hemoglobin adsorption to foreign surfaces has not previously been considered in studies of blood-material interactions, despite the fact that hemoglobin is the most abundant protein present in blood. A hemoglobin-like protein was detected on a number of surfaces exposed to blood plasma, serum, and red cell suspensions. Hemoglobin adsorption to polyethylene from plasma was found to approximately equal the amount of adsorption of albumin and fibrinogen. The high relative affinity of hemoglobin for polyethylene was further confirmed by adsorption isotherm and direct competition experiments. The data from all four experimental methods support the following ranking of plasma protein affinity for polyethylene: Hemoglobin greater than fibrinogen greater than albumin congruent to gamma-globulin.     

Spot hemoglobin. Studies on the Root effect hemoglobin of a marine teleost.

The Spot, Leiostomus xanthrus, has a single tetrameric hemoglobin. Structural studies indicate the presence of alpha- and beta-like chains with COOH-terminal sequences of --Arg and --TYR-His, respectively, the same as is found in human hemoglobin. Spot hemoglobin possesses a Root effect: a heterotropic control mechanism like the Bohr effect but with more extreme pH dependence in the equilibria and kinetics of O2 and CO binding. The Root effect seems to be a molecular adaptation, in that pH- and anion-sensitive hemoglobins may help fish achieve neutral buoyancy by facilitating O2 delivery to the swim bladder. Changes in the kinetics of both "on" and "off" processes contribute to the greatly decreased ligand affinity of Spot hemoglobin at low pH. The time course ofligand combination at low pH is biphasic and wavelength dependent, suggesting a differential effect of pH on the alpha- and beta-like chains. The change in the shape of the ligand-binding curve with pH may be interpreted in terms of a proton-dependent transition between low (T) and high (R) affinity conformations. However, this may not be the only mechanism, since differential pH effects on the two types of chains may also contribute to the observed pH dependence.     

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.     

Mouse hemoglobin during gestation. Absence of fetal hemoglobin

A "fetal hemoglobin' has been reported to exist during mouse gestation, Investigations using CMC chromatography, starch gel electrophoresis or isoelectric focusing have shown a hemoglobin band from fetal tissues, and blood was obtained which was different from the adult hemoglobin and designated a "fetal hemoglobin'. In the current study isoelectric focusing was used to study the hemoglobins existing in the tissues and blood during fetal and neonatal development and the results suggest there is no "fetal hemoglobin' present during gestation. It appears that the hemoglobin designated as "fetal' in our laboratory was a methemoglobin formed by an incomplete reaction of KCN with the hemoglobin. The additional hemoglobin bands which were obtained from fetal liver or neonatal spleen tissues appeared to be a modified adult hemoglobin.     

The reaction between hemoglobin alpha-subunit and haptoglobin.

The interaction between human hemoglobin alpha-subunit and porcine haptoglobin was investigated by polyacrylamide gel electrophoresis, gel filtration chromatography, sedimentation through excess alpha-subunit and gel filtration in an alpha-subunit-containing medium. No interaction was detected by the first two methods indicating dissociation of the complex during the application of these separation techniques. The latter two methods, in which the complex is studied in a medium of excess subunits, showed that haptoglobin became saturated with the binding of two alpha-subunits.     

Arctic life adaptation--I. The function of reindeer hemoglobin

1. The functional properties of hemoglobin from the reindeer (Rangifer tarandus tarandus L.) are characterized as a function of pH, temperature and organic phosphate concentration. 2. Alongside overall similarities shared with most vertebrate hemoglobins, hemoglobin from the reindeer shows significant differences with respect to the effect of both organic phosphates and chloride anions. 3. The limited effect of temperature on oxygen binding (delta H = -4 kcal/mol O2) could be regarded as an interesting case of molecular adaptation to extreme environmental conditions.     

The reaction of deoxy-sickle cell hemoglobin with hydroxyurea.

In addition to its capacity to increase fetal hemoglobin levels, other mechanisms are implicated in hydroxyurea's ability to provide beneficial effects to patients with sickle cell disease. We hypothesize that the reaction of hemoglobin with hydroxyurea may play a role. It is shown that hydroxyurea reacts with deoxy-sickle cell hemoglobin (Hb) to form methemoglobin (metHb) and nitrosyl hemoglobin (HbNO). The products of the reaction as well as the kinetics are followed by absorption spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. Analysis of the kinetics shows that the reaction can be approximated by a pseudo-first order rate constant of 3.7x10(-4) (1/(s.M)) for the disappearance of deoxy-sickle cell hemoglobin. Further analysis shows that HbNO is formed at an observed average rate of 5.25x10(-5) (1/s), three to four times slower than the rate of formation of metHb. EPR spectroscopy is used to show that the formation of HbNO involves the specific transfer of NO from the NHOH group of hydroxyurea. The potential importance of this reaction is discussed in the context of metHb and HbNO being able to increase the delay time for sickle cell hemoglobin polymerization and HbNO's vasodilating capabilities through conversion to S-nitrosohemoglobin.