Association-dissociation of molecules of hemoglobin and polymeric hemoglobin in solutions

The process of association-dissociation of hemoglobin molecules into dimers of its subunits in water and water-saline solutions is studied by the method of gel-penetrating chromatography and ultrafiltration. The quantitative assessment of stabilization of quaternary structure of hemoglobin in chemically bound polymer derivative in comparison with native peptide on the basis of building differential concentration curves is conducted for the first time. By the method of atomic-force spectroscopy, the morphology of nanoparticles of hemoglobin and its modified polymeric derivative is studied.     

Alternative diaspirins for modification of hemoglobin and sickle hemoglobin

Studies of modification of hemoglobin and of sickle hemoglobin by alternative aspirins have been extended to a series of new bis esters with a variety of substituted bridging diacids and to a group of mono esters with polar acyl groups. Rates of hydrolysis of these alternative aspirins have also been examined, and they reveal that a careful balance between stability and reactivity is essential for optimal activity. Four-carbon bridging groups have been found to be particularly effective, two of these raising the minimum gelling concentration of sickle hemoglobin by as much as 100%.     

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.     

Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin. Solution and X-ray studies of carboxymethylated hemoglobin and X-ray studies of carbamino hemoglobin

Hemoglobin can be specifically carboxymethylated at its NH2-terminal amino groups (i.e. HbNHCH2COO-) to form the derivatives alpha 2Cm beta 2, alpha 2 beta 2Cm, and alpha 2Cm beta 2Cm, where Cm represents carboxymethyl. Previous studies (DiDonato, A., Fantl, W. J., Acharya, A. S., and Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895) suggested that these derivatives could be used as stable analogues of the corresponding carbamino (Hb-NHCOO-) forms of hemoglobin, adducts that are generated reversibly in vivo when CO2 combines with alpha-amino groups. In this paper we present x-ray diffraction studies of both carbamino hemoglobin and carboxymethylated hemoglobin that verify this proposal and we use the carboxymethylated derivatives to study the functional consequences of placing a covalently bound carboxyl group at the NH2 terminus of each hemoglobin subunit. Our studies also provide additional information concerning the oxygen-linked binding of anions and protons to Val-1 alpha. Difference electron density analysis of deoxy alpha 2Cm beta 2Cm versus the unmodified deoxyhemoglobin tetramer (deoxy alpha 2 beta 2) shows that the covalently bound carboxyl moieties replace inorganic anions that are normally bound to the free NH2-terminal amino groups in crystals of native deoxyhemoglobin grown from solutions of concentrated (2.3 M) ammonium sulfate. In the case of the beta-subunits, the carboxymethyl group replaces an inorganic anion normally bound between the alpha-amino group of Val-1 beta, the epsilon-amino group of Lys-82 beta, and backbone NH groups at the NH2-terminal end of the F'-helix. In the case of the alpha-subunits, the carboxymethyl group replaces an anion that is normally bound between the alpha-amino group of Val-1 alpha and the beta-OH group of Ser-131 alpha. A corresponding difference electron map of carbamino deoxyhemoglobin in low-salt (50 mM KCl) crystals shows that CO2 bound in the form of carbamate occupies the same two anion binding sites. The alkaline Bohr effect of alpha 2Cm beta 2 is only marginally lower (approximately 7%) than that of alpha 2 beta 2. Previous studies (Kilmartin, J. V., 1977) have shown that about 30% of the alkaline Bohr effect is the result of an oxygen-linked change in the pK alpha of Val-1 alpha, and O'Donnell et al., 1979, found that this portion of the Bohr effect is the result of the oxygen-linked binding of chloride to Val-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of polymerization of deoxyhemoglobin S and mixtures of hemoglobin A and hemoglobin S at high hemoglobin concentrations

Transverse water proton relaxation times (T₂) have been measured as a function of time after deoxygenation of solutions containing hemoglobin S. The shortened T₂ values observed upon deoxygenation of hemoglobin S result from an increase in the correlation time (τ_c) of the water fraction irrotationally bound to deoxyhemoglobin S as it polymerizes. Therefore, the change in τ_c as a function of time after deoxygenation can be used to measure the rate of polymer formation. The change in τ_c observed is reasonably fit by the first-order equation τ = τ₀ (1 ? e^?kt) + τ_oxy. At a total hemoglobin concentration of approximately 300 mg/ml, the pseudo-first-order rate constant in a heterozygous AS sample is 25 times slower than in a homozygous S sample, k = 0.019 and 0.47 s^?1, respectively. Since the transit time for an erythrocyte in vivo is approximately 15 s, these results suggest that the heterozygous A/S erythrocyte would traverse the circulation and become reoxygenated before extensive polymerization and, therefore, cell sickling could occur. For the homozygous S/S erythrocyte, there is ample time for polymerization and for cell sickling during circulation.     

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.     

Xenopus laevis hemoglobin and its hybrids with hemoglobin A+

Isolated alpha and beta chains from Xenopus laevis hemoglobin have been purified. The isolation procedure yields native alpha chains whose functional behavior has been characterized and compared with that of human alpha chains. Isolated beta chains in the presence of oxygen are characterized by low stability, and hence their functional characterization was limited to the CO binding kinetics. When stoichiometric amounts of the isolated alpha and beta chains are mixed, a tetramer characterized by heme-heme interactions and oxygen affinity comparable to that of the native molecule is readily reconstituted. Moreover, both chains, under appropriate conditions, form stable hybrid tetramers with the partner subunits from human hemoglobin; results on the functional properties of these hybrid hemoglobins are presented and discussed in relation to the stereochemical model of the Root effect.     

Conformation and cooperativity in hemoglobin.

19-F and 31-P nuclear magnetic resonance (NMR) spectroscopy have been used to study the ligand binding process in human hemoglobin. 19-F nuclear magnetic resonance studies of hemoglobin specifically trifluoroacetonylated at cysteine-beta93 have permitted observation and characterization of molecular species containing two and three ligands. The behavior of these intermediate species in response to changes in pH and organic phosphate concentration is not completely consistent with any of the current theories of allostery. A model consistent with the 19-F and 31-P NMR data is proposed.     

Methylations in human hemoglobin

Levels of N-Methylvaline (MeVal) and N tau-methylhistidine (MeHis) were measured in male smokers and non-smokers in a program aimed at mapping background alkylations of hemoglobin (Hb) as potential indicators of doses of exogenous and endogenous genotoxic agents. MeVal was also determined in Hb from rats, Syrian golden hamsters, mice and chickens. MeVal was found to occur at levels around 0.5 nmole/g Hb, with relatively little variation between individuals and species. MeVal was not significantly affected by smoking. This result contrasts with elevated levels of N-hydroxyethylvaline (HOEtVal) measured in the same persons (T?rnqvist et al., 1986b). Levels of S-methylcysteine (MeCys) (Bailey et al., 1981) and MeHis were much higher than those of MeVal. The high levels of MeCys and MeHis may be due partly to misincorporation during protein synthesis and to artifacts. S-Adenosylmethionine and formaldehyde are possible endogenous sources of MeVal. One individual (smoker) out of 21 selected for measurement of MeVal was an outlier, with raised levels of both MeVal and HOEtVal, as would be expected in case of a defective detoxification system.     

Iron nitrosyl hemoglobin formation from the reactions of hemoglobin and hydroxyurea

Hydroxyurea represents an approved treatment for sickle cell anemia and acts as a nitric oxide donor under oxidative conditions in vitro. Electron paramagnetic resonance spectroscopy shows that hydroxyurea reacts with oxy-, deoxy-, and methemoglobin to produce 2-6% of iron nitrosyl hemoglobin. No S-nitrosohemoglobin forms during these reactions. Cyanide and carbon monoxide trapping studies reveal that hydroxyurea oxidizes deoxyhemoglobin to methemoglobin and reduces methemoglobin to deoxyhemoglobin. Similar experiments reveal that iron nitrosyl hemoglobin formation specifically occurs during the reaction of hydroxyurea and methemoglobin. Experiments with hydroxyurea analogues indicate that nitric oxide transfer requires an unsubstituted acylhydroxylamine group and that the reactions of hydroxyurea and deoxy- and methemoglobin likely proceed by inner-sphere mechanisms. The formation of nitrate during the reaction of hydroxyurea and oxyhemoglobin and the lack of nitrous oxide production in these reactions suggest the intermediacy of nitric oxide as opposed to its redox form nitroxyl. A mechanistic model that includes a redox cycle between deoxyhemoglobin and methemoglobin has been forwarded to explain these results that define the reactivity of hydroxyurea and hemoglobin. These direct nitric oxide producing reactions of hydroxyurea and hemoglobin may contribute to the overall pathophysiological properties of this drug.     

Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes — these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.     

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.     

Unusual CO bonding geometry in abnormal subunits of hemoglobin M Boston and hemoglobin M Saskatoon

To clarify the role of the proximal histidine (F8-His), distal His (E7-His), and E11 valine (E11-Val) in ligand binding of hemoglobin (Hb), we have investigated the resonance Raman (RR) spectra of the carbon monoxide adduct of Hbs M (COHb M) in which one of these residues was genetically replaced by another amino acid in either the alpha or beta subunit. In the fully reduced state, all Hbs M gave v3 at approximately 1472 cm-1 and vFe-His at 214-218 cm-1, indicating that they have a pentacoordinate heme and the heme iron is bound to either E7-His or F8-His. The porphyrin skeletal vibrations of the COHb M were essentially unaltered by replacements of E7- or F8-His with tyrosine (Tyr) and of E11-Val by glutamic acid (Glu). The vCO, vFe-CO, and delta Fe-C-O frequencies of COHb M Iwate (alpha F8-His----Tyr), COHb M Hyde Park (beta F8-His----Tyr), and COHb M Milwaukee (beta E11-Val----Glu) were nearly identical with those of COHb A. In contrast, the RR spectra of COHb M Boston (alpha E7-His----Tyr) and COHb M Saskatoon (beta E7-His----Tyr) gave two new Raman bands derived from the abnormal subunits, vFe-CO at 490 cm-1 and vCO at 1972 cm-1, in addition to those from the normal subunits at 505 cm-1 (vFe-CO) and 1952 cm-1 (vCO). The CO adduct of the abnormal subunits exhibited apparently no photodissociation upon illumination of CW laser with a stationary cell under which the normal subunit exhibited complete photodissociation.(ABSTRACT TRUNCATED AT 250 WORDS)