Pseudo cross-link of human hemoglobin with mono-(3,5-dibromosalicyl)fumarate

The reaction of human oxyhemoglobin with mono(3,5-dibromosalicyl)fumarate, produces a derivative specifically acylated at the two lysines beta 82, which can be purified with a 70% yield. The oxygen affinity of this derivative at 37 degrees C at pH 7.4, 0.1 M Cl- is of 12 mm Hg, and is not affected by organic phosphate. In the presence of 5% CO2, the oxygen affinity decreases to 25 mm Hg. In all cases the cooperativity is lowered, with a value of n in the Hill plots near 2. Sedimentation velocity measurements indicate that, contrary to normal hemoglobin, this derivative fails to dissociate into dimers upon exposure to pH 5.5. The stability of the tetrameric structure is probably due to a modification of the beta-beta interface, resulting from electrostatic and hydrophobic interactions introduced in the beta cleft by the fumaryl residues. These new interactions are probably the origin of a new reverse Bohr effect group at alkaline pH. Consistent with the stabilization of the tetrameric structure, the half-time of retention of this compound in the rat is increased 4-fold with respect to that of normal hemoglobin. These characteristics cast a favorable light on the usage of this compound as an oxygen carrier in transfusional and perfusional fluids.     

Equilibrium oxygen binding to human hemoglobin cross-linked between the alpha chains by bis(3,5-dibromosalicyl) fumarate

Oxygen equilibrium curves of human hemoglobin Ao (HbAo) and human hemoglobin cross-linked between the alpha chains (alpha alpha Hb) by bis(3,5-dibromosalicyl) fumarate were measured as a function of pH and chloride or organic phosphate concentration. Compared to HbAo, the oxygen affinity of alpha alpha Hb was lower, cooperativity was maintained, although slightly reduced, and all heterotropic effects were diminished. The major effect of alpha alpha-cross-linking appears to be a reduction of the oxygen affinity of R-state hemoglobin under all conditions. However, while the oxygen affinity of T-state alpha alpha Hb was slightly reduced at physiologic chloride concentration and in the absence of organic phosphates, KT was the same for both hemoglobins in the presence of 2,3-diphosphoglycerate (or high salt) and higher for alpha alpha Hb in the presence of inositol hexaphosphate. The reduced O2 affinity arises from smaller binding constants for both T- and R-state alpha alpha Hb rather than through stabilization of the low affinity conformation. All four Adair constants could be determined for alpha alpha Hb under most conditions, but a3 could not be resolved for HbAo without constraining a4, suggesting that the cross-link stabilizes triply ligated intermediates of hemoglobin.     

Thermal stability of hemoglobin crosslinked in the T-state by bis(3,5-dibromosalicyl) fumarate

Bis(3,5-dibromosalicyl) fumarate was used to crosslink oxyhemoglobin between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxyhemoglobin between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R.Y., et al. (1986) J. Biol. Chem. 261, 9929). Thermal denaturations demonstrated that alpha crosslinked hemoglobin (alpha 99XLHb A) has the same stability as the beta crosslinked one (beta 82XLHb A). Both alpha and beta crosslinked methemoglobins have a denaturation temperature in 0.9 M guanidine of 57 degrees C compared to 41 degrees C of Hb A. The second product from the T-state crosslinking reaction was found to be crosslinked between the beta chains by chain separation and amino acid analysis. The possible positions for this crosslink are limited to the bisphosphoglycerate binding site in the three-dimensional structure. Its stability is comparable to that of the alpha 99XLHb A or beta 82XLHb A. These modified hemoglobins are potential blood substitutes.     

Oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between 82 beta 1 and 82 beta 2 lysyl residues by bis(3,5-dibromosalicyl) fumarate

We investigated oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between lysine-82 beta 1 and lysine-82 beta 2 by a fumaryl group, which is prepared by reaction of the CO form with bis(3,5-dibromosalicyl) fumarate. The cross-linked hemoglobin preparation isolated by the previous purification method, namely, gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography, was found to be contaminated with about 20% of an electrophoretically silent impurity that shows remarkably high affinity for oxygen. This impurity was separated from the desired cross-linked hemoglobin by a newly developed purification method, which utilizes a difference between the authentic hemoglobin and the impurity in reactivity of the sulfhydryl groups of cysteine-93 beta toward N-ethylmaleimide under a deoxygenated condition. After this purification procedure, the oxygen equilibrium properties of purified cross-linked hemoglobin in the absence of organic phosphate became very similar to those of unmodified hemoglobin with respect to oxygen affinity, cooperativity, and the alkaline Bohr effect. The functional similarity between the cross-linked hemoglobin and unmodified hemoglobin allows us to utilize this cross-linking for preparing asymmetric hybrid hemoglobin tetramers, which are particularly useful as intermediately liganded models. Previous studies on this type of cross-linked hemoglobin should be subject to reexamination due to the considerable amount of the impurity.     

Structural features required for the reactivity and intracellular transport of bis(3,5-dibromosalicyl)fumarate and related anti-sickling compounds that modify hemoglobin S at the 2,3-diphosphoglycerate binding site

Bis(3,5-dibromosalicyl)fumarate (I) reacts preferentially with oxyhemoglobin to cross-link the two beta 82 lysine residues within the 2,3-diphosphoglycerate (DPG) binding site and as a result markedly increases the solubility of deoxyhemoglobin S. The cross-link acts by perturbing the acceptor site for Val 6 within the sickle cell fiber (Chatterjee, R., Walder, R. Y., Arnone, A., and Walder, J. A. (1982) Biochemistry 21, 5901-5909). In the present studies we have compared a large number of analogs of I to determine the structural features of the reagent required for specificity and for transport into the red cell. Both electrostatic and hydrophobic interactions contribute to the binding of these compounds at the DPG site. The optimal position for the negatively charged groups on the cross-linking agent for productive binding is adjacent to the ester as in the original salicylic acid derivatives. There is a direct correlation between the reactivity toward hemoglobin and the hydrophobicity of the substituent attached at the para position. Phenyl and substituted phenyl derivatives as in the analgesic, antiinflammatory drug diflunisal are particularly effective. These groups probably interact with hydrophobic residues of the amino-terminal tripeptide and the EF corner of the beta chains adjacent to the DPG binding site. Although bis(3,5-dibromosalicyl)fumarate is very reactive toward hemoglobin in solution, it is much less effective in modifying hemoglobin within the red cell. The reaction with intracellular hemoglobin was shown to be limited by competing hydrolysis of the reagent catalyzed at the outer surface of the erythrocyte membrane. Inactivation of the red cell membrane acetylcholinesterase with phenylmethylsulfonyl fluoride did not inhibit this reaction. Introduction of a single methyl group onto the carbon-carbon double bond of the fumaryl moiety decreases the lability of the ester 10-fold, due to steric effects, and allows the reagent to be taken up by the red cell and modify intracellular hemoglobin. The kinetics of transport of the methylfumarate derivative, bis(3,5-dibromosalicyl)mesaconate, are first-order, consistent with passive diffusion. The attachment of larger alkyl groups onto the cross-link bridge further enhances the transport of the reagent into the red cell. The solubility of deoxyhemoglobin S cross-linked with the butylfumarate derivative was found to be increased by almost 10% compared to the original fumarate diester.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of crosslinking on the thermal stability of hemoglobins. II. The stabilization of met-, cyanomet-, and carbonmonoxyhemoglobins A and S with bis(3,5-dibromosalicyl) fumarate

Hemoglobins A and S were crosslinked between Lys 82 beta 1 and Lys 82 beta 2 using bis (3,5-dibromosalicyl) fumarate (J. A. Walder et al. (1979) Biochemistry 18, 4265). Thermal denaturation experiments were used to compare the stabilities of the met, cyanomet, and carbonmonoxy forms of these crosslinked hemoglobins to the corresponding uncrosslinked proteins. Uncrosslinked carbonmonoxy- and cyanomethemoglobins had transition temperatures about 11 degrees C higher than the corresponding met samples. The increase in denaturation temperature (Tm) due to crosslinking was 15 degrees C for the methemoglobins, 10 degrees C for the cyanomethemoglobins, and 4 degrees C for the carbonmonoxy ones. There was no significant difference in stability between the met and carbonmonoxy crosslinked proteins. In order of increasing stability the samples were: met Hb S less than met Hb A less than CO Hb S less than CO Hb A = CN-met Hb A less than met XL-Hb S = CO XL-Hb S less than met XL-Hb A = CO XL-Hb A less than CN-met XL-Hb A. The slight decrease in the stability of Hb S (beta 6 Glu----Val) compared to Hb A can be explained by the replacement of an external ionic group by a hydrophobic residue in Hb S. In mixtures of crosslinked and normal Hb A, the Tm of the uncrosslinked material was slightly increased by the presence of the more stable crosslinked hemoglobin. The effects of both crosslinking and cyanide or carbon monoxide binding can be explained by Le Chatelier's principle since both would favor the native form of the protein.     

Enzyme electrokinetics: energetics of succinate oxidation by fumarate reductase and succinate dehydrogenase

Protein film voltammetry is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory flavoenzyme--the membrane-extrinsic catalytic domain of Escherichia coli fumarate reductase (FrdAB). The activity as a function of the electrochemical driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover conditions reveal the reduction potentials of the FAD, the stability of the semiquinone, relevant protonation states, and pH-dependent succinate--enzyme binding constants for all three redox states of the FAD. Fast-scan experiments in the presence of substrate confirm the value of the two-electron reduction potential of the FAD and show that product release is not rate limiting. The sequence of binding and protonation events over the whole catalytic cycle is deduced. Importantly, comparisons are made with the electrocatalytic properties of SDH, the membrane-extrinsic catalytic domain of mitochondrial complex II.     

Mouse alpha chains inhibit polymerization of hemoglobin induced by human beta S or beta S Antilles chains

A murine model of sickle cell disease was tested by studying the polymerization of hybrid hemoglobin tetramers between alpha mouse and human beta S or beta S Antilles chains were prepared from Hb S Antilles, which was a new sickling hemoglobin inducing a sickle cell syndrome more severe than Hb S. The hybrid molecules did not polymerize in solution, indicating that the mouse alpha chains inhibited fiber formation. Consequently, a mouse model for sickle cell disease requires the transfer and expression of both alpha and beta S or beta S Antilles genes.     

Alpha and beta chains of hemoglobin inhibit production of Staphylococcus aureus exotoxins

Prior studies suggest Staphylococcus aureus exotoxins are not produced when the organism is cultured in human blood. Human blood was fractionated into plasma and water-lysed red blood cells, and it was demonstrated that mixtures of alpha and beta globins of hemoglobin (as low as 1 mug/mL) inhibited S. aureus exotoxin production while increasing production of protein A and not affecting bacterial growth. Pepsin but not trypsin digestion destroyed the ability of alpha and beta globin to inhibit exotoxin production. Exotoxin production by both methicillin-resistant and methicillin-susceptible organisms was inhibited. Production of streptococcal pyrogenic exotoxin A by Streptococcus pyogenes was unaffected by alpha and beta globin chains but was inhibited when produced in S. aureus. Use of isogenic S. aureus strains suggested the targets of alpha and beta globin chains, leading to inhibition of staphylococcal exotoxins, included the two-component system SrrA-SrrB. delta hemolysin production was also inhibited, suggesting the two-component (and quorum sensing) system AgrA-AgrC was targeted. The alpha and beta globin chains represent promising molecules to interfere with the pathogenesis of serious staphylococcal diseases.     

Kinetic resolution of ligand binding to the alpha and beta chains within human hemoglobin.

Nitric oxide has been used as a chain-specific, spin label of unliganded heme groups present in kinetic mixtures of human hemoglobin and n-butyl isocyanide. In these experiments, deoxyhemoglobin was reacted with n-butyl isocyanide for a controlled time and then mixed rapidly with a high concentration of nitric oxide to fill residual, unoccupied heme sites. The final mixture was frozen immediately after formation to prevent any displacement of bound isonitrile. The EPR spectrum of the frozen sample was resolved into alpha and beta nitric oxide components; these reflect the relative proportions of alpha- and beta-heme sites which were unoccupied by n-butyl isocyanide. Individual time courses for the alpha and beta subunits were obtained by varying the time between the formation of the isonitrile/hemoglobin mixture and its reaction with nitric oxide. At pH 7.0 only the beta chain time course exhibits an initial rapid phase; the alpha chain time course is monophasic, exhibiting almost, exponential behavior. This result shows unequivocally that the beta-hemes within deoxyhemoglobin react much more rapidly with n-butyl isocyanide than the alpha hemes.     

Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase.

Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E. coli strain. Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated. Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.     

UV resonance Raman study of beta93-modified hemoglobin A: chemical modifier-specific effects and added influences of attached poly(ethylene glycol) chains.

The reactive sulfhydryl group on Cys beta93 in human adult hemoglobin (HbA) has been the focus of many studies because of its importance both as a site for synthetic manipulation and as a possible binding site for nitric oxide (NO) in vivo. Despite the interest in this site and the known functional alterations associated with manipulation of this site, there is still considerable uncertainty as to the conformational basis for these effects. UV resonance Raman (UVRR) spectroscopy is used in this study to evaluate the conformational consequences of chemically modifying the Cys beta93 sulfhydryl group of both the deoxy and CO-saturated derivatives of HbA using different maleimide and mixed disulfide reagents. Included among the maleimide reagents are NEM (n-ethylmaleimide) and several poly(ethylene glycol) (PEG)-linked maleimides. The PEG-based reagents include both different sizes of PEG chains (PEG2000, -5000, and -20000) and different linkers between the PEG and the maleimide. Thus, the effect on the conformation of both linker chemistry and PEG size is evaluated. The spectroscopic results reveal minimal perturbation of the global structure of deoxyHbA for the mixed disulfide modification. In contrast, maleimide-based modifications of HbA perturb the deoxy T state of HbA by "loosening" the contacts associated with the switch region of the T state alpha(1)beta(2) interface but do not modify the hinge region of this interface. When the NEM-modified HbA is also subjected to enzymatic treatment to remove the C-terminal Arg alpha141 (yielding NESdes-ArgHb), the resulting deoxy derivative exhibits the spectroscopic features associated with a deoxy R state species. All of the CO-saturated derivatives exhibit spectra that are characteristic of the fully liganded R structure. The deoxy and CO derivatives of HbA that have been decorated on the surface with large PEG chains linked to the maleimide-modified sulfhydryl through a short linker group all show a general intensity enhancement of the tyrosine and tryptophan bands in the UVRR spectrum. It is proposed that this effect arises from the osmotic impact of a large, close PEG molecule enveloping the surface of the protein.     

Use of heme spin-labeling to probe heme environments of alpha and beta chains of hemoglobin.

A spin label attached to a propionic acid group of the heme has been used to probe the heme environment of the alpha and beta chains of hemoglobin in both the subunit and tetrameric forms. The electron paramagnetic resonance (EPR) studies of hemoglobin hybrids in which the spin label is attached to either the alpha- or beta-heme (alpha2SLbeta 2 or alpha2beta2SL) and spin-labeled isolated chains (alphaSL and betaSL) show that: 1) alpha- and beta-hemes have different environments in the tetrameric forms of oxy-, deoxy-, and methemoglobins as well as in isolated single chains; 2) when isolated subunits associate to form hemoglobin tetramers, the environment of the alpha-heme changes more drastically than that of the beta-heme; 3) upon deoxygenation of hemoglobin, the structure in the vicinity of the alpha-heme changes more drastically than that of the beta-heme; and 4) upon the addition of organic phosphates to methemoglobin, the change in the spin state of the heme irons mainly arises from beta-heme. The results demonstrate conclusively that the alpha and the beta subunits of hemoglobin are structurally nonequivalent as are their structural changes as the result of ligation. The relationship of EPR spectrum and structure of hemoglobin is discussed.     

Molecular cloning and characterization of hemoglobin alpha and beta chains from plateau pika (Ochotona curzoniae) living at high altitude

Hemoglobin (Hb) plays an important role in oxygen transfer from lung to tissues. Possession of a Hb with high oxygen affinity helps highland animals to adapt to high altitude, has been studied profoundly. Plateau pika (Ochotona curzoniae), a native species living at 3,000-5,000 m above sea level on Qinghai-Tibet Plateau, is a typical hypoxia and low temperature tolerant mammal. To investigate the possible mechanisms of plateau pika Hb in adaptation to high altitude, the complete cDNA and amino acid sequences of plateau pika hemoglobin alpha and beta chains have been described. Compared with human Hb, alterations in important regions can be noted: alpha111 Ala-->Asn, beta35 Tyr-->Phe, beta112 Cys-->Val, beta115 Ala-->Ser, and beta125 Pro-->Gln. Phylogenetic analysis of alpha and beta chains shows that plateau pika is closer to rabbit than to other species. This study provides essential information for elucidating the possible roles of hemoglobin in adaptation to extremely high altitude in plateau pika.