The hemoglobin system of the serpent eel Ophisurus serpens: structural and functional characterization

The hemoglobin system of the serpent eel Ophisurus serpens was structurally and functionally characterized with the aim of comparing it to the hemoglobin system of other fish species, as oxygen loading under the severe habitat conditions experienced by O. serpens could have necessitated specific adaptation mechanisms during evolution. The hemoglobin system of O. serpens includes one cathodic and four anodic components. The molecular mass of the α and β chains of the cathodic component as well as the 2 α and 4 β of the anodic components were determined. Analysis of the intact α and β chains from cathodic hemoglobin and their proteolytic digestion products by high-resolution MS and MS/MS experiments resulted in 92 and 95 % sequence coverage of the α and β globins, respectively. The oxygen binding properties of both hemoglobin components were analyzed with respect to their interactions with their physiological effectors. Stripped cathodic hemoglobin displayed the highest oxygen affinity among Anguilliformes with no significant effect of pH on O₂-affinity. In the presence of both chloride and organic phosphates, O₂-affinity was strongly reduced, and cooperativity was enhanced; moreover, cathodic hemoglobin contains two indistinguishable GTP-binding sites. Stripped anodic hemoglobins exhibited both low O₂-affinity and low cooperativity and a larger Bohr effect than cathodic hemoglobin. The cathodic hemoglobin of O. serpens and the corresponding component of Conger conger share the greatest structural and functional similarity among hemoglobin systems of Anguilliformes studied to date, consistent with their phylogenetic relationship.     

Structural basis of heme reactivity in myoglobin and leghemoglobin: thermal difference spectra

Thermal pertubation difference spectra of sperm whale myoglobin (Mb) and soybean leghemoglobin a (Lb a) in the near-ultraviolet reveal similarities in the tryptophan environment of the two proteins. Of the two tryptophans in each protein, one has its indolyl NH group fully exposed to aqueous solvent, while the other behaves as if it were surrounded by motile but nonpolar residues with little access to water. These environments are not significantly altered by removal of the heme group. Assuming conformational homology, the helix-spacing role of Trp-A12 in Mb (Kendrew, J.C. (1962), Brookhaven Symp. Biol. 15, 216-228) may be taken over, in Lb a, by Trp-H8 which, though remote in linear sequence, would occupy a suitable spatial location. Thermal difference spectra in the Soret and visible regions of pure high-spin (fluoroferric) and pure low-spin (cyanoferric) complexes showed a red shift on cooling Mb complexes, reflecting a predominantly nonpolar environment around the heme, but a blue shift on cooling Lb complexes, reflecting a more solvent-exposed environment. Thermal difference spectra using rose bengal as a probe of the heme pockets in the two apoproteins supported these conclusions. Thermal difference spectra for the high-spin complexes of both Mb and Lb are slightly larger in magnitude than in the low-spin complexes. This may reflect a more flexible heme pocket in the high-spin state, as suggested by recent circular dichroic results. A structural basis for the high oxygen affinity of Lb compared with Mb is proposed, based upon the observed differences in polarity and flexibility of the heme pocket and in amino acid substitutions.     

Deferiprone: structural and functional modulating agent of hemoglobin fructation

Diabetic complication arises from the presence of advanced glycation end products in different sites of the body. Great attention should be paid to recognizing anti-glycation compounds. Here, deferiprone as an oral iron chelator drug administrated in treatment of β-thalassemic patients was selected to find its effect on the fructation of hemoglobin (Hb). Our results indicated that deferiprone could prevent the AGE and carbonyl formation via inhibition of structural changes in the structure of Hb during the fructation process. Moreover, deferiprone can preserve peroxidase and esterase activities of fructated Hb similar to native Hb. Therefore, deferiprone can be introduced as an anti-glycation drug to prevent the AGE formation.     

Killing of Trypanosoma brucei by concanavalin A: structural basis of resistance in glycosylation mutants

Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered oligosaccharides compared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.     

Tyrosine B10 and heme-ligand interactions of Lucina pectinata hemoglobin II: control of heme reactivity

The distal pocket of hemoglobin II (HbII) from Lucina pectinata is characterized by the presence of a GlnE7 and a TyrB10. To elucidate the functional properties of HbII, biophysical studies were conducted on HbII and a HbI PheB10Tyr site-directed mutant. The pH titration data at neutral conditions showed visible bands at 486, 541, 577 and 605 nm for both proteins. This suggests the possible existence of a conformational equilibrium between an open and closed configuration due to the interactions of the TyrB10, ligand, and heme iron. The kinetic behavior for the reaction of both ferric proteins with H2O2 indicates that the rate for the formation of the ferryl intermediates species varies with pH, suggesting that the reaction is strongly dependent on the conformational states. At basic pH values, the barrier for the reaction increases as the tyrosine adopts a closed conformation and the ferric hydroxyl replaces the met-aquo species. The existence of these conformers is further supported by resonance Raman (RR) data, which indicate that in a neutral environment, the ferric HbII species is present as a possible mixture of coordination and spin states, with values at 1558 and 1580 cm(-1) for the nu2 marker, and 1479, 1492, and 1503 cm(-1) for the nu3 mode. Moreover, the presence of the A3 and A(o) conformers at 1924 and 1964 cm(-1) in the HbII-CO infrared spectra confirms the existence of an open and closed conformation due to the orientation of the TyrB10 with respect to the heme active center.     

Resonance raman studies of heme structural differences in subunits of deoxy hemoglobin

Low frequency resonance Raman (RR) spectra are reported for deoxy hemoglobin (Hb), its isolated subunits, its analogue bearing methine-deuterated hemes in all four subunits (Hb-d(4)), and the hybrids bearing the deuterated heme in only one type of subunit, which are [alpha(d4)beta(h4)](2) and [alpha(h4)beta(d4)](2). Analyzed collectively, the spectra reveal subunit-specific modes that conclusively document subtle differences in structure for the heme prosthetic groups in the two types of subunits within the intact tetramer. Not surprisingly, the most significant spectral differences are observed in the gamma(7) mode that has a major contribution from out of plane bending of the methine carbons, a distortion that is believed to relieve strain in the high-spin heme prosthetic groups. The results provide convincing evidence for the utility of selectively labeled hemoglobin hybrids in unraveling the separate subunit contributions to the RR spectra of Hb and its various derivatives and for thereby detecting slight structural differences in the subunits.     

Novel mutants of 23S RNA: characterization of functional properties.

Single point mutations corresponding to the positions G2505 and G2583 have been constructed in the gene encoding E.coli 23S rRNA. These mutations were linked to the second mutation A1067 to T, known to confer resistance to thiostrepton (1). Mutant ribosomes were analyzed in vitro for their ability to direct poly(U) dependent translation, their missence error frequency and in addition their sensitivity to peptidyltransferase inhibitors. It was evident that the mutated ribosomes had an altered dependence on [Mg2+] and an increased sensitivity to chloramphenicol during poly(U) directed poly(Phe) synthesis. In a transpeptidation assay mutated ribosomes were as sensitive to chloramphenicol as wild-type ribosomes. However, the mutant ribosomes exhibited an increased sensitivity to lincomycin. An increase in translational accuracy was attributed to the mutations at the position 2583: accuracy increased in the order G less than A less than U less than C.     

Behavioral mutants of Euglena gracilis: functional and spectroscopic characterization

Three mutant strains of the phytoflagellate Euglena gracilis Z have been characterized in order to analyze the signal perception and signal transduction pathways involved in photo- and gravitaxis. Using the fluorescence of the chromophoric groups believed to be involved in photoperception (flavins and pterins) a method was developed for an in situ and in vivo detection of the paraxonemal body, the proposed location of the photoreceptor molecules. Two of the mutant strains, 1224-5/9f and 1224-5/1f, do not possess a stigma and also lack a paraxonemal body, as indicated by fluorescence measurements. The third strain, FB, has a small stigma, but only some cells contain a paraxonemal body. In contrast to the present hypothesis on photoorientation of Euglena, all strains were able to orient with respect to the light direction. However, the mutant strains did not show any orientation at low irradiances. At medium and high irradiances the strains 1224-5/9f and 1224-5/1f oriented perpendicular to the light direction (diaphototaxis) while cells of strains of FB showed partly negative phototaxis and partly diaphototaxis. Diaphototaxis was never observed in the wild type strain. Strains 1224-5/9f and 1224-5/1f showed normal graviresponses compared with the wild type. Astasia longa, a nonphtototactic relative of E. gracilis, as well as strain FB were both negative and positive gravitactic at all culture ages tested. This result confirmed the hypothesis that the paraxonemal body is not directly involved in graviperception.     

Bishistidyl heme hexacoordination, a key structural property in Drosophila melanogaster hemoglobin

Hemoglobins at high concentration have been isolated long ago from some insect larvae living in hypoxic environments. Conversely, a monomeric hemoglobin has been discovered recently in the fruit fly Drosophila melanogaster as intracellular protein expressed both in larvae and in the adult fly. Such a finding indicates that the oxygen supply in insects may be more complex than previously thought, relying not only on O2 diffusion through the tubular tracheal system, but also on carrier-mediated transport and storage. We present here the crystal structure of recombinant D. melanogaster hemoglobin at 1.20 A resolution. Spectroscopic data show that the protein displays a hexacoordinated heme, whose axial ligands are the proximal and distal His residues. Such bis-His ligation of the heme has sizable effects on the protein local structure. Three protein matrix cavities, comparable in size but not in topological locations with those of sperm whale myoglobin, are spread through the protein matrix; one of these can host a xenon atom. Additionally, D. melanogaster hemoglobin binds one molecule of 3-(cyclohexylamino)propanesulfonic acid (CAPS) buffer at a surface pocket, next to the EF hinge. Despite the high resolution achieved, no sequence/structure features specifically supporting the heme hexa- to pentacoordination transition required for diatomic ligand binding could be recognized.     

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.     

The hemoglobin of the common sting-ray, Dasyatis sabina: structural and functional properties.

1. The hemoglobin of the sting-ray, Dasyatis sabina, is both polymorphic and heterogeneous; three components predominate. 2. One major component has two kinds of polypeptide chain, of which one, presumably an alpha-chain, has a blocked NH2-terminus and an arginyl COOH-terminus, whereas carboxypeptidases A and B release tyrosine and histidine from the COOH-terminus of the beta-chain. 3. The amino acid sequence of the beginning NH2-terminal segment of the beta-chain of the major component has been determined. 4. The hemoglobin of the sting-ray, Dasyatis sabina, is highly resistant to urea and does not dissociate readily into subunits. 5. Oxygen binding by the hemoglobin is not affected by organic phosphates or high concentrations of either NaCl or urea. 6. The hemoglobin does not polymerize beyond tetramers. 7. Cooperativity, as monitored by n in the Hill equation, is pH-dependent and maximal between pH 8.5 and 9.0. 8. The hemoglobin has a large Bohr effect; the oxygen affinity is 16 times higher at pH 10 than at pH 6.5.     

Complexity of FGF receptors: genetic basis for structural diversity and functional specificity.

Since 1989, the receptors for fibroblast growth factors (FGFs) were cloned and characterized as a subgroup of the family of receptor tyrosine kinases. Four FGF receptor genes were identified, all of which encode membrane-bound glycoproteins containing three immunoglobulin (Ig) -like domains at the extracellular region, where only two of these domains are involved in ligand binding. Three unique features characterize the FGF receptors: 1) overlapping recognition and redundant specificity, where one receptor may bind with a similar affinity several of the seven known FGFs and one FGF may bind similarly to several distinct receptors. 2) The binding of FGFs to their receptors is dependent on the interaction of FGF with cell surface heparan sulfate proteoglycans. 3) A multitude of isoforms of cell-bound or secreted receptors are produced by the same gene. The gene structure of these receptors revealed two major mechanisms that are responsible for the formation of the diverse forms: alternative mRNA splicing, resulting in deletions or alternate exons usage, and internal polyadenylation, resulting in truncated products. These are reminiscent of mechanisms that also operate in the immunoglobulin family to generate diversity and to produce either secreted or cell-bound molecules. Tissue-specific alternative splicing in FGF receptors allows for the generation of two distinct receptors from a single gene because alternative exons determine the sequence of the COOH-terminal half of the third Ig-like domain involved in ligand binding. This represents a novel genetic mechanism to generate receptor diversity and specificity and to increase receptor repertoire.     

Tris-mercaptophenylphosphine complexes of rhenium: Synthesis, reactivity and structural characterization

The reactions of various rhenium(III) precursers with the tetradentate umbrella ligand tris-(ortho-mercaptophenyl)phosphine (PS₃ for the tri-anionic form) followed by various neutral donor ligands is the focus of this work.The addition of a solution of P(SLi)₃ in THF to [ReBr₃(THT)₃] in THF followed by the addition of excess isonitrile gives the octahedral complex [PS₃Re(CNiPr)₂]. This reaction also allows isolation of [PS₃Re(CNiPr)] which is separated from the bis-isonitrile complex via column chromatography. Both of these complexes were structurally characterized with the five-coordinate species displaying trigonal bipyramidal geometry and the six-coordinate species displaying a distorted octahedral geometry. The decomposition product [(S₂PS-SPS₂)Re] is also isolated from the chromatographic separation of the two isonitrile complexes. This neutral octahedral complex displays coordination through two phosphine and four thiolate moieties, with a non-coordinated disulfide bridge that links the remaining sulfurs.The addition of a solution of H₃PS₃ and iPr₂NEt in THF to [ReBr₃(THT)₃] followed by the addition of dimethylphenylphosphine yields [PS₃Re(PMe₂Ph)₂]. This complex, similar to the bis-isonitrile species, displays a distorted octahedral geometry.These complexes and others in the series have been fully characterized with NMR spectroscopy, infrared and mass spectrometry and elemental analyses.     

Radiation-induced changes of structural and functional properties of human hemoglobin

Gel filtration and SDS-PAGE separation of hemoglobin (Hb) irradiated under argon or N2O show formation of covalent-aggregated Hb molecules. The production of covalent bonds is attributed mainly to the action of hydroxyl radicals, because addition of ethanol, a scavenger of these radicals, suppresses this reaction to a great extent. The oxidized heme iron forming metHb or hemichromes is found in all the separated fractions of irradiated Hb. It is also found that the radiation-modified Hb molecules exhibit a decrease of co-operative binding of oxygen.     

The facilitated diffusion of oxygen by hemoglobin and myoglobin.

We have clarified the use of Wyman's differential equation for the facilitated oxygen flux through a slab of solution of myoglobin or hemoglobin by showing that there is a unique choice of boundary condition on the carrier concentration to be employed in conjunction with it. The singular perturbation solution of Wyman's equation, due to Murrayand Mitchell and Murray, has been extended. By means of it, the paradox of Wittenberg, that the facilitated oxygen flux per mole of heme is apparently independent of the protein carrier, has been resolved.     

Radiation-induced changes of structural and functional properties of human hemoglobin

Structural changes of human deoxyhemoglobin (deoxyHb) induced by water and ethanol radicals were investigated in this study using absorption spectroscopy. Spectra of deoxyHb samples irradiated under various conditions (the atmosphere of argon or N2O in the absence or presence of ethanol) indicate their conversion into methemoglobin (metHb), hemichrome- and cholehemichrome-like products. The absorbance at the characteristic maxima of these derivatives and also of the oxidized or reduced samples following irradiation depends on the dose of radiation and the conditions employed.     

Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.     

Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains.

Human hepatocyte growth factor (hHGF) consists of characteristic structural domains. In this study, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of Meth A cell growth, and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by competition analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the N-terminal, the first kringle, or the second kringle domain were not biologically effective and could not compete with hHGF bound to the c-met/HGF receptor. The results indicate that these domains are necessary for the biological activities of hHGF mediated by binding to the c-met/HGF receptor. The mutant proteins lacking the third or fourth kringle domain moderately retained biological activities and the receptor binding. The relative levels of the tyrosine phosphorylation of the c-met/HGF receptor by these mutant proteins correlated well with the relative potencies of the biological activities when compared with that of the wild-type hHGF. The mutant protein lacking the light chain was not effective in the biological activities and tyrosine phosphorylation of the c-met/HGF receptor, but competed with hHGF bound to the c-met/HGF receptor. These results suggest that the heavy chain plays an important role in the interaction of hHGF with the c-met/HGF receptor and that the light chain is further required for the tyrosine phosphorylation of the c-met/HGF receptor.