Study of the conversion of GDP-mannose into GDP-fucose in Nereids: a biochemical marker of oocyte maturation

The high-resolution proton nuclear magnetic resonance spectra of carp hemoglobin have been compared to those of human normal adult hemoglobin. Carp deoxy and carbonmonoxy hemoglobins in the deoxy-type quaternary state exhibit two downfield exchangeable proton resonances as compared to four seen in human normal adult deoxyhemoglobin. This suggests that two of the hydrogen bonds present in human normal adult deoxyhemoglobin are absent or occur in very different environments in carp hemoglobin. One of the exchangeable proton resonances of carp hemoglobin, while present in the deoxy-type quaternary state of the carbonmonoxy and deoxy derivatives, is absent in the oxy-type quaternary state of both, in agreement with the assignments of these quaternary structures by other methods. The ring-current-shifted proton resonances (sensitive tertiary structural markers) of carp carbonmonoxyhemoglobin are substantially different from those of human normal adult hemoglobin. The aromatic proton resonance region of carp hemoglobin has fewer resonances than that of human normal adult hemoglobin, consistent with its much reduced histidine content. The hyperfine-shifted proximal histidyl NH-exchangeable proton resonances of carp hemoglobin suggest that during the transition from the oxy to the deoxy quaternary structure, there is a greater alteration in the heme pocket of one type of subunits (presumably the beta chain) than that in the other subunit. The present results suggest that there are differences in both tertiary and quaternary structures between carp and human normal adult hemoglobins which could contribute to the great differences in the functional properties between these two proteins.     

Radioimmunochemical characterization of hemoglobins Lepore and Kenya: unique antigenic determinants located on hybrid hemoglobins.

Antisera were produced in rabbits to the three known types of Lepore hemoglobins, which contain hybrid delta-beta non-alpha-chains, and to hemoglobin Kenya, which has a hybrid gamma-beta non-alpha-chain. By using a sensitive radioimmunoassay technique, the absorbed antisera were shown to contain an antibody population that was specific for the hybrid hemoglobin and did not cross-react with normal hemoglobins. However, with the absorbed Lepore-specific antisera, the three known types of Lepore hemoglobins were antigenically indistinguishable from each other, suggesting that antibodies are not produced to the primary structural differences which define the three non-alpha-chains of the Lepore hemoglobins. These studies demonstrate that the non-alpha-subunits of hemoglobins Lepore and Kenya possess unique antigenic determinant sites, evidently resulting from an altered polypeptide conformation.     

Structural and functional properties of hemoglobins from unicellular organisms as revealed by resonance Raman spectroscopy

Hemoglobins have been discovered in organisms from virtually all kingdoms. Their presence in unicellular organisms suggests that the gene for hemoglobin is very ancient and that the hemoglobins must have functions other than oxygen transport, in view of the fact that O2 delivery is a diffusion-controlled process in these organisms. Based on sequence alignment, three groups of hemoglobins have been characterized in unicellular organisms. The group-one hemoglobins, termed truncated hemoglobins, consist of proteins with 110-140 amino acid residues and a novel two-over-two alpha-helical sandwich motif. The group-two hemoglobins, termed flavohemoglobins, consist of a hemoglobin domain, with a classical three-over-three alpha-helical sandwich motif, and a flavin-containing reductase domain that is covalently attached to it. The group-three hemoglobins consist of myoglobin-like proteins that have high sequence homology and structural similarity to the hemoglobin domain of flavohemoglobins. In this review, recent resonance Raman studies of each group of these proteins are presented. Their implications are discussed in the context of the structural and functional properties of these novel hemoglobins.     

Oxygen equilibrium characteristics of hemoglobins of baboons, Theropithecus gelada,Papio hamadryas and Papio anubis

Hemoglobins of three baboons, Theropithecus gelada, Papio hamadryas- and Papio anubis, were purified and their oxygen equilibrium characteristics were studied. (a) Oxygen affinity, as expressed by P₅₀, oxygen partial pressure for 50% oxygen binding, was in the order of gelada hemoglobin > anubis hemoglobin > hamadryas hemoglobin although the differences were small. (b) The presence of 2,3-diphosphoglycerate reduced their oxygen affinity in a similar manner. The effect on baboon hemoglobins was greater than that on human and Japanese monkey hemoglobins. (c) The intensity of the Bohr effect, as expressed by $\text{?Δlog}\text{P}{}_{50}\text{ΔpH}$, at pH 7·4 agreed well with each other and the value was 0·62 in the presence of 2 mm diphosphoglycerate and 0·52 in its absence. These results indicate that phenotypic adaptation (acclimatory) may play an important role in the adaptation of gelada baboon to high altitudes.     

Conformation of bovine nitrosylhemoglobins: an ESR study

The structural properties of nitrosylhemoglobins from two bovine species, namely cow and buffalo, have been investigated using electron spin resonance spectroscopy. Bovine hemoglobins show sensitivity to the presence of chloride ions and organic phosphates. ESR spectral features indicate a stable deoxy quaternary conformation of the molecule when compared to normal adult human hemoglobin A. Amino acid substitutions at the amino terminal end of the beta chain and at other sites of the alpha and beta chains seems to shift the allosteric equilibrium towards the T state in bovine hemoglobins. The results also confirm the intrinsically low oxygen affinity of bovine hemoglobins under physiological conditions.     

Hemoglobins of baboons, Papio cynocephalus, P. gelada and P. hamadryas

Major hemoglobins of adult Papio cynocephalus, P. gelada, and P. hamadryas and of newborn P. cynocephalus were purified; globins were prepared; and α and β or α and γ chains were separated. The amino acid compositions and aminoterminal groups were determined. These were compared with analogous data for human hemoglobins, other baboon hemoglobins and macaque hemoglobins.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

Comparisons of the immunological properties of two structural polypeptides of type C RNA viruses endogenous to old world monkeys.

Immunologically very closely related type C RNA viruses are endogenous to the domestic cat and to an old world primate, the baboon. In the present studies, radioimmunological techniques have been developed for detection of the 15,000 and 30,000 molecular weight (MW) polypeptides of each virus. The much more pronounced type-specific antigenic determinants of the lower MW polypeptides made it possible to readily differentiate these viruses from each other as well as from a type C virus isolate from a second baboon species. Normal rhesus monkey tissues were partially purified and shown to contain a reactivity with MW and immunological properties similar to that of the baboon virus 30,000 MW polypeptide. Despite a similar degree of purification, antigenic reactivity like that of the baboon virus 15,000 MW polypeptide was undetectable even in the brodest immunological tests available for this polypeptide. The present findings indicate that the immunological properties of two structural polypeptides of closely related viruses endogenous to primate and feline species have undergone different rates of antigenic change in the course of evolution within their respective host cell genome.     

Oxygen infrared spectra of oxyhemoglobins and oxymyoglobins. Evidence of two major liganded O2 structures

The dioxygen stretch bands in infrared spectra for solutions of oxy species of human hemoglobin A and its separated subunits, human mutant hemoglobin Zurich (beta 63His to Arg), rabbit hemoglobin, lamprey hemoglobin, sperm whale myoglobin, bovine myoglobin, and a sea worm chlorocruorin are examined. Each protein exhibits multiple isotope-sensitive bands between 1160 and 1060 cm-1 for liganded 16O2, 17O2, and 18O2. The O-O stretch bands for each of the mammalian myoglobins and hemoglobins are similar, with frequencies that differ between proteins by only 3-5 cm-1. The spectra for the lamprey and sea worm hemoglobins exhibit greater diversity. For all proteins an O-O stretch band expected to occur near 1125 cm-1 for 16O2 and 17O2, but not 18O2, appears split by approximately 25 cm-1 due to an unidentified perturbation. The spectrum for each dioxygen isotope, if unperturbed, would contain two strong bands for the mammalian myoglobins (1150 and 1120 cm-1) and hemoglobins (1155 and 1125 cm-1). Two strong bands separated by approximately 30 cm-1 for each oxy heme protein subunit indicate that two major protein conformations (structures) that differ substantially in O2 bonding are present. The two dioxygen structures can result from a combination of dynamic distal and proximal effects upon the O2 ligand bound in a bent-end-on stereochemistry.     

Implication of the alpha 1 beta 1 interface in the hemoglobin affinity changes. A comparative study between normal and San Diego fully ligated hemoglobins

The alpha 1 beta 1 interface of normal and mutated San Diego hemoglobins in their fully liganded form was investigated, through the SH vibrational absorption of beta-112 cysteine, by Fourier-transform infrared spectroscopy. The center frequency of this thiol group was significantly shifted in San Diego hemoglobin compared with normal human hemoglobin. Different dimer organization between the two proteins was also revealed by circular dichroism of the heme. These findings agree well with assessment that the alpha 1 beta 1 interface, far from being inert, is involved in the affinity changes of the hemoglobin molecule.     

A ubiquitously expressed human hexacoordinate hemoglobin

We have identified a new human hemoglobin that we call histoglobin because it is expressed in a wide array of tissues. Histoglobin shares less than 30% identity with the other human hemoglobins, and the gene contains an intron in an unprecedented location. Spectroscopic and kinetic experiments with recombinant human histoglobin indicate that it is a hexacoordinate hemoglobin with significantly different ligand binding characteristics than the other human hexacoordinate hemoglobin, neuroglobin. In contrast to the very high oxygen affinities displayed by most hexacoordinate hemoglobins, the biophysical characteristics of histoglobin indicate that it could facilitate oxygen transport. The discovery of histoglobin demonstrates that humans, like plants, differentially express multiple hexacoordinate hemoglobins.     

The stability of the heme-globin linkage in some normal, mutant, and chemically modified hemoglobins.

The rates and equilibria of heme exchange between methemoglobin and serum albumin were measured using a simple new spectrophotometric method. It is based on the large difference between the spectrum of methemoglobin and that of methemealbumin at pH 8-9. The rate of heme exchange was found to be independent of the albumin concentration and inversely proportional to the hemoglobin (Hb) concentration. Taken together with the finding that the rate was 10 times greater for Hb Rothschild, which is completely dissociated into alpha beta dimers and 10 times smaller for two cross-linked hemoglobins, the subunits of which cannot dissociate, this showed that the rate of dissociation of heme from alpha beta dimers is very much greater than from tetramers. Conditions were found for the attainment of an equilibrium distribution of hemes between beta globin and albumin. The equilibrium distribution ratio, R = methemealbumin/albumin/methemoglobin/apohemoglobin, for hemoglobin A was 3.4 with human and 0.005 with bovine serum albumin. Both the rates of exchange and the R values of HbS and HbF were the same as that for HbA. The equilibrium distribution ratio for Hb Rothschild was 7 times greater than that for HbA whereas that of one but not the other of the cross-linked hemoglobins was 10 times smaller. The structural bases for these differences are analyzed.     

Proton nuclear magnetic resonance studies of hemoglobin Malm?: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malm? (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.     

Subunit dissociation in fish hemoglobins.

The tetramer-dimer dissociation equilibria (K 4,2) of several fish hemoglobins have been examined by sedimentation velocity measurements with a scanner-computer system for the ultracentrifuge and by flash photolysis measurements using rapid kinetic methods. Samples studied in detail included hemoglobins from a marine teleost, Brevoortia tyrannus (common name, menhaden); a fresh water teleost, Cyprinus carpio, (common name, carp); and an elasmobranch Prionace glauca (common name, blue shark). For all three species in the CO form at pH 7, in 0.1 M phosphate buffer, sedimentation coefficients of 4.3 S (typical of tetrameric hemoglobin) are observed in the micromolar concentration range. In contrast, mammalian hemoglobins dissociate appreciably to dimers under these conditions. The inability to detect dissociation in three fish hemoglobins at the lowest concentrations examined indicates that K 4,2 must have a value of 10(-8) M or less. In flash photolysis experiments on very dilute solutions in long path length cells, two kinetic components were detected with their proportions varying as expected for an equilibrium between tetramers (the slower component) and dimers (the faster component); values of K 4,2 for the three fish hemoglobins in the range 10(-9) to 10(-8) M were calculated from these data. Thus, the values of K 4,2 for liganded forms of the fish hemoglobins appear to be midway between the value for liganded human hemoglobin (K 4,2 approximately 10(-6) M) and unliganded human hemoglobin (K 4,2 approximately 10(-12) M). This conclusion is supported by measurements on solutions containing guanidine hydrochloride to enhance the degree of dissociation. All three fish hemoglobins are appreciably dissociated at guanidine concentrations of about 0.8 M, which is roughly midway between the guanidine concentrations needed to cause comparable dissociation of liganded human hemoglobin (about 0.4 M) and unliganded human hemoglobin (about 1.6 M). Kinetic measurements on solutions containing guanidine hydrochloride indicated that there are changes in both the absolute rates and the proportions of the fast and slow components, which along with other factors complicated the analysis of the data in terms of dissociation constants. Measurements were also made in solutions containing urea to promote dissociation, but with this agent very high concentrations (about 6 M) were required to give measureable dissociation and the fish hemoglobins were unstable under these conditions, with appreciable loss of absorbance spectra in both the sedimentation and kinetic experiments.     

A resonance Raman study of Ambystoma tigrinum hemoglobins: evidence for intraspecies hemepocket variations

Using resonance Raman difference spectroscopy, the Raman-active vibrational modes of hemoglobins from adult, neotenic, and larval forms of the salamander, Ambystoma tigrinum have been compared to each other and to human hemoglobin. The local heme environment of the adult and neotenic proteins were identical and differed from that of the larval protein. Differences were observed in modes sensitive to porphyrin pi electron density and axial ligation. Systematic differences were also observed between human and adult salamander hemoglobins particularly in modes sensitive to the heme vinyl environment. The relationship between these environmental differences, oxygen binding affinity, and the effects of allosteric modulators are discussed.     

Hemoglobins: Diversity of structures and functions

This review briefs the modern concepts of the diversity of hemoglobin functions. The hemoglobins discovered in the representatives of all kingdoms of living nature are described. Specific structural features of various groups of these proteins, including flavohemoglobins and truncated hemoglobins, are discussed. The transport, catalytic, and sensory functions of these proteins are described as well as their roles in oxidative, nitrosative, and carbonyl stresses and the changes in their functions caused by modifications of the molecule. The issues of hemoglobin origin and evolution are discussed.     

Body temperature-related structural transitions of monotremal and human hemoglobin

In this study, temperature-related structural changes were investigated in human, duck-billed platypus (Ornithorhynchus anatinus, body temperature T(b) = 31-33 degrees C), and echidna (Tachyglossus aculeatus, body temperature T(b) = 32-33 degrees C) hemoglobin using circular dichroism spectroscopy and dynamic light scattering. The average hydrodynamic radius (R(h)) and fractional (normalized) change in the ellipticity (F(obs)) at 222 +/- 2 nm of hemoglobin were measured. The temperature was varied stepwise from 25 degrees C to 45 degrees C. The existence of a structural transition of human hemoglobin at the critical temperature T(c) between 36-37 degrees C was previously shown by micropipette aspiration experiments, viscosimetry, and circular dichroism spectroscopy. Based on light-scattering measurements, this study proves the onset of molecular aggregation at T(c). In two different monotremal hemoglobins (echidna and platypus), the critical transition temperatures were found between 32-33 degrees C, which are close to the species' body temperature T(b). The data suggest that the correlation of the structural transition's critical temperature T(c) and the species' body temperature T(b) is not mere coincidence but, instead, is a more widespread structural phenomenon possibly including many other proteins.     

The structure and function of plant hemoglobins.

Plants, like humans, contain hemoglobin. Three distinct types of hemoglobin exist in plants: symbiotic, non-symbiotic, and truncated hemoglobins. Crystal structures and other structural and biophysical techniques have revealed important knowledge about ligand binding and conformational stabilization in all three types. In symbiotic hemoglobins (leghemoglobins), ligand binding regulatory mechanisms have been shown to differ dramatically from myoglobin and red blood cell hemoglobin. In the non-symbiotic hemoglobins found in all plants, crystal structures and vibrational spectroscopy have revealed the nature of the structural transition between the hexacoordinate and ligand-bound states. In truncated hemoglobins, the abbreviated globin is porous, providing tunnels that may assist in ligand binding, and the bound ligand is stabilized by more than one distal pocket residue. Research has implicated these plant hemoglobins in a number of possible functions differing among hemoglobin types, and possibly between plant species.     

WAXS studies of the structural diversity of hemoglobin in solution

Specific ligation states of hemoglobin are, when crystallized, capable of taking on multiple quaternary structures. The relationship between these structures, captured in crystal lattices, and hemoglobin structure in solution remains uncertain. Wide-angle X-ray solution scattering (WAXS) is a sensitive probe of protein structure in solution that can distinguish among similar structures and has the potential to contribute to these issues. We used WAXS to assess the relationships among the structures of human and bovine hemoglobins in different liganded forms in solution. WAXS data readily distinguished among the various forms of hemoglobins. WAXS patterns confirm some of the relationships among hemoglobin structures that have been defined through crystallography and NMR and extend others. For instance, methemoglobin A in solution is, as expected, nearly indistinguishable from HbCO A. Interestingly, for bovine hemoglobin, the differences between deoxy-Hb, methemoglobin and HbCO are smaller than the corresponding differences in human hemoglobin. WAXS data were also used to assess the spatial extent of structural fluctuations of various hemoglobins in solution. Dynamics has been implicated in allosteric control of hemoglobin, and increased dynamics has been associated with lowered oxygen affinity. Consistent with that notion, WAXS patterns indicate that deoxy-Hb A exhibits substantially larger structural fluctuations than HbCO A. Comparisons between the observed WAXS patterns and those predicted on the basis of atomic coordinate sets suggest that the structures of Hb in different liganded forms exhibit clear differences from known crystal structures.