Allosteric transitions in cobalt hemoglobins.

Circular dichroism and difference ultraviolet visible spectra were obtained for cobalt hemoglobin derivatives. At 287 nm the ellipticity difference between the oxy- and deoxycobaltohemoglobin is about one-half as great as that for the native proteins indicating smaller quaternary conformational changes for the former. Deoxygenation increases the Soret rotational strengths of both iron and cobalt hemoglobins to comparable degrees suggesting similar conformational changes for their aromatic residues near the "heme." Deoxygenation causes a much larger decrease of L band ellipticity for iron than cobalt hemoglobin. Circular dichroism spectra of nitrosylcobaltohemoglobin indicate the molecule to have a T quaternary structure. The circular dichroism spectra of cobaltihemoglobin do not seem to fit the patterns of the other cobalt derivatives and its 287 nm ellipticity is pH-dependent. From the shape of the Soret circular dichroism spectra, it is estimated that the transition dipole makes an angle with the line joining the two opposing pyrrole nitrogens of about 60 degrees for oxy- and deoxycobaltohemoglobin, 80 degrees for cobaltihemoglobin, as compared to 70 degrees for the native oxy- and deoxyhemoglobins. Inositol hexaphosphate has little or no effect on the circular dichroism spectra of cobalt hemoglobins in the 287 nm region, but it significantly increases the Soret rotational strength and decreases the L band ellipticity. The results are interpreted to mean that polyphosphates modify primarily the protein structure of hemoglobins at the tertiary level, and that the intersubunit interactions are weak in cobalt hemoglobins.     

Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobin with molecular oxygen.

The pH dependence of the electron paramagnetic resonance (EPR) spectrum and oxygen affinity of cobaltous porphyrin-containing myoglobin (CoMb) have been examined. The hyperfine structures of the EPR spectrum of oxy-CoMb undergo small, reversible pH-dependent changes with pK values of 5.33, 5.55, and 5.25 +/- 0.05 for proto-, meso-, and deutero-CoMb's, respectively, whereas deoxy-CoMb does not exhibit any pH dependence of its EPR spectrum. The partial pressure of oxygen at half-saturation of proto-CoMb decreases from 26 to 42 Torr on lowering the pH from 7.0 to 4.8. For comparison, we have prepared cobaltous porphyrin-containing monomeric Glycera hemoglobin (CoHb (Glycera)), in which the distal histidyl group of myoglobin is replaced by a leucyl residue, and examined the equilibria and kinetics of its oxygenation and EPR spectrum. CoHb (Glycera) has exhibited a very low oxygen affinity (p50 = 7 X 10(2) Torr at 5 degrees) and a large dissociation rate constant (more than 8 X 10(4) S-1 at 5 degrees). The EPR spectrum of oxy-CoHb (Glycera) was affected by neither pH nor replacement of H2O with D2O. Low temperature photodissociation studies by EPR and spectrophotometry have shown that the photolyzed form of the ligated hemoglobin (Glycera) is similar to its deoxy form, in contrast to myoglobin which gives a new intermediate states as the photolyzed form. These differences between CoMb and CoHb (Glycera) are interpreted with relation to the possible role of the distal histidyl residue in CoMb.     

Evolution of ruminant hemoglobins. Thermodynamic divergence of ox and buffalo hemoglobins.

The ligand-binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed. Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion. In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the delta H for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (delta H = -30.5 kJ/mol O2). This discrepancy with the other ruminant hemoglobins (e.g. ox, delta H = -10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.     

The structure of annelid and mollusc hemoglobins.

The polypeptide chain composition and the chemical properties of several annelid hemoglobins and chlorocruorins are presented. In agreement with earlier studies on the hemoglobin from Arenicola cristata (Waxman, L. (1971) J. Biol. Chem. 246, 7318-7327), nearly all of the pigments which have been examined consist of one or more different disulfide-linked polypeptide chains of 13,000 to 16,000 daltons, and the heme-protein stoichiometry suggests that more than one polypeptide is associated with each heme. Except for the prosthetic group, there is no outstanding chemical difference between the chlorocruorins and the hemoglobins, nor is ther any apparent differnce between those hemoglobins which show cooperative oxygen binding properties and those which do not. The results suggest that all these hemoglobins have similar structures. On the other hand, the polypeptide chains of mollusc hemoglobins have molecular weights of greater than 220,000. Each polypeptide binds many heme groups. Thus, annelids use small polypeptide chains while molluscs use giant polypeptides to carry O2.     

Comparative solubilities and electrophoresis of microtine hemoglobins.

1. The electrophoretic mobilities of the hemoglobins of 7 taxa of microtines were compared. Microtus oeconomus, M. pennsylvanicus pullatus and M. xanthognatus showed identical 2-band patterns on electrophoresis of their hemoglobins while M. pennsylvanicus tananaensis showed only a single hemoglobin corresponding to the major band of the others. Dicrostonyx rubricatus and D. stevensoni exhibited identical patterns different from the Microtus species. Lemmus sibiricus had a slow hemoglobin component with mobility slightly different from the slow ones of the Microtus species while the fast component appeared the same. 2. Electrophoresis of individual globin chains from hemolysates, purified hemoglobins, and isolated chains indicated a large degree of similarity between the species studied, although there were significant differences in hemoglobin patterns. 3. The minor hemoglobin band in Microtus seems to be the result of a second alpha chain locus as determined from the hemoglobins from hybrids of two subspecies. 4. Salting-out studies indicated differences between hemoglobins that were not detectable by electrophoresis of either whole hemoglobins or isolated chains. 5. M. xanthognathus hemolysate was considerably less soluble than those of M. oeconomus and M. pennsylvanicus pullatus which had essentially the same solubility. 6. The major hemoglobin components of M. pennsylvanicus pullatus and M. xanthognathus were considerably less soluble than either the corresponding unfractionated hemolysates or purified minor components.     

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.     

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.     

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.     

Glutathione mixed disulfides and heterogeneity of chicken hemoglobins.

1. Adult chicken hemoglobins Hb A and Hb D interact with glutathione disulfide, GSSG. The major hemoglobin, Hb A, forms at least two new components, termed GHb AI and GHb AII, and Hb D forms at least one, GHb DI. 2. At pH 8.0 and 5 degrees C, glutathione disulfide (GSSG) in a molar excess of 50 x took 6 days to complete the reaction, although at pH 8.6 and 41 degrees C only 1 hr was needed, where the hemoglobins Hb A and Hb D were converted to their most mobile forms GHb AII and GHb DI. 3. Slight molar excess (2.7 GSSG/Hb, pH 7.4, 41 degrees C), reacting for 1 hr, showed extensive formation of GHb AI and some GHb AII. 4. Electrophoretic patterns, from the reaction products of 54 GSSG/Hb excess at different times, showed a marked pH dependence. 5. Titration with pCMB (p-chloromercuribezoic acid) of DTE (dithioerythrytol)-reduced samples showed 8.0 +/- 0.4 (N = 5) -SH (sulfhydryl) per tetramer. In hemolysates not reacted with DTE, 6.0 +/- 0.4 (N = 3) -SH were detected. 6. DTE-reduced and GSSG-reacted hemoglobins showed 4.6 +/- 0.5 (N = 7) -SH and 1.5 +/- 0.4 (N = 6) -SH, respectively, as titrated by DTNB, pH 8.0. DTE-reduced hemoglobins showed four fast-reacting -SH groups, no longer present in GSSG-reacted hemoglobins. 7. Our data indicate that chicken GHb AI and GHb DI probably have two glutathionyl residues per tetramer whereas GHb AII has four.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit dissociations in natural and recombinant hemoglobins.

A precise and rapid procedure employing gel filtration on Superose-12 to measure the tetramer-dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val-6(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.