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.     

Carbamoylated hemoglobins A and S: physical properties.

Dimer-tetramer association constants (K2,4) of derivatives of CO-hemoglobins A and S specifically carbamoylated at the NH2-terminal valine residues were measured. Reactivites of the beta-93 sulfhydryls of the hemoglobin A derivatives were also investigated. As compared with the association constants of the parent molecules, the values of K2,4 of both hemoglobin types are raised by carbamoylation of the alpha-chain NH2 terminus, lowered by carbamoylation of the beta-chain NH2 terminus, and raised by carbamoylation of both termini. The apparent second-order rate constant for reaction of p-mercuribenzoate (PMB) with the beta-93 sulfhydryls is, however, unchanged by carbamoylation. These two observations are interpreted to indicate that in the liganded molecule structural changes are produced at the interface between dimers but not in the region of the beta-93 sulfhydryls. From the combination of the K2,4 measurements with ligand-binding data for the same derivatives (Kilmartin, J. V., et al. (1973), J. Biol. Chem. 248, 4039; Nigen, A. M., et al. (1974), J. Biol. Chem. 249, 6611) the carbamoylation-induced changes in the dimer-tetramer association constants of the unliganded derivatives were estimated to be of magnitude equal to or smaller than those in K2,4. It is concluded that much of the change in oxygen affinity that occurs upon carbamoylation of hemoglobins A and S can be accounted for without invoking extensive structural changes in the unliganded molecule.     

A consensus sequence of plant hemoglobins

From the study of plant hemoglobin protein and gene sequences, a consensus sequence was constructed and analysed by computer methods; analysis comprised the amino acid composition, hydropathy profile, and match degree with other plant and non-plant hemoglobin sequences. Resulting consensus sequence shows the main features of hemoglobin genes and proteins in plants.     

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.     

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.     

Hybrid formation for liganded hemoglobins A and C at subzero temperatures.

The kinetics of formation of the asymmetric carbonmonoxyhemoglobin hybrid (alpha beta)A(alpha beta)C from the parent molecules alpha 2 beta 2A and alpha 2 beta 2C have been studied by electrophoresis at subzero temperatures (down to -40 degrees C) using as supporting media gels of acrylamide/methylacrylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid formation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH greater than 10, t = -40 degrees C, the hybrid between alpha 2 beta 2A and alpha 2 beta 2C is stable for several hours. A rapid thermal quenching of a mixture of alpha 2 beta 2A and alpha 2 beta 2C prevented hybrid formation during the time required to separate the 2 molecules.     

Comparative immunology of Galapagos iguana hemoglobins.

The antigenic properties of the major hemoglobin component of the Galapgaos iguanas were studied using second-approximation qualitative and quantitative immunochemical techniques. Phylogenetic distances, relative to the Galapagos marine iguana. Amblyrhynchus cristatus, were established on the basis of immunological cross-reactions.     

Ligand-dependent Bohr effect of Chrionomus hemoglobins.

The O2 and CO Bohr effects of monomeric and dimeric hemoglobins of the insect Chironomus thummi thummi were determined as proton releases upon ligation. For the O2 Bohr effect of the monomeric hemoglobin III a maximum value of 0.20 H+/heme was obtained at pH 7.5. Upon ligation with CO, however, only 0.04 H+/heme were released at the same pH. In agreement with this finding isoelectric focusing experiments revealed different isoelectric points for O2-liganded and CO-liganded states of hemoglobin III. Analogous results were obtained in the cases of the monomeric hemoglobin IV and the dimeric hemoglobins of Chironomus thummi thummi; here O2 Bohr effects of 0.43 and 0.86 H+/heme were observed. For the corresponding CO Bohr effects values of 0.08 and 0.31 H+/heme were obtained respectively. On the basis of the available structural data the reduced CO Bohr effect in hemoglobin III is discussed as arising from a steric hindrance of the CO ligand by the side chain of isoleucine-E11, obstructing the movement of the heme-iron upon reaction with carbon monoxide. It should, however, be noted that ligands, according to their different electron donor and acceptor properties, may generally induce different conformational changes and thus different Bohr effects, in those hemoglobins in which distinct tertiary and/or quaternary constraints have not evolved. The general utilization of CO instead of O2 as allosteric effector is ruled out by the results reported here.     

植物的血红蛋白

近几年来,植物血红蛋白的研究进展十分迅速,豆科植物中与共生固氮无关的血红蛋白基因和包括禾本科植物在内的许多非豆科植物血红蛋白基因的发现使人们对植物血红蛋白有了新的认识,进而把植物血红蛋白分为共生血红蛋白和非共生血红蛋白两种类型。对这两种血红蛋白的性质、功能、基因结构及表达等方面的研究不仅对共生固氮中植物与微生物的相互关系和固氮工程研究;而且对植物细胞的呼吸代谢和耐涝机理等研究有重要价值。