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.     

Studies on the oxidation of hemoglobin Zurich (beta 63 E7 Arg)

Autoxidation and chemically-induced oxidation of hemoglobin Zurich (beta 63 E7 Arg) have been investigated by electron paramagnetic resonance and optical absorption spectroscopy. The results show that the replacement of the distal histidine of the hemoglobin beta chains by an arginine greatly enhances the susceptibility of the heme-iron to oxidative challenge. Both the kinetics and the products of the oxidation are pH dependent. Thus, at acidic and neutral pH, treatment of the protein with ferricyanide leads to a fast conversion of the oxy-protein to aquo-methemoglobin, which, eventually, is slowly converted to hemichromes. In contrast, the hydroxy-met derivative, formed upon chemical oxidation at high pH, is rapidly converted to hemichromes. The electron paramagnetic resonance features of the ferric derivatives of hemoglobin Zurich are somewhat singular, reflecting the modifications of the heme environment in the distal region of the abnormal chains. However, they can be related to heme complexes having their structural counterparts in oxidation products of hemoglobin A.     

Hemoglobins. XXVII. The amino acid sequence of hemoglobin III from Myxine glutinosa L. A new hemecomplex: E7 glutamine, E11 isoleucine]

The sequence analysis of the main component, "HbIII", of the hemoglobins from the hagfish (Myxine glutinosa L.) is described. The hagfish belongs to the Cyclostomata, the most primitive class of the vertebrates. The hagfish hemoglobin displays a great heterogeneity, as described earlier. It consists of several monomeric hemoglobins. The globin of HbIII was isolated and used for the sequence analysis. The tryptic peptides as well as the cyanogen bromide and the BNPS-skatol fragments were separated. The sequences of the peptides were determined automatically by the help of a sequenator. Compared with other hitherto analyzed vertebral hemoglobins, also including other Cyclostomata, the primary structure of "HbIII" differs by more than 50%. The differences are so many that one can refer the Myxine hemoglobin neither as an alpha- nor as a beta-chain (of the tetrameric hemoglobins). The hagfish hemoglobin like other Cyclostomata has an additional segment of 9 residues at the amino terminus end compared with the mammalian hemoglobins. In the F-helix there is an insertion of 3 amino acid residues and in the interhelical gap, GH, there is a deletion of 9 residues. The substitutions of the residues forming the heme complex are of special interest. The distal histidine, E7, is substituted for glutamine. The proximal histidine, F8, is invariable. The valine E11 is substituted by isoleucine and the leucine FG3 by phenylalanine. These positions are involved in the contact with the heme group. This complex has never been described before.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

The primary structure of hemoglobins of the rock hyrax (Procavia habessinica, Hyracoidea): insertion of glutamine in the alpha chains

The chromatography of the hemoglobin of the rock hyrax (Procavia habessinica) gives two components (73% HbI and 27% HbII). The amino-acid analysis and the sequences of the globin chains elucidated with the phenylthiohydantoin method, did not show any differences between the alpha I and alpha II or beta I and beta II chains, respectively. The different chromatographical behaviour cannot be explained. After chain separation by chromatography on CM-52 cellulose, all four primary structures were elucidated automatically in a sequenator on the chains and the tryptic peptides. In 20% of the beta I chains the N-terminal valine was blocked by acetyl. The alignment was performed by homology with the chains of human adult hemoglobin. The alpha chain of the rock hyrax has 142 amino-acid residues, i.e. one residue more than normal mammalian alpha chains, caused by an insertion of glutamine in the GH region supposed between positions 115 and 116. A comparison of human and hyrax hemoglobins shows an exchange of 21 amino-acid residues in the alpha chains and of 24 in the beta chains. Some substitutions in alpha 1 beta 1 contacts and in the surrounding of the heme are not supposed to effect the function of the hemoglobin. The phylogenetic relationship between the rock hyrax and the Indian elephant (Elephas maximus) on the one hand and with some Perissodactyla on the other, is discussed. Up to now the exchanges of alpha 110(G17)Ala leads to Ser and beta 56(D7)Gly leads to His have only been found in hyrax and elephant. This indicates a certain relationship between Hyracoidea and Proboscidea.     

Administration of keratinocyte growth factor (KGF) modulates the pulmonary expression of nicotinic acetylcholine receptor subunits alpha7, alpha9 and alpha10

Administration of recombinant human keratinocyte growth factor (rHuKGF, Delta23N-KGF, palifermin) protects the lung against a variety of injurious stimuli. The exact mechanisms leading to lung protection are unknown. Alterations in the non-neuronal cholinergic system of the lung might be involved, as vital pulmonary functions are regulated by acetylcholine. Here, we investigated the effect of KGF on the expression of nicotinic acetylcholine receptor subunits alpha7, alpha9 and alpha10 in rat lungs. Adult rats were treated via intratracheal instillation with rHuKGF or with an equivalent volume of PBS. The expression of nicotinic acetylcholine receptor subunits was analyzed by real-time RT-PCR, immunoblotting and immunohistochemistry. Treatment with rHuKGF led to a decreased expression of nicotinic receptor subunit alpha7 in the total lung. In contrast, the expression of the receptor subunits alpha9 and alpha10 was up-regulated. In conclusion, nicotinic acetylcholine receptors are differentially regulated by KGF treatment in vivo, which might result in changes in the biological effects of acetylcholine.     

Intrinsic oxygen affinity of hemoglobins: the hemoglobin of bisons (Bison bonasus,Bovidae)

The hemoglobin from a European Bison (Bison bonasus) was analysed and the complete primary structures of the alpha I-, alpha II-and beta-chains have been determined. The alpha I- and alpha II-chains differ only at position alpha 19 (Asp----Gly). The beta-chains are homogeneous. The sequences are compared with the globin chains of Bison bison and bovine and the polymorphism of the alpha-chains is discussed. On the basis of the primary structure it may be concluded that the hemoglobin of Bison bonasus belongs to the group of hemoglobins with intrinsically low oxygen affinity.