Fumito Taketa

Binding of 2-formylpyridine monothiosemicarbazonato copper II to cat and normal human hemoglobins.

The antitumor agent 2-formylpyridine monothiosemicarbazonato copper(II) forms adducts with sulfur and nitrogen donor atoms from cat hemoglobin but only nitrogen donor atoms from human hemoglobin. Improved resolution of the mI = 1/2 lines in the g parallel region at S-band not only confirms the number of nitrogen donor atoms in the square planar configuration but provides evidence for strong coupling from a proton. Adduct formation results in an increase in the oxygen affinity of hemoglobin. Thus, it is suggested that allosteric enzyme inhibition may be a mechanism for the action of this agent.

Effects of 2-formylpyridine monothiosemicarbazonato copper II on red cell components

1-Formylpyridine monothiosemicarbazonato copper II (CuL+) is readily taken up by red cells and is initially bound to glutathione and hemoglobin. Glutathione was depleted within 5 hr of incubation, presumably by oxidation mediated by CuL+ and O2 with concomitant generation of toxic oxygen species. Cupric ion was slowly transferred from CuL+ to hemoglobin within about 7 hr and hemoglobin was oxidized until the major form prevailing after 10 hr was alpha 2 beta 2+. Little increase in hemolysis due to addition of CuL+ dissolved in the radical scavenger dimethyl sulfoxide was observed with prolonged incubation. Strong inhibition of red cell hexokinase by CuL+ was observed when the enzymes in red cell lysates and hemoglobin-free red cell lysates were examined. CuL+ was also an effective inhibitor of yeast hexokinase. However, the inhibitory effect of CuL+ within the red cells was less pronounced. It is suggested that even though intracellular accumulation of CuL+ creates an oxidizing environment and is potentially capable of inhibiting thiol enzymes such as hexokinase, protective effects are exerted in the red cell by the presence of hemoglobin, of radical scavengers, and of high levels of enzymes that detoxify toxic oxygen species.

Interaction of triethyltin with cat hemoglobin: Identification of binding sites and effects on hemoglobin function☆

Abstract Binding of triethyltin to the cat hemoglobins (HbA and HbB) results in the “masking” of two of the freely reactive sulfhydryl groups (SH) within the hemoglobin tetramer. That the “masked” SH groups occur in position 13α of each α-subunit was demonstrated by the lack of labeling of cysteine 13α with [14C]N-ethylmaleimide when triethyltin is present. Studies with cat-human hybrid hemoglobins indicate that the α-subunit of the cat hemoglobins alone is involved in the formation of a complex with triethyltin. Using available data on the primary as well as three dimensional structures of animal hemoglobins, it is suggested the cysteine 13α and histidine 20α serve as axial ligands in the formation of a pentacoordinate triethyltin cat hemoglobin complex. The binding of triethyltin results in an increase in the oxygen affinity of the two cat hemoglobins.

Hemoglobin wood β97(FG4) His → Leu: A new high-oxygen-affinity hemoglobin associated with familial erythrocytosis

Abstract The characterization of hemoglobin Wood (β97(FG4) His → Leu), a high oxygen affinity hemoglobin with reduced Hill constant is described. The amino acid substitution occurs at the α1β2 interface, in the same position as in hemoglobin Malmo (β97(FG4) His → Gln) and in an homologous position when compared with hemoglobins Chesapeake (α92(FG4) Arg → Leu) and J. Capetown (α92(FG4) Arg → Gln).

The oxidation of cat, human, and the cat-human hybrid hemoglobins α2Humanβ2Cat and α2Catβ2Human by copper(II)

The heme iron of the beta chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the beta globin chain. The presence of freely reactive -SH groups on the alpha chains of cat and sheep hemoglobins does not alter the course of this reaction; only the beta hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two beta chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, alpha 2 Human beta 2 Cat and alpha 2 Cat beta 2 Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the beta subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid alpha 2 Cat beta 2 Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or alpha 2 Human beta 2 Cat. Cat beta globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2 beta. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the beta chain and is associated with histidine 2 beta.

The effect of hemoglobin phenotype on whole blood oxygen saturation and erythrocyte organic phosphate concentration in the domestic cat (Felis catus)

Abstract 1. 1. The oxygen saturation curves and erythrocyte concentrations of ATP and 2,3-DPG of blood samples from domestic cats of different hemoglobin phenotypes have been obtained. 2. 2. The P O 2 at half-saturation and the Hill constant are the same for all cats examined and average 31·5 ± 1·0 and 2·60 ± 0·16 mm Hg, respectively. 3. 3. The 2,3-DPG and ATP concentrations in the various cats were also similar. The average concentration of 2,3-DPG was 2·73 ± 0·30 μmole/g Hb while that of ATP was 1·15 ± 0·29 μmole/g Hb.

Electron spin resonance spectra of feline no-hemoglobins

Organic phosphate and pH-dependent hyperfine structures (hfs) at g = 2.009 have recently been reported for the ESR spectrum of human adult nitric oxide hemoglobin (HHbA) [l-3]. The hfs consists primarily of the development of a three line spectrum (triplet) with spacing and intensity indicating the localization of the unpaired electron on the nitrogen nucleus of nitric oxide. Since both protons and organic phosphates are known to interact with deoxy-hemoglobin at sites that are far removed from the hemeligand binding site [4, 51, the development of hfs in HHbA-NO has been interpreted as resulting from an altered spatial environment of the heme-bound NO caused by conformational transitions in the protein [ 11. The intensity of the induced hypertine triplet is maximal under conditions that lower the oxygen affinity of the protein, such as low pH and/or saturating concentrations of phosphate effecters. Hence, it is possible that hfs may be characateristic of a low affinity conformation and may be observed in a hemoglobin with a low oxygen affinity even in the absence of organic phosphates or at neutrality. The major hemoglobin components of the domestic cat (CHbA) and (CHbB), provide a system that permits examination of the relationships between oxygen affinity, hfs intensity and protein structure. Both cat hemoglobins exhibit lower intrinsic oxygen affinities than other mammalian hemoglobins [7]. Furthermore, since CHbA is functionally regulated by organic phosphates while CHbB is not, it is of interest to see what influence phosphate effecters might have on the ESR spectra of the NO derivatives of these proteins. Hence, X-band and Q-band ESR spectra of CHbA-NO and CHbB-NO have been obtained and are compared with the corresponding spectra of HHbA-NO.

Effects of organic phosphates on oxygen equilibria and kinetics of SH reaction in feline hemoglobins

Abstract A recent report indicates that the response of frog and tadpole hemoglobin to inositol hexaphosphate (IHP) varies from observations made on hemoglobins of other species ( Araki, T., Kajita, A., and Shukuya, R. (1971) Biochem Biophys. Res. Commun.43, 1179). Since frog hemoglobins are known to be acetylated, investigation of the effects of IHP on the oxygen affinity of the acetylated (HbB) and nonacetylated (HbA) feline hemoglobins was of interest. The effect of IHP was of particular concern since it was previously shown that the oxygen affinity and SH reactivity of HbA is lowered by 2,3-diphosphoglycerate (2,3-DPG) and ATP while that of HbB is unaffected ( Taketa, F., Mauk, A. G., and Lessard, J. L. (1971) J. Biol. Chem.246: 4471). In contrast to the relatively weak effect of 2,3-DPG on the oxygen affinity and SH reactivity of cat HbA compared with its effect on human HbA, the effect of IHP on cat HbA is much greater and is comparable to its effect on human HbA. However, both phosphate compounds are ineffective with cat HbB; insensitivity of cat HbB presumably arises from the occurrence of N-acetylated β-chain amino termini as well as substitutions at or near histidine 143-β of this protein. Thus, properties of the two cat hemoglobins are sharply differentiated in the presence of IHP. Consequently the response of cat hemolyzates that contain a 1:1 mixture of HbA and HbB is such that the shape of the oxygen saturation curve changes drastically in the presence of this effector. Good correlation was found with respect to the effects of 2,3-DPG and IHP on the oxygen affinities and SH reactivities in the cat hemoglobins. The results suggest that the organic phosphates induce conformational changes in these proteins that stabilize the deoxy form of the protein.

Biosynthesis and amino terminal acetylation of cat hemoglobin B.

Abstract Acetylation of the amino-terminal serine of the β chains of cat hemoglobin B (HbB) occurs during synthesis of hemoglobin in a mRNA-dependent protein synthesizing system from rabbit reticulocyte lysate in the presence of acetyl-CoA and cat reticulocyte mRNA. The process occurs after peptide chain growth of about 30 amino acid residues. When endogenous acetyl-CoA was removed from the rabbit reticulocyte lysate by pretreatment with oxalacetate and citrate synthase, nonacetylated HbB (HbB′) was synthesized. Thus, β B globin chain synthesis goes to completion in the absence of acetylation even though the latter normally occurs during nascent chain growth. When HbB′ was incubated with acetyl-CoA in a rabbit reticulocyte lysate, hemoglobin with properties identical to those of HbB was produced. Thus, the selective amino terminal acetylation of β B globin also occurs in the completed hemoglobin.

Biosynthesis of cat hemoglobins A and B: Amino terminal acetylation of the β chain of HbB

Abstract Acetylation of the amino terminal serine of the β chains of cat HbB occurs during synthesis of hemoglobin in a mRNA dependent rabbit reticulocyte lysate protein synthesizing system in the presence of acetyl-CoA and cat reticulocyte mRNA. Both of the major cat hemoglobins, the nonacetylated HbA and acetylated HbB, are synthesized efficiently in the rabbit lysate system. The acetylation of HbB-β chains occurs during the biosynthesis of these proteins. Radioautography of tryptic peptide maps reveals that acetylation occurs specifically at the amino terminal serine of HbB-β globin, and not on HbA-β globin or on the α chain common to both hemoglobins A and B. Because of the similarity of the structures of HbA-β and HbB-β globin, it is suggested that the amino terminal residue determines whether the peptide chain is recognized for acetylation by a ribosomal acetyltransferase.