Involvement of superoxide anion in the reaction mechanism of haemoglobin oxidation by nitrite.

The sigmoidal time course of haemoglobin oxidation by nitrite, involving an initial slow reaction accompanied by a subsequent rapid reaction, was extensively explored. The initial slow reaction was much prolonged by the addition of superoxide dismutase to the reaction mixture. On the other hand, in the presence of superoxide anion generated by xanthine oxidase systems, the slow phase disappeared and the reaction changed to first-order kinetics. The oxidation of intermediate haemoglobins [defined as haemoglobin tetramer in which different chains (alpha- or beta-) are in the ferric state and in the ferrous state] such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 also proceeded in a sigmoidal manner. Similar effects of superoxide anion on these reactions were observed. Since the intermediate haemoglobins such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 were found to be produced by the oxidation of haemoglobin by nitrite, the changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin during the reaction were followed by isoelectric-focusing electrophoresis. The amounts of (alpha 2+ beta 3+)2 were larger than those of (alpha 3+ beta 2+)2 at the initial stages of the reaction, suggesting that there is a functional difference between alpha- and beta-chains in the oxyhaemoglobin tetramer. On the basis of these results, a reaction model of the haemoglobin oxidation by nitrite was tentatively proposed. The changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin were well fitted to the simulation curves generated from the reaction model. Details of the derivation of the equations used for kinetic analysis have been deposited as Supplement SUP 50112 (5 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K. from whom copies may be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Thermal stability of hemoglobin crosslinked in the T-state by bis(3,5-dibromosalicyl) fumarate

Bis(3,5-dibromosalicyl) fumarate was used to crosslink oxyhemoglobin between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxyhemoglobin between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R.Y., et al. (1986) J. Biol. Chem. 261, 9929). Thermal denaturations demonstrated that alpha crosslinked hemoglobin (alpha 99XLHb A) has the same stability as the beta crosslinked one (beta 82XLHb A). Both alpha and beta crosslinked methemoglobins have a denaturation temperature in 0.9 M guanidine of 57 degrees C compared to 41 degrees C of Hb A. The second product from the T-state crosslinking reaction was found to be crosslinked between the beta chains by chain separation and amino acid analysis. The possible positions for this crosslink are limited to the bisphosphoglycerate binding site in the three-dimensional structure. Its stability is comparable to that of the alpha 99XLHb A or beta 82XLHb A. These modified hemoglobins are potential blood substitutes.     

Diffusion of peroxynitrite in the presence of carbon dioxide.

Peroxynitrite, the reactive species formed in vivo by the reaction of nitric oxide with superoxide anion, is capable of diffusing across erythrocyte membranes via anion channels and passive diffusion (A. Denicola, J. M. Souza, and R. Radi, Proc. Natl. Acad. Sci. USA 95, 3566-3571, 1998). However, peroxynitrite diffusion could be limited by extracellular targets, with the reaction with CO(2) (k(2) = 4.6 x 10(4) at 37 degrees C and pH 7.4) the most relevant. Herein, we studied the influence of physiological concentrations of CO(2) on peroxynitrite diffusion across intact red blood cells. The presence of CO(2) inhibited the oxidation of intracellular oxyhemoglobin by externally added peroxynitrite. However, the inhibition by CO(2) decreased at increasing red blood cell densities. At 45% hematocrit, 1.3 mM CO(2) (in equilibrium with 24 mM bicarbonate, at pH 7.4 and 25 degrees C) only inhibited 30% of intracellular oxyhemoglobin oxidation. This partial inhibition was also observed in red blood cells pretreated with the anion exchanger inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, ruling out a competition between peroxynitrite and bicarbonate for the transport through the anion channel. A theoretical model was developed to estimate the diffusion distance and half-life of extracellular peroxynitrite before reacting with intracellular oxyhemoglobin, at different red blood cell densities, and in the presence or absence of CO(2). The theoretical model correlated well with the experimental data. Our results indicate that, even in the presence of CO(2), peroxynitrite is able to diffuse and reach the inside of the erythrocyte.     

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates.

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.     

Kinetics of ligand binding and quaternary conformational change in the homodimeric hemoglobin from Scapharca inaequivalvis

The kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis has been extensively investigated by flash and dye-laser photolysis, temperature jump relaxation, and stopped flow methods. The results indicate that cooperativity in ligand binding, already observed for oxygen at equilibrium, finds its kinetic counterpart in a large decrease of the oxygen dissociation velocity in the second step of the binding reaction. In the case of carbon monoxide, cooperativity is clearly evident in the increase of the combination velocity constant as the reaction proceeds. Therefore, the ligand-binding kinetics of this dimeric hemoglobin shows the characteristic features of the corresponding reactions of tetrameric hemoglobins. Analysis of the data in terms of the allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) has shown that the values of the allosteric parameters cannot be fixed uniquely for a dimeric hemoglobin. The rapid changes in absorbance observed at the isosbestic points of unliganded and liganded hemoglobin following laser photolysis provided a value of 7 X 10(4) S-1 at 20 degrees C for the rate of the ligand-free quarternary conformational change, postulated on the basis of cooperative ligand binding. Comparison of the rapid absorbance changes observed during ligand rebinding in this hemoglobin with those observed in tuna hemoglobin indicate that, at full photolysis, binding to the T state is followed by further binding and conversion to the liganded R state; at partial photolysis, population of the liganded T state occurs immediately and is followed by a decay to the liganded R state upon further ligand binding. These new results, in conjunction with previous equilibrium data on the same system, show unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas.     

Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin.

The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte.     

Direct generation of superoxide anions by flash photolysis of human oxyhemoglobin.

The results presented in this report suggest that human oxyhemoglobin can directly form methemoglobin and superoxide anion when flashed with low intensity (38 joules) white light. The effect only occurred in quartz but not glass (cut off lambda approximately equal to 300 nm) cuvettes. The formation of O2 was established by observing the reduction of oxidized cytochrome c concomitant with MetHb formation at pH 9, and by showing that superoxide dismultase and catalse inhibit cytochrome c reduction at that pH. The inhibition of cytochrome c reduction by catalase led us to explore the possibility that H2O2 might reduce oxidized cytochrome c at pH 9. We show that H2O2 does reduce oxidized cytochrome c at that pH but not at pH 7. Furthermore, catalase but not superoxide dismutase, almost completely inhibited this reduction process. These experiments serve to confirm our interpretation of the effect of catalase on the reduction of oxidized cytochrome c in the photolytic experiments, thus establishing that H2O2 was also formed. In addition, we were able to identify the production of O2 and H2O2 due to the photolysis of water in agreement with the results of McCord and Fridovich ((1973) Photochem. Photobiol. 17, 115-121). Production of O2 from this source was considerably less than that observed when HbO2 was present. Addition of MetHb to aerated solutions of oxidized cytochrome c did not cause additional reduction, unlike addition of HbO2. The production of MetHb was found to have at least two components. One component was the primary photolytic process, and the second was a strongly pH-dependent reattack of HbO2 by O2. Addition of superoxide dismutase inhibited this second component, but did not significantly effect the primary photolytic process.     

The mechanism of superoxide anion generation by the interaction of phenylhydrazine with hemoglobin.

The mechanism by which superoxide anion is generated by the interaction of phenylhydrazine with either oxy- or methemoglobin was investigated. Rather than superoxide anion generation resulting from an accelerated autooxidation of oxyhemoglobin, it was found that both oxy- and methemoglobin function as peroxidases toward phenylhydrazine with the resultant oxidation of this compound to phenyldiazine. Generation of phenyldiazine from the oxidation of phenylhydrazine by hemoglobin or by the hydrolysis and subsequent decarboxylation of methyl phenylazoformate (C6H5N=NCOOCH3) resulted in the production of superoxide anion. It is suggested that under certain conditions hemoglobin may function as a drug-metabolizing peroxidase.     

Oxygen equilibrium study and light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins

Ni(II)-Fe(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunit are substituted with Ni(II) protoporphyrin IX, have been prepared and characterized. Since Ni(II) protoporphyrin IX binds neither oxygen nor carbon monoxide, the oxygen equilibrium properties of the Fe subunit in these hybrid hemoglobins were specifically determined. K1 values, namely the equilibrium constants for the first oxygen molecule to bind to hemoglobin, agreed well for these hybrid hemoglobins with the K1 value of native hemoglobin A in various conditions. Therefore, Ni(II) protoporphyrin IX in these hybrid hemoglobins behaves like a permanently deoxygenated heme. Both Ne-Fe hybrid hemoglobins bound oxygen non-co-operatively at low pH values. When the pH was raised, alpha 2 (Fe) beta 2 (Ni) showed co-operativity, but the complementary hybrid, alpha 2 (Ni) beta 2 (Fe), did not show co-operativity even at pH 8.5. The light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins indicated that the coordination states of Ni(II) protoporphyrin IX in the alpha subunits responded to the structure of the hybrid, whereas those in the beta subunits were hardly changed. In a deoxy-like structure (the structure that looks like that observed in deoxyhemoglobin), four-co-ordinated Ni(II) protoporphyrin IX was dominant in the alpha (Ni) subunits, while under the conditions that stabilized an oxy-like structure (the structure that looks like that observed in oxyhemoglobin), five-co-ordinated Ni(II) protoporphyrin IX increased. The small change observed in the absorption spectrum of the beta (Ni) subunits is not related to the change of the co-ordination number of Ni(II) protoporphyrin IX. Non-co-operative binding of oxygen to the beta subunits in alpha 2 (Ni) beta 2 (Fe) accompanied the change of absorption spectrum in the alpha (Ni) subunits. We propose a possible interpretation of this unique feature.     

Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Nucleotide binding sites

The photoaffinity analogs 2-azidoadenosine 5'-tri(di)-phosphate (2-N3AT(D)P) and 8-azidoadenosine 5'-triphosphate (8-N3ATP) have been used to probe the substructural organization of the nucleotide binding pockets within the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Both 2-N3ATP and 8-N3ATP are competitive inhibitors of dynein ATP hydrolysis, and both analogs are themselves hydrolyzed by the alpha-beta dimer. Following vanadate-dependent photolysis at the V1 site (by UV irradiation in the presence of Mg2+, ATP, and vanadate), both probes exclusively labeled the larger fragment from the alpha chain. In contrast, within the beta chain the predominant insertion sites for the two analogs were located on opposite sides of the V1 site. Therefore, the hydrolytic pockets of these two molecules have different substructures. Vanadate-dependent photolysis of the alpha and beta chains at the V2 sites (by UV irradiation in the presence of vanadate and Mn2+) profoundly affected the predominant modification sites; for example, following photolysis at the V2a site neither fragment of the alpha chain was photolabeled by 2-N3ATP or 8-N3ATP. Based on the photolabeling patterns obtained, the single V2 site within the beta chain is predicted to be analogous to the V2b site within the alpha chain. The results support the hypothesis that the V2 sites occur within the ATP binding pockets, and indicate that these functional domains are composed of portions of the heavy chains which are linearly separated by up to at least 100,000 daltons. Thus, the central region of each dynein heavy chain must be extensively folded so as to bring the widely separated photocleavage and photolabeling sites together within a single catalytic unit.     

Antioxidant properties of natural hydroquinones from the marine colonial tunicate Aplidium californicum.

Antioxidant prenylated hydroquinones and non active chromene or chroman extracted from the marine colonial tunicate Aplidium californicum have been studied in order to throw some light on their biological activity. It has been found that the active compounds inhibit superoxide anion production in rat alveolar macrophages and in the xanthine/xanthine oxidase system. The antioxidant activity may be ascribed rather to a direct reaction of the superoxide anion with the hydroquinones than to an enzymatic inhibition or a membrane signal transfer. A physiological activity close to that of alpha tocopherol can be considered.     

Spectral and oxygen-release kinetic properties of human hemoglobin bound to the cytoplasmic fragment of band 3 protein in solution

Binding of the cytoplasmic fragment of band 3 protein to oxyhemoglobin in solution caused a spectral change in the absorbance of the hemoglobin beta chain at a ratio of one monomer of band 3 protein per alpha beta dimer of hemoglobin. This spectral change was reversed at higher ratios of cytoplasmic fragment to hemoglobin. The unusual dependence on protein concentration was interpreted as indicating the formation of higher aggregates of the complex between hemoglobin and the cytoplasmic fragment of band 3 protein. Oxygen-release kinetic measurements also showed marked changes as a function of the concentration of the cytoplasmic fragment of band 3 protein. The higher ratio mixture had significantly different kinetic properties as compared with the lower ratio one, which in turn was different from oxyhemoglobin in solution. The significance of the formation of aggregates of band 3 protein containing oxyhemoglobin dimers is discussed in context with evidence suggesting that band 3 protein may exist as an equilibrium mixture of tetramers and dimers in the membrane.     

The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect.

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.     

EPR detection of glutathiyl and hemoglobin-cysteinyl radicals during the interaction of peroxynitrite with human erythrocytes

Peroxynitrite, which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of human diseases. An initial controversy about the possibility of free radical production from peroxynitrite in test tubes has been resolved, and presently it is important to establish whether peroxynitrite produces radicals in cells. Here we employed the EPR spin trapping methodology with 5,5-dimethylpyrroline N-oxide (DMPO) to study the interaction of peroxynitrite with human erythrocytes. The results confirmed previous findings in demonstrating that oxyhemoglobin is the main target of peroxynitrite in erythrocytes. As we first show here, the produced ferryl-hemoglobin oxidizes its own amino acids and, most probably, amino acids from other hemoglobin monomers to produce hemoglobin-tyrosyl and hemoglobin-cysteinyl radicals. In parallel, ferryl-hemoglobin also oxidizes intracellular glutathione to produce the glutathiyl radical. The EPR spectrum of both DMPO/(*)cysteinyl-hemoglobin (a(beta)(H) = 15.4 G) and DMPO/(*)tyrosyl-hemoglobin (a(beta)(H) = 8.8 G) radical adducts was characterized. It is proposed that erythrocytes can be efficient peroxynitrite scavengers in vivo through the coupled action of oxyhemoglobin and glutathione. Overall, the results indicate that, through the intermediacy of carbon dioxide and/or hemoproteins, oxidation of glutathione to the glutathiyl radical is likely to be an important consequence of peroxynitrite production in vivo.     

Photogeneration of superoxide by adriamycin and daunomycin. An electron spin resonance and spin trapping study

The hydroxyl and superoxide anion spin adducts of DMPO and 4-MePyBN, respectively, were obtained during photoirradiation of adriamycin and daunomycin solutions with visible light. Ethanol and dimethyl sulfoxide did not scavenge hydroxyl radicals in the photoirradiated drug solutions. Furthermore, the hydroxyl-DMPO spin adduct is not formed in the photolysis of air-free drug solutions, indicating that hydroxyl radicals are not directly produced in the photochemical reactions. Instead, the observed hydroxyl-DMPO is formed from the decay of the superoxide anion-DMPO spin adduct. The mechanism for generating the superoxide anion radical appears to be a direct electron transfer from the photoexcited adriamycin and daunomycin to dissolved oxygen.     

The kinetics of assembly of normal and variant human oxyhemoglobins

The kinetics of assembly have been monitored spectrophotometrically for normal and variant human oxyhemoglobins in 0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, at 21.5 degrees C. Oxyhemoglobin versus oxy chain static difference spectra were performed and revealed subtle but significant absorption changes in both the visible and Soret regions. Kinetic experiments were performed by rapidly mixing equivalent (in heme) concentrations of alpha and beta A chains and following the change in absorbance at 583 nm with time. Over a protein concentration range of 10-100 microM in heme prior to mixing, these time courses were homogeneous and followed first-order kinetics, yielding a value of 0.069 s-1 for the apparent rate constant of dissociation of oxygenated beta A chain tetramers. Under these conditions, the overall assembly of oxyhemoglobins S (beta 6Glu----Val) and N-Baltimore (beta 95Lys----Glu) were also governed by the rates of dissociation of their respective oxygenated beta S and beta N-Baltimore chain tetramers with the apparent first-order rate constants of 0.044 and 0.15 s-1, respectively. In the Soret region, the alpha, beta monomer combination reaction could be observed if the protein concentration (heme basis) was lowered and if protein nonequivalency (beta chain exceeded alpha chain concentration) mixing experiments were performed. A kinetic oxyhemoglobin A, oxy-alpha, oxy-beta A monomer difference spectrum could be generated, and simple second-order kinetics were observed (415 nm) yielding rate constants of 2.3, 3.3, and 4.8 X 10(5) M-1 s-1 for the assembly of oxyhemoglobins S, A, and N-Baltimore, respectively. To our knowledge, this is the first kinetic study to reveal significant differences between the rate of association of alpha and beta monomers of hemoglobin A and those of two distinctly charged hemoglobin variants.     

The kinetics of chain exchange in two-chain coiled coils: alpha alpha- and beta beta-tropomyosin.

Measurements are presented on the time course of chain exchange among two-chain alpha-helical coiled coils of rabbit tropomyosin. All experiments are in a regime (temperature, protein concentration) in which coiled-coil dimers are the predominant species. Self-exchange in alpha alpha-tropomyosin was investigated by mixing alpha alpha species with alpha* alpha*, the asterisk designating an alpha-chain whose lone sulfhydryl (C190) has been blocked by carboxyamidomethylation. The overall process alpha alpha + alpha* alpha* in equilibrium with 2 alpha alpha* is followed by measurement of the fraction (h) of alpha alpha* species as a function of time. Similarly, self-exchange in beta beta-tropomyosin is examined by measurements of the overall process: beta beta + beta* beta* in equilibrium with 2 beta beta*, in which beta* signifies a beta-chain blocked at both sulfhydryls (C36 and C190). The observed time course for both chains is well fit by the first-order equation: h (t) = h (infinity) (1-e-k1t), with h (infinity) congruent to 0.5. This long-time limit is as expected for self-exchange, and agrees with experiments that attain equilibrium after slow cooling of thermally dissociated and unfolded chains. The simplest consonant mechanism is chain exchange by rate-limiting dissociation of dimers followed by random reassociation. Kinetic analysis shows k1 to be the rate constant for the chain dissociation step, a quantity not previously measured for any coiled coil. This rate constant for beta beta species is about an order of magnitude greater than for alpha alpha. In both, the activation enthalpy and entropy are very large, suggesting that activation to an extensively (greater than 50%) unfolded species necessarily precedes dissociation. Experiments are also reported for overall processes: alpha alpha + beta* beta* in equilibrium with 2 alpha beta* and alpha* alpha* + beta beta in equilibrium with 2 alpha* beta. Results are independent of which chain is blocked. Again h (infinity) congruent to 0.5, in agreement with equilibrium experiments, and the time course is first order. The rate constants and activation parameters are intermediate between those for self-exchange.     

Iron(III)-mediated photolysis of outer arm dynein ATPase from sea urchin sperm flagella

Irradiation of outer arm dynein ATPase from sea urchin sperm tail flagella at 365-410 nm in the presence of Fe(III)-gluconate complex and ATP produces photolytic cleavage at two distinct sites on the beta heavy chain, located approximately 250 and approximately 230 kDa from its amino terminus. The former cut is close to or identical with the V1 site of the vanadate-mediated photocleavage (Gibbons, I.R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J.Y., and Gibbons, B.H. (1987) J. Biol. Chem. 262, 2780-2786. The rate of photolysis shows a hyperbolic dependence on Fe(III)-gluconate concentration with half-maximal rate occurring at 23 microM at pH 6.3. In the presence of 0.1-0.5 mM Fe(III)-gluconate-ATP, approximately 58% of the beta chain becomes cleaved with a half-time of about 34 s; the remainder of the beta chain and almost all of the alpha chain are resistant to cleavage. This photolytic cleavage of the beta chain is accompanied by an approximately parallel loss of the dynein latent ATPase activity, whereas the Triton-activated ATPase is lost to a somewhat greater extent. Mg2+ concentrations above approximately 3 mM inhibit photolysis. Substitution of ADP for ATP changes the pattern of cleavage so that both the alpha and beta heavy chain undergo scission but at the 250-kDa site only. AMP, adenyl-5'-yl imidodiphosphate and Fe(II) do not support cleavage at either site. Trivalent rhodium-ATP complexes, as models of MgATP, can also catalyze photolysis of the beta chain at the 250-kDa site. These results suggest that photolysis results from the activation of an Fe(III)-ATP complex bound to the hydrolytic ATP binding site of the beta chain and that both Fe(III) cleavage sites are located close to the nucleotide binding site in the tertiary folding of the beta heavy chain. The cleavage reaction possibly involves initial photoreduction of Fe(III) bound at the Mg2+ binding site in the dynein.Fe.ATP complex, followed by covalent modification of an amino acid side chain that leads to eventual peptide scission.     

Interaction of subunits of polypeptide chain elongation factor I from pig liver. Formation of EF-1alpha.EF-1betagamma and EF-1beta complexes

In the preceding papers, we showed that one of the two complementar factors of polypeptide chain elongation factor 1 (EF-1) from pig liver, EF-1alpha, functionally corresponds to bacterial EF-Tu (Nagata, S., Iwasaki, K., and Kaziro, Y. (1976) Arch. Biochem. Biophys. 172, 168), while the other, EF-1betagamma, as well as one of its subunits, EF-1beta, corresponds to bacterial EF-Ts (Motoyoshi, K. and Iwasaki, K. (1977) J. Biochem. 82, 703). Therefore, the interaction between EF-1alpha and EF-1 betagamma or EF-1beta was was examined and the following results were obtained. i) EF-1betagamma catalytically promoted the exchange of [14C]GDP bound to EF-1alpha with exogenous [3H]GDP. ii). In the absence of the exogenous guanine nucleotide, EF-1betagamma as well as EF-1beta could displace GDP bound to EF-1alpha to form an EF-1alpha.EF-1betagamma as well as an EF-1alpha.EF-1beta complex. iii) The occurrence of EF-1alpha.EF-1betagamma and EF-1alpha.EF-1beta complexes was demonstrated by gel filtration on Sephadex G-150. These results strongly indicate that the mechanism of the action of EF-1betagamma or EF-1beta in converting EF-1alpha.GDP into EF-1alpha.GTP is analogous to bacterial EF-Ts, and the reaction is accomplished by the following reactions; EF-1alpha.GDP + EF-1betagamma (or EF-1beta) in equilibrium EF-1alpha.EF-1betagamma (or EF-1beta) + GDP; EF-1alpha.EF-1beta (or EF-1beta) + GTP IN EQUILIBRIUM EF-1alpha.GTP + EF-1betagamma (or EF-1beta).     

Production of superoxide anion during the oxidation of hemoglobin by menadione.

Menadione in the presence of oxyhemoglobin will accelerate the formation of methemoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.