Difference in rates of the reaction of various mammalian oxyhemoglobins with phenylhydrazine.

Second order rate constants for the initial reaction of 12 mammalian oxyhemoglobins (Hb) with equimolar phenylhydrazine (PHZ), a compound inducing Heinz body hemolytic anemia, were determined by recording continuous changes in absorbance with time at 577 nm. The rate constants were varied in a range from 43 m-1.s-1 with pig Hb to 255 m-1.s-1 with dog Hb. On the other hand, isosbestic points at 526 and 587 nm were common to all the reaction processes. The aerobic reaction of Hb with PHZ resulted in denaturation of hemoprotein, and final reaction products were determined to be beta-meso-phenylbiliverdin IX alpha and N-phenylprotoporphyrin IX. These results suggest that the reactivity of PHZ to Hb is influenced by the globin molecule, and the oxidative cleavage of the porphyrin ring causes the denaturation of hemoprotein.

Bonding of nitrosobenzene and phenyl isocyanide to chelated mesoheme, hemoglobin and ferrous phthalocyanine.

Reactions of nitrosobenzene, phenyl isocyanide and their ring-substituted analogues with hemoglobin, ferrous phthalocyanine and a synthetic model compound of hemoglobin were investigated by optical, 1H-NMR and infrared spectroscopy. Complexes of chelated ferromesoheme, the model compound, with 2-methyl-, 2-ethyl, 2-isopropyl- or 2,6-disubstituted nitrosobenzene were less stable than its complex with nitrosobenzene. Formation of a complex of the model compound with 2-tert-butylnitrosobenzene was incomplete. Previous studies showed that 2,6-disubstituted nitrosobenzenes are not ligands of ferrohemoglobin. In the present work 2,6-dimethylphenyl isocyanide was found to be a ligand of ferrohemoglobin. These results are consistent with binding of the nitrogen of the nitroso group of nitrosobenzene and of the carbon of the isocyanide group of phenyl isocyanide to ferroheme. The same bonding modes of these ligands to ferrous phthalocyanine were inferred from ring-current-induced shifts in the 1H-NMR spectra of the respective complexes.

Radical intermediates in the oxidation of octaethylheme to octaethylverdoheme.

Iron(III) oxyoctaethylporphyrin was isolated and purified as a dimer. The addition of tosylmethyl isocyanide to a solution of the dimer produced a monomer species, which was isolated and identified as bis(tosylmethyl isocyanide)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. The product of dissociation of the dimer by imidazole was bis(imidazole)iron(III) 5-oxyoctaethylporphyrin. The spectral properties of the product of dissociation of the dimer by pyridine and published data on bis(pyridine)oxymesoheme and bis(pyridine)oxyprotoheme were consistent with its identification as bis(pyridine)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. When this product was exposed to oxygen, a weak radical signal appeared in its electron spin resonance spectrum, which was attributed to the displacement of one of its pyridine ligands by O2 to form (pyridine)(dioxygen)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. The pyridine oxygen radical converted spontaneously to octaethylverdohemochrome, which was purified and identified as bis-(tosylmethyl isocyanide)iron(II) octaethylverdohemochrome hydroxide. The yield of verdohemochrome from iron oxyporphyrin was increased by the addition of phenylhydrazine or ascorbate. A scheme for the oxidation of iron(III) oxyporphyrin to iron(II) verdoheme by O2 that proposes a mechanism for the expulsion of CO and the replacement of a methene bridge of the porphyrin ring by an oxa bridge is presented.

Formation of aryl and aryldiazenyl complexes in reactions of arylhydrazines and aryldiazenes with a synthetic model compound of haemoprotein.

The anaerobic reaction of chelated protohaemin, a synthetic model compound of ferrihaemoglobin, with phenyldiazene produced a compound with the visible-absorption spectrum of a ferrihaemochrome. The compound reacted with CN-, which is a ligand of both ferric and ferrous porphyrins, to produce the complex of the synthetic ferrihaemoglobin with CN-. Though the spectrum of the compound formed by the addition of phenyldiazene to chelated protohaemin is characteristic of a ferric porphyrin complex, this compound reacted with both toluene-p-sulphonylmethyl isocyanide and CO, which are strong ligands of ferrous porphyrins, to produce the corresponding ferrous complexes. These ligand-binding reactions indicated that the complex of chelated protohaem with phenyldiazene can behave either as a complex of a ferric porphyrin with phenyldiazenyl anion (C6H5N = N-) or a complex of a ferrous porphyrin with phenyldiazenyl radical (C6H5N = N.). Para substituents on phenyldiazene were without effect on the formation of 4-substituted phenyldiazenyl complexes with chelated protohaem. Ortho substituents resulted in less-stable complexes. The phenyl complex of chelated protohaem was prepared by the aerobic reaction of phenylhydrazine with chelated protohaemin, and its structure was confirmed by its n.m.r. spectrum. The ligand-binding properties, n.m.r. spectrum and absorption spectrum of this complex differed from those of the phenyldiazenyl complex. The phenyl complex also was produced when the phenyldiazenyl complex was exposed to O2.

Urinary excretion of isomers of biliverdin after destruction in vivo of haemoproteins and haemin.

The amount and isomeric composition of urinary biliverdin in rabbits were analysed by h.p.l.c. Physiological values were maintained after the injection of haemin. On the other hand, when haemoglobins from several mammalian species were injected into rabbits, the excretion of biliverdin-IX alpha and biliverdin-IX beta were increased 6-18-fold and 32-66-fold respectively over physiological excretion. Injection of myoglobin resulted in a 44-fold increase in excretion of the IX alpha-isomer. Coupled oxidation with ascorbate of haemoglobin and myoglobin by oxygen produced mainly the IX alpha- and IX beta-isomers from haemoglobin and the IX alpha-isomer from myoglobin. The destruction of part of the haem from injected haemoproteins by non-enzymic chemical degradation would account for the observed respective increases in the excretion of biliverdin isomers. The excretion of biliverdin isomers after the injection of phenylhydrazine into rabbits was similar to that after the injection of haemoglobin.

A two-molecule mechanism of haem degradation.

Coupled oxidation of octaethylhaemin and phenylhydrazine hydrochloride with 16,16O2 and 18,18O2 produced octaethyl[16O]verdohaemochrome and octaethyl[18O]-verdohaemochrome respectively. Reactions of these products with 16,16O2 in the presence of phenylhydrazine hydrochloride yielded octaethyl[16O, 16O]biliverdin and octaethyl[18O, 16O]biliverdin. The same reactions with 18,18O2 yielded octaethyl[16O, 18O]biliverdin and octaethyl[18O, 18O]biliverdin. Accordingly, the two oxygen atoms of biliverdin are incorporated from different O2 molecules in separate reactions, namely the formation of verdohaemochrome and the conversion of verdohaemochrome into biliverdin. These reactions account for a "two-molecule mechanism' of biliverdin formation from haem with verdohaemochrome participating as an intermediate product.

Mechanism of initial reaction of phenylhydrazine with oxyhemoglobin and effect of ring substitutions on the biomolecular rate constant of this reaction.

Phenylhydrazine in the presence of oxygen causes the oxidative denaturation of hemoglobin. The initial step in this process is a bimolecular reaction, probably a two-electron transfer from phenylhydrazine to oxyhemoglobin. The product of this reaction is neither methemoglobin nor deoxyhemoglobin. Superoxide dismutase and catalase eliminate side reactions that increase the apparent rate of this reaction as measured spectrophotometrically at 577 nm; scavengers for the hydroxyl radical and singlet oxygen do not affect this rate either in the presence or in the absence of these enzymes. Halogen atoms and alkyl groups decrease the rate when ortho and increase the rate when meta or para to the hydrazino group. Chlorine atoms at both ortho positions or the carboxylate group at the ortho or the para position block the reaction. In the presence of phenylhydrazine under air, methemoglobin is converted to the same complex as that produced when phenyldiazene is added to methemoglobin anaerobically. Under N2 or CO, phenylhydrazine reduces methemoglobin to deoxyhemoglobin or carbonmonoxyhemoglobin.

Verdohemochrome IX alpha: preparation and oxidoreductive cleavage to biliverdin IX alpha.

Several studies have shown that both terminal oxygen atoms of biliverdin are derived from molecular oxygen. Since the conversion of verdohemochrome to biliverdin has been assumed to be hydrolytic, these findings seemed to exclude verdohemochrome as an intermediate in the degradation of heme to biliverdin. Coupled oxidation of myoglobin and ascorbate yielded a pure preparation of verdohemochrome IX alpha. The structure and ferrous state of this product were determined from its composition, ligand reactions, 1H NMR spectrum, and conversion to biliverdin IX alpha dimethyl ester. Reaction with ascorbate and 18O2 converted this compound to biliverdin that contained an atom of 18O. Successive treatment of verdohemochrome, first oxidation with H2O2 and then reduction with phenylhydrazine, yielded the iron complex of biliverdin. These results showed that hydrolysis is not an obligatory step in the conversion of verdohemochrome to biliverdin and, moreover, indicated how heme can be converted, with verdohemochrome as an intermediate, into biliverdin in which the two terminal oxygen atoms are derived from different O2 molecules.

beta-meso-Phenylbiliverdin IX alpha and N-phenylprotoporphyrin IX, products of the reaction of phenylhydrazine with oxyhemoproteins.

Oxyhemoglobin and oxymyoglobin were allowed to react aerobically with phenylhydrazine and p-tolylhydrazine. The chloroform extract of each reaction mixture, after treatment with H2SO4/methanol, yielded a blue pigment and a green pigment, which were identified by electronic absorption, mass, and proton NMR spectroscopy as the dimethyl esters of beta-meso-arylbiliverdin IX alpha and N-arylprotoporphyrin IX, respectively. N-Phenylprotoporphyrin IX dimethyl ester formed complexes with Zn2+, Cd2+, and Hg2+ but not with other cations. The proton NMR spectrum of the zinc complex suggested binding of the phenyl group to one of the two pyrrole rings of protoporphyrin IX with a propionic acid substituent. The effectiveness of phenylhydrazine as an inducer of Heinz body formation may be due to destabilization of the hemoglobin molecule by the replacement of heme with phenyl adducts of biliverdin and protoporphyrin.

The reaction of halophenylhydrazines with oxygen in the presence of oxyhemoglobin and metal ions.

We have compared the rates of reaction of ortho and para substituted halophenylhydrazines with oxygen, and we have found that the reaction rates of these phenylhydrazines are accelerated by metal ions and oxyhemoglobin. Stimulation of the reaction rate by oxyhemoglobin was 20-times that by Fe3+ at the same concentration. In the presence of oxyhemoglobin, the initial decrease in the concentration of oxygen was followed by an increase. We propose that phenyldiazene produced from the oxidation of phenylhydrazine by oxyhemoglobin reduced oxygen to superoxide and caused the initial rapid decrease in oxygen concentration. The partial restoration of oxygen in the reaction mixture could be accounted for by the dismutation of superoxide to oxygen and hydrogen peroxide, and of hydrogen peroxide to oxygen and water.

Induction of Heinz body formation by sodium dithionite.

Incubation of normal erythrocytes with sodium dithionite resulted in the formation of Heinz bodies, but incubation with sodium metabisulfite did not. Addition f superoxide dismutase to the incubation medium increased the formation of Heinz bodies by sodium dithionite. Addition of catalase to suspensions of erythrocytes in the presence and absence of superoxide dismutase inhibited the formation of Heinz bodies. These findings indicate that hydrogen peroxide, not superoxide, is the active oxidant in Heinz body formation.

The initiation of fetal hemoglobin biosynthesis.

Biosynthesis of the alpha and beta chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the delta chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent gamma chain at the time of removal of its N-terminal methionine was estimated to be 40--60 from the relative yields of labeled tryptic peptides.

Oxidative degradation of haemoglobin by nitrosobenzene in the erythrocyte.

Substitutions on the benzene ring of nitrosobenzene did not have the same effect on oxidative haemolysis as substitutions on phenylhydrazine. We previously found that the haemolytic effect of arylhydrazines paralleled their oxidative conversion into ligands of ferrihaemoglobin. In contrast, although most substituted nitrosobenzenes that are ligands of ferrohaemoglobin caused haemolysis and most that are not ligands failed to cause nitrosoarenes appeared to be related more closely to the ease of their reduction to arylhydroxylamines than to their properties as ligands. We propose a mechanism of oxidative degradation whereby the cyclic formation of phenylhydroxylamine from nitrosobenzene within an erythrocyte leads to the accumulation of H2O2, which then reacts with ferrohaemoglobin to initiate the oxidative cleavage of haem. The posulated active intermediate in this reaction is the same as that previously proposed in the oxidative degradation of haemoglobin by phenylhydrzine and in the coupled oxidation of ascorbic acid and haemoglobin.

Influence of ring substituents on the binding of nitrosobenzene by ferrohemoglobin.

The affinity of a substituted nitrosobenzene for ferrohemoglobin and the visible absorption spectrum of the resulting compound was influenced by the nature, position, and number of substituents on its benzene ring. Alkyl ring substituents inhibited the binding of nitrosobenzene to ferrohemoglobin, and binding was blocked by an ortho tertbutyl group or by a pair of ortho methyl groups. A single halogen atom increased binding affinity except that iodine decreased affinity, more at the ortho than at the para position. Binding occurred with a pair of ortho fluorine atoms but not with a pair of ortho chlorine or bromine atoms. These results favor a model of nitrosoarene ferrohemoglobin in which the iron of ferroheme is bonded to the nitrogen atom of the nitroso group since a bond to the oxygen atom would not be hindered by ortho substituents. The presence of a carboxylate substituent resulted in prevention of binding, which was reversed by esterification of the group. Large neutral para substituents, which cannot directly affect formation of the Fe-NO bond, inhibited binding, although not to the same degree as ortho substituents. It thus appears that the affinity of a substituted nitrosobenzene for ferrohemoglobin depends on interactions of the nonbonding part of the ligand molecule with the heme crevice as well as on the ability of its nitroso group to form a bond with the iron of heme. Nitrosoarenes also exhibited in varying degrees the property of removing an electron from ferrohemoglobin to form ferrihemoglobin.

Ligands and oxidants in ferrihemochrome formation and oxidative hemolysis.

We have investigated the effect of size of a single neutral ring substituent on the induction of hemolytic anemia and the formation of a ferrihemochrome by substituted phenylhydrazines. The severity of induced anemia decreased with increase in size of a halogen atom or an alkyl group ortho to the hydrazino group, little anemia resulting from 2-iodophenylhydrazine and no anemia from 2-tert-butylphenylhydrazine. The size of a halogen atom or an alkyl group at the meta or para position had relatively little effect on the severity of induced anemia. The ability of an arylhydrazine to induce hemolytic anemia paralleled its ability to produce a ferrihemochrome with an exogenous ligand, probably the corresponding aryldiazene. In general, rapid and complete formation of ferrihemochrome occurred with arylhydrazines that induced severe anemia. The degree of hemolysis induced by an arylhydrazine was not related to its rate of autooxidation, i.e., the rate at which oxidants are formed by the reduction of oxygen. We propose a mechanism of arylhydrazine-induced oxidative denaturation based on the simultaneous formation of hydroxyl radical and aryldiazene ferrihemochrome in a reaction of oxyhemoglobin with arylhydrazine. We suggest that after oxidation of the porphyrin ring is initiated by a hydroxyl radical, oxidative cleavage of the ring is facilitated by the presence of a large ligand in the heme crevice. Thus, aryldiazene ferrihemochrome may contribute to instability in a hemoglobin molecule, whereas globin ferrihemochrome results from instability.

In vitro translation of globin: effect of proteins purified by affinity chromatography on polyadenylate-Sepharose.

By means of affinity chromatography on poly(adenylic acid) (poly(A))-fixed Sepharose, protein fractions having strong affinity to poly(A) were prepared from postribosomal supernatants of rabbit reticulocyte and rat liver. These fractions contained several proteins similar by electrophoretic analysis to rabbit globin messenger ribonucleoprotein. Protein fractions from both sources were shown to form ribonucleoprotein complexes with rabbit globin mRNA, and these complexes sedimented at the same rate as native globin messenger ribonucleoprotein. Binding of the proteins to RNA was not highly specific, since not only poly(A) but also other polynucleotides as poly(C) or poly(U) were bound to these proteins. Ribosomal RNAs, tRNA, or DNAs did not bind the proteins. In order to ascertain the function of the poly(A)-Sepharose purified proteins, their effects on translation of globin mRNA was studied in vitro. Addition of rabbit reticulocyte protein to globin mRNA resulted in no more than a slight stimulation of both alpha- and beta-chain synthesis. Poly(A)-Sepharose purified protein from rat liver, however, caused a marked preferential reduction of alpha-chain synthesis. These results showed that at least some proteins in the poly(A)-Sepharose purified proteins affect the translation of globin. This inference suggested a possibility that protein moiety in globin mRNP might be involved in control of globin synthesis.

Reactions of phenyldiazene and ring-substituted phenyldiazenes with ferrihemoglobin.

Two simultaneous reactions take place between ferrihemoglobin and phenyldiazene in the absence of excess ferricyanide or of oxygen, namely the reduction of ferrihemoglobin to ferrohemoglobin and the binding of an exogenous ligand by ferrihemoglobin to form a compound with the optical spectrum of a ferrihemochrome. In the presence of excess ferricyanide, only the formation of a ferrihemochrome is observed. This compound differs from the ferrihemochrome induced by salicylate or by benzoate with respect to optical spectrum, concentration of inducer, and stability. One phenyl group is bound per heme, probably as phenyldiazene. Phenyl groups are also bound to globin, but phenyldiazene does not react anaerobically with thiols. Each ring-substituted isomer of methylphenyldiazene or bromophenyldiazene yields a different ferrihemochrome spectrum with ferrihemoglobin in the presence of ferricyanide. Only reduction of ferrihemoglobin occurs with 2- or 4-diazenylbenzoic acid in the absence of excess ferricyanide, but partial formation of ferrihemochrome occurs with 4-diazenylbenzoic acid in excess ferricyanide. The ability of an aryldiazene to bind quantitatively to ferrihemoglobin parallels the ability of the corresponding arylhydrazine to induce in vivo hemolysis.