Purification of uroporphyrinogen decarboxylase from human erythrocytes. Immunochemical evidence for a single protein with decarboxylase activity in human erythrocytes and liver.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 4419-fold to a specific activity of 58.3 nmol of coproporphyrinogen III formed/min per mg of protein (with pentacarboxyporphyrinogen III as substrate) from human erythrocytes by adsorption to DEAE-cellulose, (NH4)2SO4 fractionation, gel filtration, phenyl-Sepharose chromatography and polyacrylamide-gel electrophoresis. Progressive loss of activity towards uroporphyrinogens I and III occurred during purification. Experiments employing immunoprecipitation, immunoelectrophoresis and titration with solid-phase antibody indicated that all the uroporphyrinogen decarboxylase activity of human erythrocytes resides in one protein, and that the substrate specificity of this protein had changed during purification. The purified enzyme had a minimum mol.wt. of 39 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gel filtration gave a mol.wt. of 58 000 for the native enzyme. Isoelectric focusing showed a single band with a pI of 4.60. Reaction with N-ethylmaleimide abolished both catalytic activity and immunoreactivity. Incubation with substrates or porphyrins prevented inactivation by N-ethylmaleimide. An antiserum raised against purified erythrocyte enzyme precipitated more than 90% of the uroporphyrinogen decarboxylase activity from human liver. Quantitative immunoprecipitation and crossed immunoelectrophoresis showed that the erythrocyte and liver enzymes are very similar but not identical. The differences observed may reflect secondary modification of enzyme structure by proteolysis or oxidation of thiol groups, rather than a difference in primary structure.     

Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P-450-mediated uroporphyrinogen oxidation.

It was previously shown that uroporphyrinogen oxidation is catalysed by a form of cytochrome P-450 induced by 3-methylcholanthrene [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We have now measured uroporphyrinogen oxidation and uroporphyrinogen decarboxylation simultaneously in 10,000 g supernatants from the livers of methylcholanthrene-treated mice and chick embryos incubated with an NADPH-generating system. We found that uroporphyrinogen oxidation is associated with inhibition of uroporphyrinogen decarboxylase activity. The decreased uroporphyrinogen decarboxylase activity was not due to depletion of substrate, since decarboxylase activity was not increased by a 2.6-fold increase in uroporphyrinogen. Uroporphyrinogen oxidation and the associated inhibition of decarboxylase activity were also observed with liver supernatant from methylcholanthrene-treated chick embryo; both actions required the addition of 3,3',4,4'-tetrachlorobiphenyl. Uroporphyrinogen oxidation catalysed by microsomes from a methylcholanthrene-treated mouse inhibited the uroporphyrinogen decarboxylase activity in the 100,000 g supernatant. Ketoconazole, an inhibitor of cytochrome P-450, prevented both uroporphyrinogen oxidation and the inhibition of uroporphyrinogen decarboxylation. The addition of ketoconazole to mouse supernatant actively oxidizing uroporphyrinogen inhibited the oxidation and restored decarboxylation. The latter finding suggested that a labile inhibitor was formed during the oxidation. These results suggest uroporphyrinogen oxidation may be important in the mechanism of chemically induced uroporphyria.     

Photodynamic inactivation of red cell uroporphyrinogen decarboxylase by porphyrins

The effects of light and porphyrins on the activity of red cell uroporphyrinogen decarboxylase were studied. Photoinactivation of uroporphyrinogen decarboxylase was dependent on uroporphyrin concentration, irradiation time and temperature. Using 40 W/m2 of UV light intensity, 40-45% decreased activity was produced with 200 microM uroporphyrin I, at 37 degrees C and after 2 hr of illumination. It has been demonstrated that porphyrins photoinactivate uroporphyrinogen decarboxylase and a mechanism for this action in relation to skin lesions is proposed.     

Reduced substrate affinity for human erythrocyte uroporphyrinogen decarboxylase constitutes the inherent biochemical defect in porphyria cutanea tarda

The pathogenesis of human porphyria cutanea tarda (PCT) is associated with an intrinsic abnormality of the uroporphyrinogen decarboxylase enzyme. To characterize this, we studied the kinetic properties of the red cell enzyme procured from patients with various forms of PCT and non-porphyric controls. The enzyme activity (units/mg hemoglobin) in the red cell hemolysate was close to normal in sporadic PCT but about 75% diminished in the familial PCT. The Michaelis constants (Km) of 200-fold purified red cell enzyme preparations, determined by using pentacarboxylic porphyrinogen I and uroporphyrinogen I as substrates, were more than 3.8-4.0 times higher, and the maximum velocity (Vmax) was about 70% diminished in familial PCT, whereas the Km was about 1.7-1.9 times higher and the Vmax was more or less normal for sporadic PCT. These observations suggest for the first time that the primary lesion in familial PCT is a genetically determined kinetic abnormality of uroporphyrinogen decarboxylase which appears to be different from the sporadic form of the disease.     

Effects of polychlorinated biphenyl compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital and iron on hepatic uroporphyrinogen decarboxylase. Implications for the pathogenesis of porphyria.

Treatment of cultured chick embryo hepatocytes with phenobarbital, polychlorinated biphenyl compounds and 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in increased delta-aminolaevulinate synthase and decreased uroporphyrinogen decarboxylase activities and porphyrin accumulation; uroporphyrin and heptacarboxyporphyrin predominated. Iron had no effect on these changes. Simultaneous treatment of cultures with dioxin and phenobarbital produced a synergistic response in delta-aminolaevulinate synthase induction, uroporphyrinogen decarboxylase inhibition and porphyrin accumulation. These data suggest that an inhibitor of uroporphyrinogen decarboxylase may be generated in the liver from polychlorinated biphenyl compounds or dioxin by metabolic activation. Additionally these findings bear on the postulated role of these and related chemicals in determining the low levels of uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients.     

Immunoreactive uroporphyrinogen decarboxylase is unchanged in porphyria caused by TCDD and hexachlorobenzene

A method has been developed for the immuno-titration of rodent liver uroporphyrinogen decarboxylase (porphyrinogen carboxy-lyase, EC 4.1.1.37) and used to show that two porphyrogenic polyhalogenated aromatic hydrocarbons, 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin and hexachlorobenzene, cause porphyria in rodents by decreasing the catalytic activity of uroporphyrinogen decarboxylase without altering the amount of immunoreactive enzyme protein. Investigation of the nature of the inactive form of uroporphyrinogen decarboxylase produced by these compounds should provide new information about the mechanism of their toxicity.     

Study of the origin of urinary porphyrins in experimental hexachlorobenzene-induced porphyria

The activity of the enzyme uroporphyrinogen decarboxylase was determined in the liver and the kidneys of C57BL/6 mice and Wistar albino rats with chronic hexachlorobenzene intoxication and the amount of the deposited uroporphyrin was measured in the both organs. In the control animals the activity of hepatic uroporphyrinogen decarboxylase was several times higher than the renal one. The administration of hexachlorobenzene led to an inhibition of the enzyme activity, which was equally expressed (about 2.5 times) in the liver and kidneys of the both species. The accumulation of uroporphyrin was more pronounced in the hepatic tissue than in the kidneys (about 9 times in mice and 5 times in rats on average). Taking into consideration the much higher uroporphyrin accumulation in the liver, the more active haem biosynthesis in this organ, as well as its larger size, one could accept that the predominant part of the urinary porphyrins in hexachlorobenzene porphyria has a hepatic and not a renal origin.     

Defective human erythrocyte uroporphyrinogen decarboxylase in familial porphyria cutanea tarda: the metabolic lesion or the result of endogenous porphyrinemia?

We have demonstrated that oral charcoal therapy is as effective as therapeutic phlebotomy in reducing porphyrinemia in porphyria cutanea tarda. The effects of immediate and sustained reduction of porphyrinemia on the catalytic properties of partially purified (approximately 200-fold) preparations of red cell uroporphyrinogen decarboxylase of a patient with familial porphyria cutanea tarda were studied. All populations of the patient's red cells exhibited defective enzyme activity, and the apparent Michaelis constants (Km) determined with penta-, hepta-, and octa-carboxylic I porphyrinogen substrates were approximately 3-4 times higher as compared to the normal controls. Mixing experiments (normal and defective enzyme), and preincubation of the normal enzyme with porphyric plasma prior to purification, yielded data supporting the concept that the catalytic defects of red cell uroporphyrinogen decarboxylase in familial porphyria cutanea tarda are independent of interactions between circulating endogenous porphyrins and the enzyme.     

Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity.

The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis. The gene was isolated by functional complementation of a hem12 mutant. Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41,348 Da. The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N-terminus of an open reading frame described in Synechococcus sp. We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme. They correspond to the amino acid changes Gly33----Asp and Gly300----Asp, located in two evolutionarily conserved regions. Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein. These results argue that a single active center exists in uroporphyrinogen decarboxylase.     

The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker''s yeast (Saccharomyces cerevisiae).

Nine new hem12 haploid mutants of baker's yeast (Saccharomyces cerevisiae), totally or partially deficient in uroporphyrinogen decarboxylase activity, were subjected to both genetic and biochemical analysis. The mutations sites studied are situated far apart within the HEM12 gene located on chromosome IV. Uroporphyrinogen decarboxylase activity in the cell-free extracts of the mutants was decreased by 50-100%. This correlated well with the decrease of haem formation and the increased accumulation and excretion of porphyrins observed in vivo. The pattern of porphyrins (uroporphyrin and its decarboxylation products) accumulated in the cells of mutants partially deficient in uroporphyrinogen decarboxylase activity did not differ significantly, although differences in vitro were found in the relative activity of the mutant enzyme at the four decarboxylation steps. The excreted porphyrins comprised mainly dehydroisocoproporphyrin or pentacarboxyporphyrin. In heterozygous hem12-1/HEM12 diploid cells, a 50% decrease in decarboxylase activity led to an increased accumulation of porphyrins as compared with the wild-type HEM12/HEM12 diploid, which points to the semi-dominant character of the hem12-1 mutation. The biochemical phenotypes of both the haploid and the heterozygous diploid resembles closely the situation encountered in porphyria cutanea tarda, the most common human form of porphyria.     

Free radical mechanism of oxidation of uroporphyrinogen in the presence of ferrous iron

Human porphyria cutanea tarda is an unusual consequence of common hepatic disorders such as alcoholic liver disease. Hepatic iron plays a key role in the expression of the metabolic lesions, i.e., defective hepatic decarboxylation of porphyrinogens, catalyzed by uroporphyrinogen decarboxylase. This prompted the present study to determine the in vitro effects of iron on the uroporphyrinogen substrate in the absence and presence of atmospheric oxygen. We observed that (i) unless oxygen is the limiting reactant, autoxidation of ferrous iron and iron-catalyzed oxidation of uroporphyrinogen occurred soon after initiating the reaction at pH 7.4 and 30 degrees C in buffers which are non- or poor chelators of iron; (ii) the rates of uroporphyrinogen oxidation were proportional to the initial concentration of ferrous ion; (iii) about 70% of the oxidations of uroporphyrinogen were accountable due to a free-radical chain reaction pathway involving superoxide radical and hence inhibitable by superoxide dismutase; (iv) uroporphyrinogen could be further oxidized to completion by the hydroxyl radical since the reaction was partially inhibited by both mannitol and catalase which prevent hydroxyl radical production; (v) the oxidizing effects of ferric ion on uroporphyrinogen were none or negligible as compared to those of ferrous ion. Ferric was reduced to ferrous ion in the presence of dithiothreitol. When the ferrous ion thus formed was reoxidized in the presence of atmospheric oxygen, minor but definite oxidations of both uroporphyrinogen and dithiothreitol were observed. The oxidations of Fe2+ and uroporphyrinogen could be blocked by 1,10-phenanthroline, a ferrous iron chelator. The data suggest that ferrous is the reactive form of iron that may contribute to pathogenic development of the disease by irreversibly oxidizing the porphyrinogen substrates to nonmetabolizable porphyrins, which accumulate in porphyric liver.     

Enzymatic and immunological studies of uroporphyrinogen decarboxylase in familial porphyria cutanea tarda and hepatoerythropoietic porphyria

Uroporphyrinogen decarboxylase activity was measured in hemoglobin-free lysates from two patients with hepatoerythropoietic porphyria (HEP) and from 12 unrelated patients with familial porphyria cutanea tarda (PCT). In HEP patients, enzyme activities were 5% of normal, and familial studies clearly confirmed that patients with HEP are cases of homozygous PCT. Immunoreactive uroporphyrinogen decarboxylase was measured by developing a direct and noncompetitive enzyme immunoassay (EIA). For the 12 familial PCT patients, we found an immunoreactive protein decreased (51%) to the same extent as the catalytic activity (48%) [cross-reactive immunological material ( CRIM ) negative]. The children from the HEP family were also CRIM negative, contrasting with another HEP family previously described as CRIM positive; our data support the hypothesis of a heterogeneity in familial uroporphyrinogen decarboxylase deficiency.     

Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an Mr approximately 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of approximately 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol X h-1 X (mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uroporphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions including Fe2+, Co2+, Cu2+, Zn2+, and Pb2+. The pattern of accumulation of intermediate (hepta-, hexa-, and pentacarboxylate porphyrinogens) and final (coproporphyrinogen) decarboxylation products is affected by the ratio of substrate (uroporphyrinogen I or III) concentration to enzyme concentration. Under physiologic conditions where the uroporphyrinogen to enzyme ratio is low, the substrate is nearly quantitatively decarboxylated, and the major product is coproporphyrinogen. If the ratio of uroporphyrinogen to enzyme is high, intermediates accumulate, and heptacarboxylate porphyrinogen becomes the major decarboxylation product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for two uroporphyrinogen decarboxylase isoenzymes in human erythrocytes

In animals and plants, uroporphyrinogen decarboxylase catalyzes the stepwise decarboxylations of uroporphyrinogen, the precursor of heme and chlorophyll. To better understand its metabolic roles, we characterized the enzyme purified to electrophoretic homogeneity (about 11,000-fold) from human erythrocytes by a novel uroporphyrin-sepharose affinity chromatographic method. Native polyacrylamide disc gel electrophoresis of the purified enzyme preparation showed two bands detected by staining either for protein or with uroporphyrin-I. Each individual protein eluted from the gel when subjected to re-electrophoresis on SDS-polyacrylamide gel, appeared as a single protein band with molecular masses of approximately 54,000 and approximately 35,000 daltons respectively. Both proteins were able to catalyze all four decarboxylation steps, though the ratios of enzyme activity using octa-, hepta-, hexa- to pentacarboxylic porphyrinogen substrates were distinctly different. Also, their kinetic analysis with heptacarboxylic porphyrinogen-I substrate provided distinctly different apparent Michaelis constants. This provides the first evidence that decarboxylations of uroporphyrinogen to coproporphyrinogen are catalyzed by two isoenzymes.     

Uroporphyrinogen decarboxylase. Purification, properties, and inhibition by polychlorinated biphenyl isomers

Uroporphyrinogen decarboxylase (EC 4.1.1.37) which converts uroporphyrinogen I or III into coproporphyrinogen I or III, respectively, was purified about 5,500-fold from chicken erythrocytes. Purification was accomplished by chromatography on DEAE-cellulose, ammonium sulfate fractionation, chromatography on Sephadex G-100, and chromatofocusing. The most purified preparation was homogeneous on polyacrylamide gel electrophoresis and had a specific activity of 1,420 units/mg of protein, the highest value so far reported. The molecular weight, as determined by Sephadex G-150 gel chromatography, is 79,000. The subunit molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 39,700, suggesting that uroporphyrinogen decarboxylase is dimeric in form. The purified enzyme had an isoelectric point of 6.2 and a pH optimum of 6.8. The SH reagents inhibited the enzyme activity, but neither metal ions nor cofactor requirements could be demonstrated. A new and simple method for the separation of free uroporphyrin, hepta-, hexa-, and pentacarboxylic porphyrins and coproporphyrin was developed using a high pressure liquid chromatograph equipped with a spectrofluorometric detector. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified enzyme were performed. 3,4,3',4'-Tetrachlorobiphenyl and 3,4,5,3',4'5'-hexachlorobiphenyl which specifically induce delta-aminolevulinic acid synthetase also strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III.     

Decarboxylation of porphyrinogens by rat liver uroporphyrinogen decarboxylase.

The decarboxylations of uroporphyrinogens I and III and of heptacarboxylic, hexacarboxylic and pentacarboxylic porphyrinogens III by rat liver uroporphyrinogen decarboxylase were compared, and the results suggest that the removal of the first carboxy group from uroporphyrinogen III is a more rapid step than that from isomer I or the other substrates investigated.     

Investigations of rat liver uroporphyrinogen decarboxylase. Comparisons of porphyrinogens I and III as substrates and the inhibition by porphyrins.

1. The decarboxylations of uroporphyrinogens, hepta-, hexa- and penta-carboxyporphyrinogens I and III by porphyrinogen carboxy-lyase (EC 4.1.1.37) in rat liver supernatant have been compared as functions of substrate concentrations. Although Km and Vmax. (for total porphyrinogens formed) were estimated, prophyrinogens and CO2 produced at 1 microM were considered to be a better indication of real relative rates, owing to substrate/product inhibitions. Uroporphyrinogen III was the best substrate by the criteria of Km/Vmax. and decarboxylation at 1 microM and was converted into coproporphyrinogen more quickly than its series-I isomer. 2. The difference between uroporphyrinogens I and III as substrates was confirmed by using a mixture of [14C8]uroporphyrinogens, the discrimination occurring principally in the first decarboxylation. 3. Porphyrins, especially oxidation products of the substrates, inhibited the enzyme. Heptacarboxyporphyrin III was the most effective inhibitor of both uroporphyrinogen III and heptacarboxyporphyrinogen III conversion into coproporphyrinogen. 4. Rapid analysis of the livers from rats made porphyric with hexachlorobenzene demonstrated that substantial quantities of the tetrapyrroles were present in vivo as the porphyrinogens (21-42%). 5. Enzymic decarboxylation of uroporphyrinogen III in 2H2O-containing buffer gave [2H4]coproporphyrinogen. 6. Rats treated with cycloheximide for 10h showed no decrease in uroporphyrinogen decarboxylase activity/mg of protein, suggesting a relatively slow turnover of the enzyme.     

Response of enzymes involved in the metabolism of polyamines to phytohaemagglutinin-induced activation of human lymphocytes.

The stimulation of lymphocyte ornithine decarboxylase and adenosylmethionine decarboxylase produced by phytohaemagglutinin was accompanied by an equally marked, but delayed, stimulation of spermidine synthase, which is not commonly considered as an inducible enzyme. In contrast with the marked stimulation of these biosynthetic enzymes, less marked changes were observed in the biodegradative enzymes of polyamines in response to phytohaemagglutinin. Diamine oxidase activity was undetectable during all stages of the transformation. The activity of polyamine oxidase remained either constant or was slightly decreased several days after addition of the mitogen. The activity of polyamine acetylase (employing all the natural polyamines as substrates) distinctly increased both in the cytosolic and crude nuclear preparations of the cells during later stages of mitogen activation. Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, although powerfully inhibiting ornithine decarboxylase, produced a gradual enhancement of adenosylmethionine decarboxylase activity during lymphocyte activation, without influencing the activities of the two propylamine transferases (spermidine synthase and spermine synthase).     

Cloning and expression of Chlorobium vibrioforme uroporphyrinogen decarboxylase gene in a Salmonella heme auxotroph

To study the post-uroporphyrin steps in heme and chlorophyll biosynthesis in Chlorobium, we attempted to clone the uroporphyrinogen decarboxylase ( hemE) gene. A Chlorobium genomic library was used to transform a restriction-minus Salmonella typhimurium strain. The recombinant DNA molecules were transduced into an auxotrophic Salmonella double mutant ( hemA(-) hemE(-)) by phage P22. Faster-growing colonies indicated complementation of the hemE mutation. Each clone was tested by backcross transduction of the mutant. Growth rates of the confirmed clones in LB medium were comparable to wild-type Salmonella. HPLC analysis of the substrate (uroporphyrinogen) and the product (coproporphyrinogen) of the decarboxylase activity was performed in one such clone. This clone showed an active hemE gene within a 4-kb insert.     

The 1986 Upjohn award lecture. Interaction of chemicals with hemoproteins: implications for the mechanism of action of porphyrinogenic drugs and nitroglycerin

The ferrochelatase inhibitory activity of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was studied in chick embryo liver cells. The ferrochelatase inhibitory activity of the 4-butyl, 4-pentyl, and 4-hexyl analogues was considered to be due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of the corresponding N-alkylporphyrins. The relative ferrochelatase inhibitory activity of the DDC analogues has implications for a postulated model of the binding of porphyrins in the ferrochelatase active site. 3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) was shown to be a potent porphyrinogenic agent and to inhibit ferrochelatase in chick embryo liver cells. A related sydnone, 3-benzyl-4-phenylsydnone did not inhibit ferrochelatase activity. These results supported the idea that the porphyrinogenicity of TTMS was due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of N-vinylprotoporphyrin which inhibits ferrochelatase. Polychlorinated biphenyls, phenobarbital, nifedipine, and a large number of structurally different chemicals which are porphyrinogenic in chick embryo liver cells inhibit uroporphyrinogen decarboxylase by an unknown mechanism. Thus drug-induced porphyrin biosynthesis in chick embryo liver cell culture appears to be caused by inhibition of either ferrochelatase or uroporphyrinogen decarboxylase. The biotransformation of nitroglycerin by human red blood cells is due to a combination of a sulfhydryl-dependent enzymatic process and an interaction with reduced hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)