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PubMed1997

Reactivity of the tyrosyl radical of Escherichia coli ribonucleotide reductase -- control by the protein.

Ribonucleotide reductase is a key enzyme for DNA synthesis. Its small component, named protein R2, contains a tyrosyl radical essential for activity. Consequently, radical scavengers are potential antiproliferative agents. In this study, we show that the reactivity of the tyrosyl radical towards phenols, hydrazines, hydroxyurea, dithionite and ascorbate can be finely tuned by relatively small modifications of its hydrophobic close environment. For example, in this hydrophobic pocket, Leu77-->Phe mutation resulted in a protein with a much higher susceptibility to radical scavenging by hydrophobic agents. This might suggest that the protein is flexible enough to allow small molecules to penetrate in the radical site. When mutations keeping the hydrophobic character are brought further from the radical (for example Ile74-->Phe) the reactivity of the radical is instead very little affected. When a positive charge was introduced (for example Ile74-->Arg or Lys) the protein was more sensitive to negatively charged electron donors such as dithionite. These results allow us to understand how tyrosyl radical sites have been optimized to provide a good stability for the free radical.

Amino Acid Substitution↗
PubMed1987

Isolation and nitrogenase activity of vesicles from Frankia sp. strain EAN1pec.

Vesicles, specialized cell structures thought to be the site of nitrogen fixation in the actinorhizal bacteria, were isolated from Frankia sp. strain EAN1pec by using French pressure disruption of mycelia followed by differential and isopycnic gradient centrifugation. The isolated vesicles reduced acetylene when incubated anaerobically with Mg2+ ions, ATP, and dithionite. No nitrogenase activity was detected in the disrupted mycelial fractions. Vesicles permeabilized by freeze-thaw or detergents showed increased rates of acetylene reduction due to increased permeability of dithionite. The effect on nitrogenase activity of different ATP concentrations was the same in normal and permeabilized vesicles. The endogenous respiratory rate of vesicles was significantly lower than that of mycelia, and the respiration rate of vesicles did not increase following the addition of succinate. The low respiratory activity of vesicles and their apparent dependence on externally supplied ATP for acetylene reduction suggest that the energy and reducing power for nitrogen fixation may be supplied from the mycelia to which they are attached.

Anaerobiosis↗
PubMed1985

Kinetics of O2 uptake and release by human erythrocytes studied by a stopped-flow technique.

The kinetics of O2 uptake into and release from human erythrocytes was investigated at 37 degrees C by a stopped-flow technique. From the time course of O2 saturation (SO2) change a specific transfer conductance of erythrocytes for O2 (GO2) was calculated. The following results were obtained: 1) GO2 decreased in the course of O2 uptake, but initial GO2 was nearly independent of SO2 at which uptake started; 2) addition of albumin to the medium reduced GO2; 3) increasing dithionite concentration in the medium in O2-release experiments progressively enhanced GO2, which became virtually constant for nearly the entire course of release; and 4) O2 uptake and O2 release (without dithoite) in the same SO2 range yielded very similar GO2. These results suggested that O2 uptake and release were importantly limited by diffusion through the external medium and that in the SO2 range between 0.3 and 0.8, chemical reaction exerted little limiting effect. Since O2 release at the highest dithionite concentration (40 mmol/l) appeared to be virtually unlimited by external diffusion, GO2 measured under these conditions, averaging 8.7 ml X min-1 X Torr-1 X ml erythrocytes-1, was considered to mainly reflect intracellular diffusion limitation. The corresponding specific transfer conductance for O2 transfer in whole blood (hematocrit, 0.45) is 3.9 ml X min-1 X Torr-1 X ml blood-1.

Adult↗
PubMedDate not supplied

Chemical modification of biliprotein chromophores.

The reaction of biliproteins with sodium dithionite has been studied. The reagent is selective towards the chromophores. In denatured phycocyanin from Spirulina platensis, all three chromophores react to form yellow "phycorubin", whereas only 1/3 of the chromophores react in native phycocyanin in a non-statistical manner. From reversion experiments, it can be shown, that the thermodynamic stability of the chromophores towards reaction with dithionite is increased in the native pigment. Similarly, native phytochrome in its Pr form reacts only partially to a pigment absorbing at both 420 and 660 nm. The same product is formed from native Pfr, indicating both a reversion to Pr and a partial reduction.

Dithionite↗
PubMed2005

Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison's disease.

OBJECTIVE: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. DESIGN: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. METHODS: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. RESULTS: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. CONCLUSIONS: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

Addison Disease↗
PubMed1990

A new method for the determination of carboxyhemoglobin in blood of newborns. Possible clinical implications of elevated levels.

Spectral differences between hemoglobin derivatives from adult-(HbA) and fetal-hemoglobin (HbF) make the accurate determination of carboxyhemoglobin (COHb) troublesome in blood of newborns. Only the newest dedicated instruments for the determination of COHb take these differences into account. However, since there are virtually no spectral differences between both hemoglobins in the deoxygenated state, reduction of neonatal blood with sodium dithionite eliminates that spectrophotometric error. Based on this principle, an easy, fast and accurate method for the determination of COHb in minimal amounts of blood was developed; equally well suited for blood of newborns and adults. The instrument used with this new method is a general purpose centrifugal clinical chemistry analyzer (COBAS-FARA, Roche, Basle, Switzerland). After the reduction of blood with sodium dithionite, a simple two component system (Hb and COHb) is formed, which can be spectrophotometrically quantitated by measuring at two suitable wavelengths, i.e., 579 nm and 534 nm. The COHb fraction is calculated with the help of: COHb % = 177.73.(A534/A579)-176.66. Comparison with the OSM3 Hemoximeter (Radiometer A/S, Denmark) for 145 adult blood samples with varying COHb levels (0-30%) yielded a mean difference in COHb % of about 0.06% (SD = 0.38). For fetal blood (N = 63) about the same difference was found when the OSM3 was used in the so-called fetal mode, while comparison with the regular adult mode yielded a difference of about 3.1%. This indicates that the new method is indeed not disturbed by the spectral differences between HbA and HbF.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxyhemoglobin↗
PubMed1999

MgATP-independent hydrogen evolution catalysed by nitrogenase: an explanation for the missing electron(s) in the MgADP-AlF4 transition-state complex.

When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution.

Adenosine Diphosphate↗
PubMed1975

Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.

Anaerobiosis↗
PubMed1977

The carbon monoxide-binding hemoprotein reducible by hydrogen peroxide in microsomal fractions of pea seeds.

There exist at least two kinds of CO-binding hemoproteins in microsomal fractions of germinating pea (Pisum sativum) seeds. One of them is cytochrome P-450 and the other is also a protoheme protein (judged from its pyridine hemochrome spectrum), which is not hitherto reported. The content of the new hemoprotein is much higher than that of cytochrome P-450 in the early stage of germination. During germination the former decreases and the latter increases. The new hemoprotein is not appreciably reduced by sodium dithionite alone within a few minutes, but, it is easily reduced by dithionite in the presence of methyl viologen and also by hydrogen peroxide when CO is present. The addition of hydrogen peroxide to pea microsomes in the absence of CO causes destruction of the hemoprotein and also decolorization of endogenous carotenoid. Destruction of these components is brought about by organic hydroperoxides independently of the presence of CO. In the presence of hydroxylamine, the addition of hydroperoxides to the microsomes results in the formation of an absorption spectrum similar to the spectra of ferrous-NO complexes of protoheme proteins. When N,N-dimethyl p-phenylenediamine is present, the reaction of pea microsomes with hydroperoxides gives a spectrum similar to that of the ferryl form of myoglobin. The reactions of the hemoprotein with hydroperoxides are inhibited by alpha,alpha'-dipyridyl and aniline, with which pea microsomes form binding spectra. The microsomes form a rather stable difference spectrum with hydroxylamine. However, the hemoprotein is destroyed when hydroxylamine is added to the microsomes in the reduced state.

Carbon Monoxide↗
PubMed1992

Catalytic properties of streptococcal NADH oxidase containing artificial flavins.

The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase.

Amino Acid Sequence↗
PubMed2004

Doxepin protects cultured neurons against oxidative stress-induced injury.

AIM: To investigate protective effects of doxepin (Dox) on cultured neuronal injury induced by oxidative stress. METHODS: Cytotoxicities of glutamate (Glu 0.5 mmol/L, 15 min), sodium dithionite (0.5 mmol/L, 24 h) or hemoglobin (Hb100 mg/L,24 h)and the protective effects of Dox were observed. RESULTS: Exposure of cultured neurons to Glu, sodium dithionite and Hb developed a neurotoxicity expressed in the thiazol blue tetrazolium bromide (MTT) assay, the increase of lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) content and intracellular [Ca2+]i accumulation, as well as the decrease of superoxide dismutase (SOD) activity. DOX 1-100 nmol/L significantly inhibited all above changes. CONCLUSION: Dox protects cultured neurons against oxidative stress-induced injury by suppressing intracellular [Ca2+]i accumulation, decreasing lipid peroxide generation and stimulating antioxidant enzyme.

Animals↗
PubMedDate not supplied

[A comparison of fluorescent methods of determining the concentration of vitamin B2 in blood].

Three methods of riboflavin assay in the human blood were comparatively studied. It has been found that firmly-bound flavin forms present in erythrocytes inhibit their complete transmission into the riboflavin form. Low specificity of the method using riboflavin fluorescence suppression with dithionite has been proved. Dithionite capacity for suppressing fluorescence of riboflavin-like substances significantly rises detectable levels. The use of riboflavin-binding protein permits one to determine only free riboflavin form. It has been established that the lumiflavin method is most reliable and specific for riboflavin assay in erythrocytes.

Dithionite↗
PubMed1991

Stopped-flow and rapid-scan studies of the redox behavior of cytochrome aco from facultative alkalophilic Bacillus.

Cytochrome aco purified from an alkalophilic bacterium grown at pH 10 contains hemes a, b, and c as prosthetic groups, and their redox behavior was examined by using stopped-flow and rapid-scan techniques. Under anaerobic conditions the reduction of both heme a and c moieties with dithionite proceeded exponentially but with different rates, usually the former being reduced about 4 times faster than the latter. The reduction of protoheme was much slower, and a time-difference spectrum for this species was of a high spin type with absorption peaks at 433, 557, and 609 nm. Only the protoheme combined with CO, fulfilling the criteria for cytochrome o. Potentiometric titrations determined a midpoint potential of c heme to be 95 mV at pH 7.0 and 25 degrees C and suggested the presence of two forms of a heme with midpoint potentials of 250 and 323 mV. Cytochrome aco utilizes ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) to reduce oxygen relatively rapidly without added cytochrome c (Qureshi, M. H., Yumoto, I., Fujiwara, T., Fukumori, Y., Yamanaka, T. (1990) J. Biochem. 107, 480-485). During the steady state, however, heme a stayed almost fully reduced in contrast to a partial reduction of heme c. Even after exhaustion of the dissolved oxygen the extent of reduction of heme c was 60-70% that attained by the dithionite reduction. When ascorbate plus TMPD-reduced cytochrome aco was exposed to oxygen the reduced heme c was oxidized rapidly whereas the oxidation of reduced a heme was negligibly slow. The full reduction of heme a during the steady state and its extremely slow oxidation rendered participation of heme a in the oxidase reaction less likely. A novel peak appearing transiently around 567 nm during the reaction was tentatively ascribed to an intermediate form of protoheme, or o heme, which was thus supposed to react directly with molecular oxygen. These results suggest strongly that the main electron transfer pathway would be c----o----oxygen. A possible role of a in regulating the electron flow through the main pathway and its functional relationship to a heme in the aa3-type cytochrome oxidase were discussed.

Ascorbic Acid↗
PubMed1975

Mass screening for sickle cell hemoglobin. Is there an optimal method?

The problem of screening large civilian and military populations for sickle cell hemoglobin and related conditions has been approached recently by three distinct methods. One method specifies hemoglobin electrophoresis as the initial screening technique, followed by a solubility (dithionite) test when a (nonspecific) "S band" is noted. Another method requires the use of the automated dithionite test, followed, when positive, by hemoglobin electrophoresis. A third method is a dual, automated technique for detecting anemia or hemoglobin S or both in the initial screening study; hemoglobin electrophoresis studies are done when indicated. All three methods provide data suitable for accurate diagnosis and genetic counseling, but substantial variations in costs and redundancy of data are found among these methods. Because it gives the "biggest diagnostic bang for the buck", the third merits widespread adoption.

Anemia, Sickle Cell↗
PubMed1989

The streptococcal flavoprotein NADH oxidase. II. Interactions of pyridine nucleotides with reduced and oxidized enzyme forms.

Anaerobic addition of 0.5 eq of NADH/FAD to the streptococcal NADH oxidase produces a redox form spectrally similar to that obtained with 0.5 eq of dithionite/FAD. The second phase of the titration, however, in addition to reducing the flavin with 1 eq of NADH/FAD, leads to the appearance of a long-wavelength absorbance band centered at 725 nm. Reductive titrations of the enzyme with 3-acetylpyridine-adenine dinucleotide, which has a redox potential 72 mV more positive than that of NADH, yield a similar reduced enzyme species. Dithionite reduction of the NADH oxidase followed by titration with NAD+ partially mimics the long-wavelength absorbance of the NADH-reduced enzyme but also leads to the oxidation of 1 FADH2/dimer. NADH is not formed, however, and a similar result is obtained when the dithionite-reduced oxidase is titrated with the nonreducible substrate analog 3-aminopyridine-adenine dinucleotide. These data indicate that the FADH2 oxidation observed is intramolecular and suggest that the active centers of the two apparently identical subunits/dimer are not equivalent. These results also demonstrate that bound pyridine nucleotides can modulate the redox manifold of the NADH oxidase and, when taken together with the effects of these ligands on pre-steady-state behavior, suggest an important regulatory aspect of the catalytic redox function of this unique flavoprotein.

Dithionite↗
PubMed1985

Magnetic circular dichroism studies of succinate dehydrogenase. Evidence for [2Fe-2S], [3Fe-xS], and [4Fe-4S] centers in reconstitutively active enzyme.

Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.

Animals↗
PubMed1987

Properties of bovine heart mitochondrial cytochrome b560.

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.

Animals↗
PubMed1985

Studies on neutrophil b-type cytochrome in situ by low temperature absorption spectroscopy.

The b-type cytochrome in porcine neutrophils in situ was studied by the low temperature absorption spectroscopy at 77 K. Absolute spectra of the dithionite-reduced cell suspension revealed the existence of a b-type cytochrome with alpha, beta, and Soret absorption maxima at 558, 528, and 426 nm, respectively. The alpha band was unsymmetrical and showed a main peak at 558 nm with a shoulder at around 556 nm. When the cells were anaerobically stimulated either by phorbol myristate acetate or arachidonate followed by reduction by dithionite, the alpha band split clearly into double peaks at 555.5 and 558 nm, suggesting the presence of at least two states or species of the b-type cytochrome(s) in the cell. By monitoring absolute spectra of neutrophils at 77 K, we examined the possibility of CO binding to the b-type cytochrome. The absorption spectra of reduced b-type cytochrome in the presence and absence of CO, however, were not distinguishable under various conditions including equilibration with CO under high pressure or CO treatments in a dark room or at pH 8.5, 7.0, or 5.5. In contrast, the spectra of the reduced cytochrome disappeared immediately after exposure to O2, whether or not the cells had been treated with CO. The results indicate that the cytochrome does not form a CO complex in situ but reacts with O2, either directly or indirectly.

Animals↗