Superoxide anion as a cofactor of dopamine-beta-hydrxylase.

Superoxide anion can serve a reducing agent for tyramine hydroxylation by dopamine-β-hydroxylase. Stable $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ solutions were obtained by dissolving KO₂ in dry dimethylsulfoxide and infused into buffered solutions of tyramine and dopamine-β-hydroxylase at constant rate. The reaction requires molecular oxygen, but differs from the ascorbate dependent hydroxylation in its alkaline pH optimum value (pH 7.5) and its low rate (9 nmol octopamine formed/min/mg of protein). In absence of tyramine $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ does not produce a stable reduced form of the enzyme.     

Mechanism of superoxide anion scavenging reaction by bis-(salicylato)-copper (II) complex.

The mechanism of the reaction of bis-(salicylato)-copper(II) with superoxide anion has been studied by utilizing electron paramagnetic resonance and polarographic techniques. The proposed reaction sequence is as follows:

$\text{Cu}\text{(II) +}\text{O}{}_2{}^{\mathrm{?}}\text{←}\text{Cu}\text{(II)}\text{O}{}_2{}^{\mathrm{?}}\text{←}\text{Cu}\text{(I)}\text{O}{}_2{}^{\mathrm{?}}\text{←}\text{Cu}\text{(I) +}\text{O}{}_2$

Using the xanthine-xanthine oxidase system as a superoxide generator, it was found that the concentration of this copper complex for 50% inhibition of the xanthine-cytochrome c reductase activity was about 1000 times more per mole of copper than that of erythrocyte superoxide dismutase.     

Involvement of superoxide in the prolyl and lysyl hydroxylase reactions

Four superoxide dismutase active copper chelates, Cu(acetylsalicylate)₂, Cu(salicylate)₂, Cu(lysine)₂ and Cu(tyrosine)₂, proved to be inhibitors of prolyl and lysyl hydroxylase. The kinetics of the inhibition are consistent with the proposal that these compounds dismutated ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{staggered?}}$ at the active site of the enzymes. The data strongly suggest that ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{staggered?}}$ is the active form of O₂ in the prolyl and lysyl hydroxylase reactions.     

Reactivity of chemically generated superoxide radical anion with peroxides as determined by competition kinetics

A steady-state competition system has been developed to investigate the reactions of the superoxide radical anion ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$) with various peroxides, including the so-called Haber-Weiss reaction. Potassium superoxide dissolved in an oxygen-free solution of DMSO containing 18-dicyclohexyl-6-crown, is the source of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. High pressure liquid chromatography is used as an assay system for $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ reactivity, to detect and quantitate the yield of anthracene, formed as a major product in the reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and 9,10-dihydroanthrancene. Decrease in anthracene yields, in the presence of peroxide, may be used to indicate a possible competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and added peroxide. Complications involving peroxide-stimulated formation of anthraquinone derivatives are discussed. No evidence for a competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and peroxide can be detected up to a 10-fold excess of peroxide over 9,10-dihydroanthracene.     

The one-electron transfer redox potentials of free radicals. I. The oxygen/superoxide system

The method of determination of Redox potentials of radicals, using the pulse radiolysis technique, is outlined. The method is based on the determination of equilibrium constants of electron transfer reactions between the radicals and appropriate acceptors. The limitations of this technique are discussed.The redox potentials of several quinones-semiquinones are calculated, as well as the standard redox potential of the peroxy radical. and the redox oxidation properties of the peroxy radical in various systems and pH are discussed. The value determined for the redox potentials of $\text{O}{}_2\text{O}{}_2{}^{\mathrm{?}}$ is higher by more than 0.2 V than earlier estimates, which has important implications on the possible role of O₂^? in biological processes of O₂ fixation.     

Catalytic properties of the resolved flavoprotein and cytochrome B components of the NADPH dependent O2−• generating oxidase from human neutrophils

The resolved flavoprotein and cytochrome b₅₅₉ components of the NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b₅₅₉, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b₅₅₉, depleted of flavoprotein, demonstrated no measureable NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b₅₅₉ was rapidly oxidized by oxygen, as was the cytochrome b₅₅₉ in the intact oxidase.     

Superoxide production by phagocytes. Another look at the effect of cytochrome c on oxygen uptake by stimulated neutrophils.

The widely held view that stimulated phagocytes liberate $\text{O}{}_2{}^{\mathrm{?}}$ into the extracellular medium is supported by the alterations in oxygen uptake which occur when ferricytochrome $\text{c}$ is added to a suspension of zymosan-treated neutrophils. An explanation consistent with this view is provided for some previously reported results $\text{(}\text{FEBS Lett}\text{. 100}$, 27) which initially appeared to conflict with the notion that $\text{O}{}_2{}^{\mathrm{?}}$ is released by phagocytes.     

A proposed mechanism for light emission by bacterial luciferase involving dissociative electron transfer

A new mechanism that involves dissociative electron transfer in the energy transducing step is set forward for bacterial luciferase catalyzed light emission. The proposal involves (1) dissociation of the 4a-hydroperoxyflavin to a flavin radical and ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}$, accounting for 570 and 620nm absorption, (2) ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}$ addition to the aldehyde carbonyl to form a peroxyl radical, (3) abstraction of H from an enzyme thiol group to form RCH(OOH)OH, (4) thiyl radical abstraction of the H on C in RCH(OOH)OH, a step which can show a $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$ of ca. 4, and (5) dissociative electron- transfer, a highly exothermic step that leads to a protonated flavin excited state, a carboxylic acid and water.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase A requiem for H2O4−

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O₂^? during the first few minutes of the reaction. H₂O₂ decreases this accumulation of O₂^? presumably because of the Haber-Weiss reaction ($\text{H}{}_2\text{O}{}_2\text{+}\text{O}{}_2{}^{\mathrm{?}}\text{→}\text{OH}{}^{\mathrm{?}}\text{+}\text{OH}\text{+}\text{O}{}_2$) and very small amounts of superoxide dismutase eliminate it. This accumulation of O₂^? was demonstrated in terms of a burst of reduction of cytochrome $\text{c}$, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H₂O₄^?, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

A reaction between the superoxide free radical and lipid hydroperoxide in sodium linoleate micelles

The oxidation of unsaturated fatty acid micelles by the superoxide free radical ($\text{O}{}^{\mathrm{?}}{}_2$), during γ irradiation in the presence of formate, is kinetically distinct from oxidation by hydroxyl free radicals (HO.). The evidence suggests that a direct reaction between ($\text{O}{}^{\mathrm{?}}{}_2$) and lipid hydroperoxide initiates a chain oxidation process in the micelles. While tetranitromethane, which reacts rapidly with ($\text{O}{}^{\mathrm{?}}{}_2$), protects the micelles from oxidation, active superoxide dismutase is no more effective than its apoprotein, due to lack of penetration of the micellar environment. We discuss these findings in the light of recent literature, and with reference to their possible significance for biological systems.     

Bacterial bioluminescence-identification of fatty acid as product, its quantum yield and a suggested mechanism

By using radioactive decanal the direct transformation of this aldehyde to decanoic acid, with a quantum yield of 0.13, has been demonstrated. A mechanism analogous to that of other better understood bioluminescent reactions is proposed, leading to a product, as yet unisolated from the enzymic reaction, whose fluorescence spectrum is an excellent match for that of the $\text{in vivo}$ luminescence.The extensive examination^1,2,3 of the isolated bacterial luminescence system has resulted in the accepted outline shown. We wish to modify it, in accordance with the previous evidence, by suggesting that ’intermediates I and II‘ in Hastings' terminology² are the same enzyme bound FMNH₂ moiety.

$\text{FMN}{}_2\text{enzyme}\text{?}\text{enzyme FMNH}{}_2$

$\text{enzyme FMNH}{}_2\text{RCHO}\text{?}\text{covalent complex}$

$\text{covalent complex O}{}_2\text{→}\text{P}{}^1\text{RCO}{}_2\text{H}$

A lively controversy has surrounded the attempts to determine whether aldehyde exerts a purely catalytic role² or is transformed in the reaction.⁴ If the aldehyde reacts, then the simplest product is the corresponding carboxylic acid, perhaps formed via the peracid. The most likely alternative reaction would involve enolistation and oxidation at the α-methylene group. We examined the second alternative fairly carefully, and found no evidence for it. We do not wish to report these results in detail at present, since we have now established that the acid corresponding to that formed in a normal autoxidation of the aldehyde is the product. Some indication of the nature of the products of the reaction is available.⁵Since the amount of product in the reaction is restricted to a very low level by the concentrations required, we labelled decanal with tritium at C-2 and thus were able to record the yield with some precision. Although recent work⁶ strongly implies that acid is formed stoichiometrically, the direct measurement of the quantum yield with respect to acid formation is necessary before a mechanism can be written. We have suggested a mechanism compatible with observations in this system, analogous to all cases of bioluminescence for which a mechanism is reasonably well established. This mechanism also leads to a product excited state with excellent agreement around pH7 in fluorescence wavelength to that of the $\text{in vivo}$ luminescence.     

The interaction of ions with phosphatidylcholine bilayers

The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in ²H₂O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions.Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes.The apparent binding constant $\text{K}{}_{\mathrm{<mtext>b</mtext>}}$ varies as a function of the quantity of metal ion bound and as a function of the Cl^? concentration. The apparent binding constant at “zero loading” is $\text{K}{}_0\text{= 1.25 · 10}{}^4\text{L}{}^2\text{·}\text{M}{}^{\mathrm{?}}\text{at}\text{0.15}\text{M KCl}$. It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl^? concentration.The interaction of lanthanides and divalent cations such as Ca²⁺ and Mg²⁺ is independent of pH in the pH range 3–7⁺ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is $\text{Cl}{}^{\mathrm{?}}\text{< Br}{}^{\mathrm{?}}\text{< NO}{}_3{}^{\mathrm{?}}\text{< SCN}{}^{\mathrm{?}}\text{< I}{}^{\mathrm{?}}\text{< ClO}{}_4{}^{\mathrm{?}}$. The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions.The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca²⁺ and Mg²⁺ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO₂²⁺ showing the highest affinity.     

Significance of O2 availability and cycling on the respiratory burst response of human PMN's exposed to cytochrome c and superoxide dismutase

In many instances the effect of superoxide ($\text{O}{}_2{}^{\mathrm{?}}$) trapping agents in suppressing the net rate of O₂ consumption of activated PMN's is not in accordance with theoretical expectations. We offer here an alternate explanation to those previously presented by Segal and Meshulam (FEBS Letters 100, 27–32) and Babior (Biochem. Biophys. Res. Comm. 91, 222–226). The paradoxical results previously presented can be explained by recognizing that shortly after activation of resting cells an O₂ diffusion layer is established at or near the outer surface of these cells. The presence of this diffusion layer can markedly alter the anticipated stochiometric relationship between $\text{O}{}_2{}^{\mathrm{?}}$ trapped and apparent O₂ consumed by these cells when they are exposed to $\text{O}{}_2{}^{\mathrm{?}}$ trapping agents.     

The relative effectiveness of .OH,H2O2,O2−, and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (^.OH, H₂O₂), ambivalent (O₂^?) and reducing free radicals (e^? and CO₂^?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ($\text{R}$(enz)) were measured and the rates of damage to membranes ($\text{R}$(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. ^.OH, O₂^? and H₂ O₂ were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e^? and CO₂^? were the least effective. $\text{R}$(enz) values of O₂^? and H₂O₂ were 10-times and of ^.OH 15-times that of e^?. $\text{R}$(mb) values were quite similar for e^? and H₂O₂ (about twice that of O₂^?), while that of ^.OH was 3-times that of O₂^?. Hence, with respect to $\text{R}$(mb): ${}^{\mathrm{.}}\text{OH}\text{>}\text{e}{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{O}{}_2{}^{\mathrm{?}}$ , and with respect to $\text{R}$(enz): ${}^{\mathrm{.}}\text{OH}\text{>}\text{O}{}_2{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{e}{}^{\mathrm{?}}$. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H₂O₂ added as a chemical reagent and H₂O₂ formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H₂O₂ but markedly susceptible to the latter.     

Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

Complete stoichiometry of free NADPH oxidation in liver microsomes

The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H⁺ and O₂ consumption and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and H₂O₂ production. $\text{ΔO}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{:ΔH}{}_2\text{O}{}_2$ ratio is approximately I indicating that about half H₂O₂ results from $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ dismutation, the second half being formed directly. ΔNADPH:ΔH₂O₂ and ΔO₂:ΔH₂O₂ ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH₂O₂):(ΔO₂-ΔH₂O₂) is 2 allows one to consider direct 4-electron O₂ reduction as the major way of water formation rather than endogenous substrate hydroxylation.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Mercaptide-anion as a trans-ligand in oxycytochrome P450

Oxygenation of heme-mercaptide as well as spectroscopic characteristics of the dioxygen complex formed have been studied. Absorption and magnetic circular dichroism spectra of the O₂ complex support the retention of mercaptide in the heme fifth position. A release of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ in the decomposition of the oxygenated complex and an independent formation of the latter from hemine-dimercaptide and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ together with electron paramagnetic resonance and Mössbauer data support the O₂ presence in the heme coordination sphere. The similarity of optical and magnetic circular dichroism spectra and the closeness of the ratio for oxy-heme-mercaptide and oxycytochrome P450 unequivocally confirm the presence of an axial cystein mercaptide ligand in oxycytochrome P450.     

Interaction of human fibrinogen with divalent metal chlorides

Precipitation of human fibrinogen in 0.15 m NaCl occurred at pH 7.4 (Tris-HCl buffer) when ZnCl₂, CuCl₂, NiCl₂, or CoCl₂ were added beyond their respective critical concentrations. The critical concentrations were about $\text{4 × 10}{}^{\mathrm{?5}}\text{m}\text{ZnCl}{}_2\text{, 6 × 10}{}^{\mathrm{?5}}\text{m}\text{CuCl}{}_2\text{, 3 × 10}{}^{\mathrm{?4}}\text{m}\text{NiCl}{}_2\text{and}\text{1 × 10}{}^{\mathrm{?3}}\text{m}\text{CoCl}{}_2$. At pH 5.8 2-(N-morpholino)-ethane sulphonic acid buffer, the critical concentrations were found only for CuCl₂ and ZnCl₂, and were about $\text{3 × 10}{}^{\mathrm{?5}}\text{and}\text{3 × 10}{}^{\mathrm{?4}}\text{m}$, respectively. CaCl₂ and MgCl₂ were not effective up to $\text{1 × 10}{}^{\mathrm{?2}}\text{and}\text{2 × 10}{}^{\mathrm{?2}}\text{m}$ at pH 7.4 and 5.8, respectively. At pH 7.4, precipitation was better in 0.015 m NaCl than in 0.15 m NaCl for both CuCl₂ and ZnCl₂. Little or no conformational change was indicated on binding Cu²⁺ ions. The fluorescence of tryptophan was quenched only by CuCl₂, while other metal ions (ZnCl₂, NiCl₂, CoCl₂ and CaCl₂) were ineffective as quenchers.