A supramolecular bifunctional artificial enzyme with superoxide dismutase and glutathione peroxidase activities

For constructing a bifunctional antioxidative enzyme with both superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, a supramolecular artificial enzyme was successfully constructed by the self-assembly of the Mn(III)meso-tetra[1-(1-adamantyl methyl ketone)-4-pyridyl] porphyrin (MnTPyP-M-Ad) and cyclodextrin-based telluronic acid (2-CD-TeO₃H) through host-guest interaction in aqueous solution. The self-assembly of the adamantyl moieties of Mn(III) porphyrin and the β-CD cavities of 2-CD-TeO₃H was demonstrated by the NMR spectra. In this supramolecular enzyme model, the Mn(III) porphyrin center acted as an efficient active site of SOD and tellurol moiety endowed GPx activity. The SOD-like activity (IC₅₀) of the new catalyst was found to be 0.116 μM and equals to 2.56% of the activity of the native SOD. Besides this, supramolecular enzyme model also showed a high GPx activity, and a remarkable rate enhancement of 27-fold compared to the well-known GPx mimic ebselen was observed. More importantly, the supramolecular artificial enzyme showed good thermal stability.     

Mn(III)-containing acid phosphatase: Properties of Fe(III)-substituted enzyme and function of Mn(III) and Fe(III) in plant and mammalian acid phosphatases

The function of Mn(III) in plant acid phosphatase has been investigated by a metal-substitution study, and some properties of the Fe(III)-substituted enzyme were compared with those of the native Mn(III) enzyme and mammalian Fe(III)-containing acid phosphatases. ¹⁹F nuclear magnetic resonance (NMR) and proton relaxation rate measurements showed that inhibitors such as F⁻ and nitrilotriacetic acid interact with paramagnetic Mn(III) active site. The ³¹P-NMR signal of the enzyme-phosphate complex was also broadened by the paramagnetic effect of Mn(III). In the metal-substitution experiments of the Mn(III)-acid phosphatase with Fe(III), Zn(II) and Cu(II), only the iron gave satisfactory substitution. The Fe(III)-substituted plant acid phosphatase exhibited an absorption maximum at 525 nm (ε = 3000), typical high spin ferric ESR signal at g = 4.39, and lower pH optimum (pH 4.8) than the native Mn(III)-enzyme (pH 5.8). The phosphatase activity of the Fe(III)-substituted enzyme was reduced to about 53% of that of the native enzyme. The substrate specificities of both metallophosphatases were remarkably similar, but different from that of the Fe(III)-containing uteroferrin. The present results indicate that Mn(III) and Fe(IIII) in the acid phosphatase play an important role on effective binding of phosphate and acceleration of hydrolysis of phosphomonoesters at pH 4–6.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

pH Dependence of the Efficiency of Binding of Iron Cations to the Donor Side of Photosystem II

Light-induced interaction of Fe(II) cations with the donor side of Mn-depleted photosystem II (PS II(–Mn)) results in the binding of iron cations and blocking of the high-affinity (HA_Z) Mn-binding site. The pH dependence of the blocking was measured using the diphenylcarbazide/2,6-dichlorophenolindophenol test. The curve of the pH dependence is bell-shaped with pK ₁ = 5.8 and pK ₂ = 8.0. The pH dependence of the O₂-evolution mediated by PS II membranes is also bellshaped (pK ₂ = 7.6). The pH dependence of the process of electron donation from exogenous donors in PS II(–Mn) was studied to determine the location of the alkaline pH sensitive site of the electron transport chain. The data of the study showed that the decrease in the iron cation binding efficiency at pH > 7.0 during blocking was determined by the donor side of the PS II(–Mn). Mössbauer spectroscopy revealed that incubation of PS II(–Mn) membranes in a buffer solution containing ⁵⁷Fe(II) + ⁵⁷Fe(III) was accompanied by binding only Fe(III) cations. The pH dependence of the nonspecific Fe(III) cation binding is also described by the same bell-shaped curve with pK ₂ = 8.1. The treatment of the PS II(–Mn) membranes with the histidine modifier diethylpyrocarbonate resulted in an increase in the iron binding strength at alkaline pH. It is suggested that blocking efficiency at alkaline pH is determined by competition between OH^– and histidine ligand for Fe(III). Because the high-affinity Mn-binding site contains no histidine residue, this fact can be regarded as evidence that histidine is located at another (other than high-affinity) Fe(III) binding site. In other words, this means that the blockage of the high-affinity Mn-binding site is determined by at least two iron cations. We assume that inactivation of oxygen-evolving complex and inhibition of photoactivation in the alkaline pH region are also determined by competition between OH^– and a histidine residue involved in coordination of manganese cation outside the high-affinity site.     

锰超氧化物歧化酶的催化原理与酶活性调节机制

锰超氧化物歧化酶(MnSOD)催化两分子超氧自由基歧化为分子氧和过氧化氢。超氧自由基被Mn³⁺SOD氧化成分子氧的反应以扩散的方式进行。超氧自由基被Mn²⁺SOD还原为过氧化氢的反应以快循环和慢循环两条途径平行进行。在慢循环途径中,Mn²⁺SOD与超氧自由基形成产物抑制复合物,然后该复合物被质子化而缓慢释放出过氧化氢。在快循环途径中,超氧自由基直接被Mn²⁺SOD转化为产物过氧化氢,快速循环有利于酶的复活与周转。本文提出温度是调节锰超氧化物歧化酶进入慢速或者快速循环催化途径的关键因素。随着在生理温度范围内的温度升高,慢速循环成为整个催化反应的主流,因而生理范围内的温度升高反而抑制该酶的活性。锰超氧化物歧化酶的双相酶促动力学特性可以用该酶保守活性中心的温度依赖性配位模型进行合理化解释。当温度降低时,1个水分子(或者OH^-)接近Mn、甚至与Mn形成配位键,从而干扰超氧自由基与Mn形成配位键而避免形成产物抑制。因此在低温下该酶促反应主要在快循环通路中进行。最后阐述了几种化学修饰模式对...     

Investigation of zinc-deprived bovine superoxide dismutase

Zinc-deprived bovine superoxide dismutase and its adducts with azide and thiocyanate ions have been investigated through water ¹H NMR relaxation measurements. The affinity constants of the anions for the modified protein have been determined and compared with those for the native enzyme. The results suggest that a histidine different from the bridging one is displaced upon anion coordination.     

Anodically generated manganese(III) acetate for the oxidation of α — amino acids in aqueous acetic acid: A kinetic study

Summary Manganese(III) acetate was prepared by the oxidation of manganese(II) acetate in aqueous acetic acid by electrolytic method. The anodically generated Mn(III) species was characterised by spectroscopic and redox potential studies. Conditions for the study of kinetics of Oxidation of-amino acids by Mn(III) in aqueous acetic acid was investigated. Plots of log[Mn(III)] versus time for the first order process, or Mn(III) versus time for zero order process were nonlinear. The rate constants computed from [Mn(III)]^1/2 versus time plots were independent of [Mn(III)]₀ indicating that the reaction goes through competitive zero and first order path-ways in [Mn(III)]₀. The kinetic order in [amino acid], [H⁺] and [Mn(II)] were found out. Effect of added anions like CH₃COO^–, F^–, Cl^– and ClO ₄ ^- were investigated. Evidence for the transient existence of the free radical reaction intermediate is given. Dependence of reaction rate on temperature is explained and activation parameters computed from Arrhenius and Eyring plots. A mechanism consistent with the observed results is proposed and discussed.     

Notice

¹⁷O NMR studies of ¹⁷O enriched water solutions containing superoxide dismutase have been performed between pH 7.5 and 11.7. Whereas T₁ measurements do not reveal any interaction between ¹⁷O and the paramagnetic copper center, the linewidth results appreciably increased with increasing pH with an apparent pK_a of 11.3. Comparison with ¹H NMR relaxation studies allows to interpret the present data as due to binding to the copper ion of an OH^? anion at high pH. The binding position should be of “equatorial” type, not involving the binding position of the coordinated water.     

Vanadate stimulates oxidation of NADH to generate hydrogen peroxide

Vanadate in the polymeric form of decavanadate, but not other forms, stimulated oxidation of NADH to NAD⁺ NADPH was also oxidized with comparable rates. This oxidation of NADH was accompanied by uptake of oxygen and generated hydrogen peroxide with the following stoichiometry: NADH + H⁺ + O₂ → NAD⁺ + H₂O₂. The reaction followed second-order kinetics. The rate was dependent on the concentration of both NADH and vanadate and increased with decreasing pH. The reaction had an obligatory requirement for phosphate ions. Esr studies in the presence of the spin trap dimethyl pyrroline N oxide indicated the involvement of Superoxide anion as an intermediate. The reaction was sensitive to Superoxide dismutase and other scavengers of superoxide anions.     

Pulse radiolytically generated superoxide and Cu(II)-salicylates.

The rate constants of the reactions between pulse radiolytically produced superoxide anions and the Cu(II) chelates of salicylate, acetylsalicylate, p-aminosalicylate and diisopropylsalicylate were determined at pH 7.5 and found to range from 0.8 to 2.4 × 10⁹ M^?1 sec^?1. It was intriguing to note that they had a superoxide dismutase activity identical with that of native cuprein-copper (k₂₄₅ = 1.3 × 10⁹ M^?1 sec^?1 per g-atom of Cu). These measurements confirm our earlier observations using indirect assays that all copper salicylates act as perfect model superoxide dismutases and favour the proposal that the activity of anti-inflammatory agents might be assigned to their in vivo formed Cu complexes.     

Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum

A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25°C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 × 10⁹ molar⁻¹·second⁻¹ at pH 7.8 and 25°C. The enzyme was not sensitive to NaCN or to H₂O₂, but was inhibited by N₃⁻. The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented.     

Pro-oxidant and antioxidant processes in gas gland and other tissues of cod (Gadus morhua)

Partial reduction of molecular oxygen produces reactive oxyradicals, including the superoxide anion radical (O _- ² ) and hydroxyl radical (·OH). The gas gland functions under hyperoxic and acidic conditions and therefore is likely to be subjected to enhanced oxidative stress. Aspects of pro- and antioxidant processes in gas gland were compared with other tissues likely to be subject to differing degrees of oxyradical production, viz. liver (site of chemically-mediated oxyradical production), gills and skeletal muscle. Antioxidant enzyme activities (superoxide dismutase, catalase, selenium-dependent and total glutathione peroxidase) per g wet weight were highest in liver and lowest in muscle. Catalase and glutathione peroxidase activies per g wet weight were higher in gills than in gas gland, whereas the reverse was seen for superoxide dismutase. Cytosolic superoxide dismutase activities per mg protein were two- and nine-fold higher in gas gland than in liver and gills. The pH characteristics of the antioxidant enzymes were generally similar in all the tissues. Glutathione, vitamin E and unsaturated (peroxidizable) lipid levels were generally highest in liver followed by gas gland. Lipid peroxidation (malonaldehyde equivalents) was evident in all tissues except gas gland. Hydrogen peroxide and O _- ² were involved in the NAD(P)H-dependent ferric/EDTA-mediated formation of ·OH (as measured by 2-keto-4-methiolbutyrate oxidation) by mitochondrial and postmitochondrial fractions of gas gland. Tissue maximal potentials for ·OH production paralled superoxide dismutase but not catalase or glutathione peroxidase activities. Overall, the results confirm the presence of effective antioxidant defences in gas gland and support previous workers' contentions of a central role for superoxide dismutase in this process.Abbreviations EDTA di-sodium ethylenediaminetetra-acetic acid - G-6-P glucose-6-phosphate - GPX total glutathione peroxidase - GSH reduced glutathione - GSSG oxidised glutathione - GST glutathion-S-transferase - HPLC high performance liquid chromatography - KMBA 2-keto-4-methiolbutyric acid - MOPS 3-[N-morpholino] propane-sulphonic acid - PMS postmitochondrial supernatant - Se-GPX selenium-dependent glutathion peroxidase - SOD superoxide dismutase - TCA trichloroacetic acid     

Competitive inhibition of Cu, Zn superoxide dismutase by monovalent anions.

Polarographic measurements showed that N₃^? and halides $\text{in}$ hibit the activity of bovine Cu, Zn superoxide dismutase in a competitive fashion, as previously demonstrated for CN^? and OH^?. All anions increase the spin-lattice nuclear magnetic relaxation time (T₁) of aqueous solutions of the enzyme as well, but the stability constants measured from T₁ data are lower than those calculated from activity data. The results suggest that substrate and anionic inhibitors bind during the catalytic action at the water coordination position of the enzyme copper, and that these inhibitors may have a greater affinity for the cuprous form of the enzyme which is generated in the catalytic cycle.     

Influence of salts and pH on the growth as well as NADH oxidase of the halotolerant bacterium A505

Growth yield of the halotolerant bacterium A505 was increased by the supplement of Na⁺, K⁺, or Rb⁺ into the culture media with pH 7.5, and inhibited by Li⁺ or Cs⁺. In the presence of less than 0.1 M NaCl or KCl alkaline growth media, pH 9.2 to 9.7, afforded optimal growth of this strain. Intracellular ion content of this microbe changed reflecting on the Na⁺ or K⁺ concentration in the media, although it tended to accumulate K⁺ and extrude Na⁺ in the media without NaCl supplemented. A 1.2 to 1.4-fold stimulation of in vitro NADH oxidase activity was obtained by supplement of salts, except for LiCl. The rate of NADH oxidation in the absence of salts correlated with the pH and showed clear maxima at pH about 8, irrespective of growth conditions. In the presence of 0.5 M NaCl or KCl, on the other hand, pH dependence was less significant and showed only a flat maximum at pH around 7. Effects of anions on NADH oxidase were realized following the lyotropic series: SO ₄ ^2- >F^->CH₃COO^->Cl^->I^->SCN^-, aside from NO ₃ ^- , which exhibited the largest stimulation on enzyme activity in all the anions examined.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - HQNO 2-heptyl-4-hydroxyquinoline-N-oxide - MES 4-morpholineethanesulfonic acid - Tris tris(hydroxy-methyl)methylamine     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a 15N nuclear magnetic resonance study

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 A, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 A. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion.     

Purification and some properties of superoxide dismutase from<Emphasis Type="Italic"> Deinococcus radiophilus</Emphasis>, the UV-resistant bacterium

The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg^–1 protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H₂O₂. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5±1 and 47.1±5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26±0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0–11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40°C, but a linear reduction in activity above 50°C. Inhibition of the purified SOD activity by -naphthoquinone-4-sulfonic acid, -diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(>D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.Communicated by G. Antranikian     

Anion conductance of frog muscle membranes: one channel, two kinds of pH dependence

Anion conductance and permeability sequences were obtained for frog skeletal muscle membranes from the changes in characteristic resistance and transmembrane potential after the replacement of one anion by another in the bathing solution. Permeability and conductance sequences are the same. The conductance sequence at pH = 7.4 is Cl^- Br^- > NO₃ ^- > I^- > trichloroacetate ≥ benzoate > valerate > butyrate > proprionate > formate > acetate ≥ lactate > benzenesulfonate ≥ isethionate > methylsulfonate > glutamate ≥ cysteate. The anions are divided into two classes: (a) Chloride-like anions (Cl^- through trichloroacetate) have membrane conductances that decrease as pH decreases. The last six members of the complete sequence are also chloride like. (b) Benzoate-like anions (benzoate through acetate) have conductances that increase as pH decreases. At pH = 6.7 zinc ions block Cl^- and benzoate conductances with inhibitory dissociation constants of 0.12 and 0.16 mM, respectively. Chloride-like and benzoate-like anions probably use the same channels. The minimum size of the channel aperture is estimated as 5.5 x 6.5 Å from the dimensions of the largest permeating anions. A simple model of the channel qualitatively explains chloride-like and benzoate-like conductance sequences and their dependence on pH.     

Investigation of cobalt(II) substituted carboxypeptidase a interacting with azide and cyanate ions

Cobalt(II)-substituted carboxypeptidase A has been found to reversibly bind N3^? and NCO^?, but not NCS^?, in the pH range 5–10, thus including the pH range of activity of the enzyme. The pH dependence of the anion binding constant is affected by two ionizations, which are assigned as those regulating k_cat and K_M. The electronic and ₁H NMR spectra are consistent with a substantially pseudotetrahedral geometry of the anion derivatives.     

Heat-stress stimulation of oxygen uptake by Photosystem I involves the reduction of superoxide radicals by specific electron donors

A Photosystem I submembrane fraction isolated from spinach was used to study the mechanism of heat-stress stimulation of oxygen uptake by the photosystem. Various artificial electron donors were shown to generate electron transport reactions with various degrees of thermally induced stimulation. A strong stimulation was observed with durohydroquinone as electron donor with a maximal effect at 50 °C. The degree of stimulation obtained was independent from the redox potential of the electron donors and from their oxidation site because the enzyme superoxide dismutase fully inhibited the stimulation. Instead, it is proposed that thermal stress causes the release of membrane bound superoxide dismutase from the thylakoids thus allowing the reduced form of electron donors with specific properties to reduce O₂ ^– radicals to H₂O₂ besides the usual disproportionation of O₂ ^– into O₂ and H₂O₂.Abbreviations: PS photosystem - DCIP 2,6-dichlorophenolindophenol - MV methylviologen - TMPD N,N,N,N-tetramethylphenylenediamine - SOD superoxide dismutase - Chl chlorophyll - DQ duroquinone - DAD N,N,N,N-tetramethyl-1,4-benzenediamine - PMS 5-methylphenazium methyl sulfate - PC plastocyanin