Pyocycanin,a Contributory Factor in Haem Acquisition and Virulence Enhancement of Porphyromonas gingivalis in the Lung

Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung.     

Mechanism of methaemoglobin breakdown by the lysine-specific gingipain of the periodontal pathogen Porphyromonas gingivalis

Abstract The R- and K-gingipain proteases of Porphyromonas gingivalis are involved in proteolysis of haemoglobin from which the defensive dimeric haem pigment is formed. Whilst oxyhaemoglobin is refractory towards K-gingipain, methaemoglobin is rapidly degraded. Ligation of methaemoglobin with N3-, which effectively blocks haem dissociation from the protein, prevented haemoglobin breakdown. Haem-free globin was rapidly degraded by K-gingipain. These data emphasise the need for haemoglobin oxidation which encourages haem dissociation and makes the haem-free globin susceptible to proteolytic attack.     

Sequential action of R- and K-specific gingipains of Porphyromonas gingivalis in the generation of the haem-containing pigment from oxyhaemoglobin

The arginine- and lysine-specific gingipains of Porphyromonas gingivalis have been implicated in the degradation of haemoglobin from which the black mu-oxo haem dimer-containing pigment is generated. Here, we examined interactions of oxyhaemoglobin (oxyHb) with the Arg-(R)-specific (HRgpA) and Lys-(K)-specific (Kgp) gingipains. Incubation of oxyHb with HRgpA resulted in formation of methaemoglobin (metHb), which could be prevented by the R-gingipain specific inhibitor leupeptin. oxyHb-Kgp interactions resulted in formation of a haemoglobin haemichrome. This was inhibited by the lysine-specific protease inhibitor Z-Phe-Lys-acyloxymethylketone (Z-FKck). metHb, formed by treatment of oxyHb with either NaNO(2) or by pre-incubation with HRgpA, was rapidly degraded by Kgp compared to oxyHb. metHb degradation by Kgp was also inhibited Z-FKck. Together these data show that R-gingipain activity is crucial for converting oxyHb into the metHb form which is rendered more susceptible to Kgp degradation for the eventual release of iron(III) protoporphyrin IX and production of the mu-oxo haem dimer. This explains previous observations [J.W. Smalley, M.F. Thomas, A.J. Birss, R. Withnall, J. Silver, Biochem. J. 379 (2004) 833-840.] of the requirement for a combination of both R- and K-gingipains for pigment production from oxyhaemoglobin by P. gingivalis.     

Haem disorder in reconstituted human haemoglobin

Degradation of the haem of haemoglobin (used as a chemical probe of the haem–protein relationship), suggests that reconstituted human haemoglobin contains significant haem disorder. This results from the insertion of haem into globin with an orientation 180° different from the natural orientation. Haem disorder also slowly occurs in methaemoglobin solutions.     

Reactions involving superoxide and normal and unstable haemoglobins.

Superoxide ions (O2-) oxidized oxyhaemoglobin to methaemoglobin and reduced methaemoglobin to oxyhaemoglobin. The reactions of superoxide and H2O2 with oxyhaemoglobin or methaemoglobin and their inhibition by superoxide dismutase or catalase were used to detect the formation of superoxide or H2O2 on autoxidation of oxyhaemoglobin. The rate of autoxidation was decreased at about 35% in the presence of both enzymes. The copper-catalysed autoxidation of Hb (haemoglobin) was also shown to involve superoxide production. Superoxide was released on autoxidation of three unstable haemoglobins and isolated alpha and beta chains, at rates faster than with Hb A. Reactions of superoxide with Hb Christchurch and Hb Belfast were identical with those with Hb A, and occurred at the same rate. Hb Koln contrasted with the other haemoglobins in that the thiol groups of residue beta-93 as well as the haem groups reacted with superoxide. Haemichrome formation from methaemoglobin occurred very rapidly with Hb Christchurch and Hb Belfast, as well as the isolated chains, compared with Hb A. The process did not involve superoxide production or utilization. The relative importance of autoxidation and superoxide production compared with haemichrome formation in the haemolytic process associated with these abnormal haemoglobins and thalassaemia is considered.     

The reaction of menadione with haemoglobin. Mechanism and effect of superoxide dismutase.

1. Menadione was found to react with both the haem groups and the beta-93 thiol groups of haemoglobin. 2. It oxidized the haem groups of oxyhaemoglobin, giving mainly methaemoglobin and a smaller amount of haemichrome. The reaction rate was decrease in the presence of catalase and markedly accelerated in the presence of superoxide dismutase. It is proposed that the overall reaction involves the initial reversible formation of methaemoglobin and the semiquinone, and that the effect of superoxide dismutase is to prevent the reverse reaction, by removing superoxide and hene O2-. E.s.r. evidence for the information of the semi-quinone and its reactions is presented. 3. The reaction of menadione with the beta-93 thiol groups of haemoglobin appeared to be similar to that with other thiols, forming the 3-thioether derivative of menadione, but it was also accompanied by reduction of methaemoglobin. This reduction was prevented by superoxide dismutase, but appeared to be caused by the semiquinone radical, which was produced as an intermediate. 4. Reduced glutathione functioned only to a limited extent as a scavenger of the menadione semiquinone. Its main reaction was directly with menadione to form the thioether. Ascorbate was a more efficient scavenger, and accelerated the oxidation of oxyhaemoglobin by menadione. 5. The significance of these findings in relation to menadione-induced erythrocyte haemolysis is discussed.     

The rate of reaction of superoxide radical ion with oxyhaemoglobin and methaemoglobin.

Superoxide radical ions (O2-) produced by the radiolytic reduction of oxygenated formate solutions and by the xanthine oxidase-catalysed oxidation of xanthine were shown to oxidize the haem groups in oxyhaemoglobin and reduce those in methaemoglobin as in reactions (1) and (2): (see articles) Reaction (1) is suppressed by reaction (8) when [O2-]exceeds 10 muM, but consumes all the O2- generated in oxyhaemoglobin solutions when [oxyhaemoglobin] greater than 160 muM and [O2-]less than 1 nM at pH 7. The yield of reaction (2) is also maximal in methaemoglobin solutions under similar conditions, but less than one haem group is reduced per O2- radical. From studies of (a) the yield of reactions (1) and (2) at variable [haemoglobin] and rates of production of O2-, (b) their suppression by superoxide dismutase, and (c) equilibria observed with mixtures of oxyhaemoglobin and methaemoglobin, it is shown that k1/k2=0.7 +/- 0.2 and k1 = (4 +/- 1) X 10(3) M-1-S-1 At pH7, and k1 and k2 decrease with increasing pH. Concentrations and rate constants are expressed in terms of haem-group concentrations. Concentrations of superoxide dismutase observed in normal erythrocytes are sufficient to suppress reactions (1) and (2), and hence prevent the formation of excessive methaemoglobin.     

Emerging strategies in microbial haem capture

Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins. These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins. Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell. Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell. This review describes recent work on the discovery and characterization of these systems. Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded). Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.     

Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue beta-93.

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.     

Mechanism of oxyhaemoglobin breakdown on reaction with acetylphenylhydrazine.

The reaction of oxyhaemoglobin and acetylphenylhydrazine, which results in haemoglobin denaturation and precipitation, was found to be influenced by H202 and superoxide (O2-.) generated during the reaction. By analysing the different haemoglobin oxidation products, it was found that by influencing the rate at which oxyhaemoglobin was oxidized, H2O2 accelerated the overall haemoglobin breakdown, and O2-. inhibited it. By adding GSH (reduced glutathione) or ascorbate, it was possible to slow down the rates of both oxyhaemoglobin oxidation and O2-. production, and the overall rate of haemoglobin breakdown. These results are compatible with a mechanism involving production of the acetylphenylhydrazyl free radical, and with GSH, ascorbate and O2-. acting as radical scavengers and preventing its further reactions. The reaction produced choleglobin, as well as acetylphenyldiazine and methaemoglobin, which combined to form a haemichrome. The haemichrome was less stable and precipitated first. It was also less stable than the haemichrome formed by direct reaction of acetylphenyldiazine with methaemoglobin, and it is proposed that this is because the methaemoglobin produced from oxyhaemoglobin and acetylphenylhydrazine was modified by the free radicals and H2O2 produced in the reaction.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate.

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin).     

Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.     

Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutinin-encoding gene of Porphyromonas gingivalis

The obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black-pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N-terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine-specific cysteine proteinase (Arg-gingipain, RGP)-encoding gene ( rgp1 ) and was also present in genes for lysine-specific cysteine proteinases ( prtP and kgp ) and a haemagglutinin ( hagA ) of P. gingivalis . The HGP15 domain protein was purified from an HGP15-overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa haemoglobin-binding protein in the envelope of P. gingivalis. P. gingivalis wild-type strain showed pH-dependent haemoglobin adsorption, whereas its non-pigmented mutants that produced no HGP15-related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis .     

Haemophore-mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules

Numerous bacteria are able to use free and haemoprotein-bound haem as iron sources because of the action of small secreted proteins called haemophores. Haemophores have very high affinity for haem, and can therefore extract haem from the haem-carrier proteins and deliver it to the cells by means of specific cell surface receptors. Haem is then taken up and the empty haemophores are recycled. Here, we report a study of the regulation of the Serratia marcescens has operon which is involved in haemophore-dependent haem acquisition. We characterized two genes encoding proteins homologous to specific ECF sigma and antisigma factors. We showed that they regulate the synthesis of the haemophore-specific outer membrane receptor, HasR, by a signal transduction mechanism similar to the siderophore surface-signalling systems. We also showed the essential role of HasR itself in this process. Using haem-loaded and haem-free haemophore, we identified the stimulus for the HasR-mediated signal transduction as being the binding of the haem-loaded haemophore to HasR. Thus, unlike siderophore-uptake systems, in which the signalling molecule is the transported substrate itself, in the haemophore-dependent haem uptake system the inducer and the transported substrate are different compounds.     

Porphyromonas gingivalis influences actin degradation within epithelial cells during invasion and apoptosis

Porphyromonas gingivalis, a Gram-negative oral pathogen, has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We have previously shown that the P. gingivalis proteases arginine and lysine gingipains, are necessary and sufficient to induce host cell apoptosis. In the present study, we demonstrate that 'P. gingivalis-induced apoptosis' is mediated through degradation of actin leading to cytoskeleton collapse. Stimulation of human gingival epithelial cells with P. gingivalis strains 33277 and W50 at moi:100 induced β-actin cleavage as early as 1 h and human serum inhibited this effect. By using gingipain-deficient mutants of P. gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time-dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P. gingivalis internalization is necessary for actin cleavage. Further, we also show that lysine gingipain from P. gingivalis can cleave active caspase 3. Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in microbial host cell invasion and apoptosis.     

Structure and functional properties of oxyhaemoglobin regenerated from methaemoglobin obtained chemically or by freeze-drying

Freeze-drying a solution of oxyhaemoglobin leads to the formation of a large amount of methaemoglobin. As this methaemoglobin is more difficult to reeduce with sodium dithionite than is mtehaemoglobin prepared chemically by the action of potassium ferricyanide on oxyhaemoglobin, the two are obviously different. Freeze-drying makes molecules of haemoglobin fragile to varying degrees. Optical circular dichroism measurements suggest that this change involves a disturbance in the structure of the globin in the vicinity of the oxidized haems; the fragility depends on the number of subunits oxidized. These disturbances are not irreversible and partially disappear after several successive treatments with sodium dithionite. The regenerated haemoglobin has characteristics resembling those of untreated control haemoglobin, but still retains a strong tendency to reoxidized. In contrast, methaemoglobin prepared chemically is completely reduced by a single treatment with sodium dithionite; regenerated haemoglobin is not tendered more fragile, and all its functional properties can be restored.     

Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin.

Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin.