Stability of a Novel PEGylation Site on a Putative Haemoglobin-Based Oxygen Carrier.

PEGylation of protein sulfhydryl residues is a common method used to create a stable drug conjugate to enhance vascular retention times. We recently created a putative haemoglobin-based oxygen carrier using maleimide-PEG to selectively modify a single engineered cysteine residue in the α subunit (αAla19Cys). However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions, with consequent cross-conjugation to endogenous plasma thiols such as those found on human serum albumin or glutathione. In previous studies mono-sulfone-PEG adducts have been shown to be less susceptible to deconjugation. We therefore compared the stability of our maleimide-PEG Hb adduct with one created using a mono-sulfone PEG. The corresponding mono-sulfone-PEG adduct was significantly more stable when incubated at 37 °C for 7 days in the presence of 1 mM reduced glutathione, 20 mg/mL human serum albumin, or human serum. In all cases haemoglobin treated with mono-sulfone-PEG retained >90% of its conjugation whereas maleimide-PEG showed significant deconjugation, especially in the presence of 1 mM reduced glutathione where <70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation of Hb seems adequate for an oxygen therapeutic intended for acute use, if longer vascular retention is required reagents such as mono-sulfone-PEG may be more appropriate.

Novel Redox Active Tyrosine Mutations Enhance the Regeneration of Functional Oxyhemoglobin from Methemoglobin: Implications for Design of Blood Substitutes.

Heme mediated oxidative toxicity has been linked to adverse side effects in Hemoglobin Based Oxygen Carriers (HBOC), initiated by reactive ferryl (FeIV) iron and globin based free radical species. We recently showed that the addition of a redox active tyrosine residue in the beta subunit (ßF41Y) of recombinant hemoglobin had the capability to decrease lipid peroxidation by facilitating the reduction of FeIV iron by plasma antioxidants such as ascorbate. In order to explore this functionality further we created a suite of tyrosine mutants designed to be accessible for both reductant access at the protein surface, yet close enough to the heme cofactor to enable efficient electron transfer to the FeIV. The residues chosen were: ßF41Y; ßK66Y; ßF71Y; ßT84Y; ßF85Y; and ßL96Y. As with ßF41Y, all mutants significantly enhanced the rate of ferryl (FeIV) to ferric (FeIII) reduction by ascorbate. However, surprisingly a subset of these mutations (ßT84Y, and ßF85Y) also enhanced the further reduction of ferric (FeIII) to ferrous (FeII) heme, regenerating functional oxyhemoglobin. The largest increase was seen in ßT84Y with the percentage of oxyhemoglobin formed from ferric hemoglobin in the presence of 100 µM ascorbate over a time period of 60 min increasing from 10% in ßF41Y to over 50% in ßT84Y. This increase was accompanied by an increased rate of ascorbate consumption. We conclude that the insertion of novel redox active tyrosine residues may be a useful component of any recombinant HBOC designed for longer functional activity without oxidative side effects.

The ßLys66Tyr Variant of Human Hemoglobin as a Component of a Blood Substitute.

It has been proposed that introducing tyrosine residues into human hemoglobin (e.g. ßPhe41Tyr) may be able to reduce the toxicity of the ferryl heme species in extracellular hemoglobin-based oxygen carriers (HBOC) by facilitating long-range electron transfer from endogenous and exogenous antioxidants. Surface-exposed residues lying close to the solvent exposed heme edge may be good candidates for mutations. We therefore studied the properties of the ßLys66Tyr mutation. Hydrogen peroxide (H2O2) was added to generate the ferryl protein. The ferryl state in ßLys66Tyr was more rapidly reduced to ferric (met) by ascorbate than recombinant wild type (rwt) or ßPhe41Tyr. However, ßLys66Tyr suffered more heme and globin damage following H2O2 addition as measured by UV/visible spectroscopy and HPLC analysis. ßLys66Tyr differed notably from the rwt protein in other ways. In the ferrous state the ßLys66Tyr forms oxy, CO, and NO bound heme complexes similar to rwt. However, the kinetics of CO binding to the mutant was faster than rwt, suggesting a more open heme crevice. In the ferric (met) form the typical met Hb acid-alkaline transition (H2O to -OH) appeared absent in the mutant protein. A biphasicity of cyanide binding was also evident. Expression in E. coli of the ßLys66Tyr mutant was lower than the rwt protein, and purification included significant protein heterogeneity. Whilst, ßLys66Tyr and rwt autoxidised (oxy to met) at similar rates, the oxygen p50 for ßLys66Tyr was very low. Therefore, despite the apparent introduction of a new electron transfer pathway in the ßLys66Tyr mutant, the heterogeneity, and susceptibility to oxidative damage argue against this mutant as a suitable starting material for a HBOC.

Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage.

Haemolytic events, such as those following rhabdomyolysis and subarachnoid haemorrhage, often result in pathological complications such as vasoconstriction. Haem-protein cross-linked myoglobin and haemoglobin are generated by ferric-ferryl redox cycling, and thus can be used as markers of oxidative stress. We have found haem-protein cross-linked myoglobin in the urine of patients suffering from rhabdomyolysis and haem-protein cross-linked haemoglobin in the cerebrospinal fluid of patients following subarachnoid haemorrhage. These findings provide strong evidence that these respiratory haem proteins can be involved in powerful oxidation processes in vivo. We have previously proposed that these oxidation processes in rhabdomyolysis include the formation of potent vasoconstrictor molecules, generated by the myoglobin-catalysed oxidation of membranes, inducing nephrotoxicity and renal failure. Haem-protein cross-linked haemoglobin in cerebrospinal fluid suggests that a similar mechanism of lipid oxidation is present and that this may provide a mechanistic basis for the delayed vasospasm that follows subarachnoid haemorrhage.

Extracellular heme peroxidases in actinomycetes: a case of mistaken identity.

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.

The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species.

Myoglobin catalyses the breakdown of lipid hydroperoxides (e.g., HPODE) during which the absorption band of the lipid conjugated diene (234 nm) is partially bleached. The constant for this process is strongly pH-dependent (k = 9.5 x 10(-3)s(-1), pH 7: k = 2.3 x 10(-1)s(-1), pH 5). This rate enhancement is not due to acid-induced changes in protein conformation or the involvement of protein-based radical species, as demonstrated by an almost identical pH dependence of the same reaction catalyzed by ferric haemin. The rate constants for ferryl formation and auto-reduction show different pH dependencies, with a pK of 8.3 for ferryl formation and a projected pK of 3.5 for ferryl auto-reduction. The pH dependence for the auto-reduction of the ferryl species is the same as that of the myoblobin catalyzed breakdown of HPODE. We propose that the protonated form of ferryl myoglobin (Fe(4+) - OH(-)) is the reactive species regulating the peroxidatic activity of myoglobin. The protonated ferryl species abstracts an electron from either the protein or porphyrin, allowing fast regeneration of the ferric species. Alkaline conditions stabilize the ferryl species, making myoglobin considerably less reactive towards lipids and lipid hydroperoxides. These findings are significant for understanding myoglobin-induced oxidative stress in vivo and the development of therapies.

A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure.

Muscle injury (rhabdomyolysis) and subsequent deposition of myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by approximately 80%. EPR and UV spectroscopy demonstrated that myoglobin was deposited in the kidneys as the redox competent ferric myoglobin and that it's concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of myoglobin-induced lipid peroxidation.

Mechanism of reaction of myoglobin with the lipid hydroperoxide hydroperoxyoctadecadienoic acid.

The reaction between myoglobin and the lipid hydroperoxide 13(S)-hydroperoxy-9,11(cis,trans)-octadecadienoic acid (HPODE) was studied kinetically by spectrophotometric, polarographic and analytical methods. Metmyoglobin catalysed the decomposition of HPODE, resulting in peroxide, oxygen and conjugated diene depletion, together with the transient production of ferryl myoglobin. The reaction stoichiometry was 2:1:1 for peroxide to oxygen to conjugated diene, whereas the myoglobin remained generally intact. This stoichiometry and the rates of change of conjugated diene and ferryl myoglobin concentrations were not completely consistent with previously proposed mechanisms. We propose a novel mechanism in which HPODE reacts with both ferric myoglobin and ferryl myoglobin to form a redox cycle. Both peroxyl and alkoxyl radicals are produced, explaining the observed stoichiometry of peroxide, oxygen and conjugated diene depletion and the transient appearance of ferryl myoglobin. Computer simulation shows that this mechanism is fully capable of reproducing the observed time courses of all components.

Pro-oxidant effects of cross-linked haemoglobins explored using liposome and cytochrome c oxidase vesicle model membranes.

The therapeutic use of cell-free haemoglobin as a blood substitute has been hampered by toxicological effects. A model asolectin (phosphatidylcholine/phosphatidylethanolamine) liposome system was utilized to study the pro-oxidant efficiency of several chemically modified haemoglobins on biological membranes. Lipid peroxidation, resulting from the interactions between haemoglobin and liposomes, was measured by conjugated diene formation and the maximal rates of oxygen uptake. Spectral changes gave insight into the occurrence of the ferryl iron species. The residual reactivity of oxidatively damaged haemoglobins with ligands during incubation with liposomes was assessed from rapid kinetic carbon monoxide-binding experiments. Liposomes in which cytochrome c oxidase was embedded show both haemoglobin and the enzyme to be oxidatively damaged during incubation. The functional state of cytochrome c oxidase was monitored in the presence and absence of a free radical scavenger. Once in contact, both unmodified and modified haemoglobins triggered and maintained severe radical-mediated membrane damage. Differences in the pro-oxidant activities among haemoglobins may be explained by either the differential population of their ferryl intermediates or disparate dimerization and transfer of haem into the membrane with subsequent haem degradation. This study may contribute to a better understanding of the molecular determinants of haemoglobin interactions with a variety of biological membranes.