METALLOPROTEIN-BASED BLOOD SUBSTITUTES: STRATEGIES FOR TACKLING OXIDATIVE AND NITROSATIVE STRESS
Assoc prof. Radu Silaghi-Dumitrescu, leader
Assoc. Prof. Monica Ioana Tosa
Assist. Prof. Luiza Gaina
PhD students Augustin Mot and Florina Deac
The blood currently used in transfusions presents problems linked to stability (requiring refrigeration and becoming outdated after about a month), availability (especially under crisis situations, such as major accidents, military situations, or major outbreaks), antigens (blood group compatibility), contamination (AIDS, hepatitis, or agents yet to be identified) or ethics (e.g., Jehovah’s witnesses). Blood substitutes are chemical or biochemical preparations aimed at being used in transfusions without the above-mentioned drawbacks and with the sole role of enhancing oxygen transport by the patient’s blood (as opposed to the more complex functions that blood normally achieves, such as transport or defense).
There are three classes of blood substitutes currently being debated in the scientific literature. The first two classes represent purely synthetic approaches, while the third is semi synthetic, employing the hemoglobin already produced by living organisms. As we shall see, our research has among its finalities the possible definition of a new class of blood substitutes, semi synthetic but not based on hemoglobin.
The first class of blood substitutes consists of organic compounds of the perfluorocarbon type, which, while not water-miscible, can dissolve molecular oxygen very efficiently and are quite stable from a biochemical point of view. One of the representatives of this class, Perftoran, has been developed and approved for human use in Russia; its limited transport capacity, problems with retaining it in the circulatory system, and side-effects characterized by flu-like symptoms have left this line of research in the shadows in recent years.
The second class of substitutes is also entirely synthetic and makes use of derivatized heme groups for transporting oxygen – following the natural example of hemoglobin, which also uses a heme group for this purpose, forming a reversible bond between the ferrous center and molecular oxygen. As heme Fe(II)-O2 adducts are relatively unstable in aqueous solutions and polar media in general (with autoooxidation yielding Fe(III) and superoxide, and further oxidative processes ensuing),and the metal center has intrinsic reactivity towards a number of molecules and ions present in vivo, class 2 substitutes employ (bio)chemically unreactive to coat the heme group, with a hydrophobic inside/hydrophilic outside environment reminiscent of a bona fide protein such as hemoglobin. This approach has only entered the scientific spotlight this year, and we do not anticipate clinical tests any time soon.
The third class of blood substitutes are those hemoglobin-based, which constitute the major research direction in the field of artificial blood. Within this class, the protein employed is hemoglobin extracted either from outdated human blood (with limited availability in certain cases, such as Romania), or from bovines (with virtually unlimited availability). Purified hemoglobin would at first glance present itself as the most obvious ingredient for a blood substitute preparation, as it is indeed the most important ingredient of blood itself. Paradoxically, hemoglobin, once released from within the protective envelope of the red blood cells (RBC) which normally transport it throughout the body, becomes a poisonous agent. Its toxic effects can be seen along three coordinates. Firstly, hemoglobin’s oxygen affinity is controlled by small effector molecules (e.g., the bisphosphoglycerate, BPG) within the RBC; once released from the RBC, hemoglobin’s affinity changes drastically, and so ]does its ability to efficiently deliver oxygen. Even more dangerous are currently accepted to be the two other coordinates of hemoglobin toxicity, namely, reactivities towards oxidative stress agents (especially hydrogen peroxide) and nitrosative stress agents (especially nitric oxide, NO). The discovery that NO is in fact the mysterious EDRF (endothelium-derived relaxing factor) and has a key role in regulating blood pressure made the object of a Nobel prize a few years ago; NO is produced in the vascular walls (in the endothelial cells) at very small steady-state levels by NO-synthase. Oxy hemoglobin is extremely reactive towards NO, as would be expected given the pronounced Fe(III)-O2- (ferric-superoxo) character of the Fe(II)-O2 entity; indeed, the reaction between superoxide and NO, two free radicals, has long been known to approach the diffusion limit. While protected inside the RBC, hemoglobin has very limited direct contact with the endothelium-generated NO; however, free hemoglobin, as it would be used as a blood substitute, can approach the endothelium much better and may in fact even be extravasated, implying a much enhanced consumption of the NO. The resulted NO deficit leads to drastic changes in blood pressure, which, uncontrolled, may even bring about the death of the patient. On the other hand, hemoglobin’s heme center is also reactive towards hydrogen peroxide (and peroxides in general), especially under conditions where due to natural autooxidation processes the hemoglobin reaches the ferric state.[4-10] The product of the reaction between ferric hemoglobin and peroxides is , as with most hemoproteins (e.g., catalases, peroxidases, cytochromes P450, to give just a few famous examples[4,9,11]), a species formally described as Fe(IV) with an oxo ligand (ferryl). Ferryl hemoglobin is constantly produced in our blood, and this process is enhanced under conditions of stress (be it physical effort or disease-related), and its unique degradation products can be detected in samples taken from any individual. Hemoglobin ferryl is a powerful and non-specific oxidant. Under normal conditions, within the RBC, hemoglobin’s reactivity towards peroxides is limited by the presence of an antioxidant system (catalase, non-heme peroxidases, ascorbate, glutathione, and others), all of which work towards removing oxidative stress agents, while hemoglobin is maintained in the less prooxidant ferrous form by the methemoglobin reductase enzyme system.[12,13] On the other hand, free hemoglobin outside of the RBC, in the form that might be used as a blood substitute, is on one hand deprived of the antioxidant system and on the other hand faster extravasated from the vascular system accumulating in the kidneys, where the peroxidatic activity is unleashed and leads to renal failure. Thus, the low pH encountered in the kidneys, favoring autooxidation and reaction with peroxides, and also enhancing the redox potential of the ferryl hemoglobin, together with the lower oxygen concentrations (resulting in more metal centers available for prooxidant reactivity, and in oxidative stress), all lead to a marked enhancement of prooxidant reactivity. One of the results of this otherwise non-specific activity is the oxidation of membrane-derived lipids to generate a class of prostaglandins normally employed by the organism to control blood flow; it is the excess of these compounds that can eventually lead to renal failure and death.[4-10] To these issues, a further problem is presented by the fact that hemoglobin is a tetramer with monomers non-covalently bound to each other; within the RBC, the hemoglobin concentration is three orders of magnitude above the dissociation constant of the tetramer into dimers, while outside of the RBC the concentration is distinctly smaller and may result in partial dissociation and subsequent extravasation or increased reactivity.
For the reasons outlined above, purified hemoglobin can only be a reasonable blood substitute candidate once its reactivities can be controlled/reduced. Such reduction has been achieved so far via five routes, to which we will add two others that so far have not been employed practically.
A first approach for containing hemoglobin’s reactivity involves covalent crosslinking of the monomers so as to avoid tetramer dissociation. As would be expected based on the above-exposed considerations on oxidative and nitrosative stress (details of which were unknown at the time), such crosslinked have maintained their toxicity, with DBBF (a product employing an aspirin derivative for crosslinking) being one of the clearest cases in this respect. [1,13]
A second approach has involved interprotein crosslinking of hemoglobin, thereby generating particles of large molecular weight/volume, which (1) have increased stability in the bloodstream and (2) avoid homogenous close contact between hemoglobin and endothelium, thereby reducing the hemoglobin-NO reactivity. Such a product is currently approved for human use in special surgery cases in South Africa. This latter preparation has the added property that it is prepared using only partially purified hemoglobin (of bovine origin), which retains catalase and superoxide dismutase activities; which ensure a dramatic increase of the resistance of this product towards oxidative stress. Cell-free bovine hemoglobin also presents the advantage of having an oxygen affinity similar to that of human RBC’s. 
A thrid approach towards reducing heomglobin’s pro-oxidant activity has involved derivatization of the protein surface with polymeric systems, with results much similar to those achieved by the protein-protein crosslinking approach described above. Polyethylene glycols have formed the focus of such strategies, although oligo and polysaccharides have also been employed.
A fourth approach has been to genetically modify human hemoglobin (hence overexpressing it in inferior organisms, as opposed to the other strategies, all of which were employing native hemoglobin). Distinct site-directed mutations have been identified that drastically reduce reactivity towards NO by modifying amino acids gating access of small molecules towards the heme site; no examples of similarly rational design of hemoglobin to remove prooxidant activity are currently available in the blood substitutes field.[1,2]
The fifth approach to containing hemoglobin’s reactivity involves encapsulation in liposomes or vesicles (where in the latter case the vesicle may be used as template for generating a polymeric capsule of dimensions compatible with those of an RBC. The disadvantage of liposomes is a markedly reduced lifetime compared to bona fide cellular membranes; virtually nothing is known about the behavior of polymer-encapsulated hemoglobin towards agents of oxidative and nitrosative stress, and it is unclear to what extent the use of non-water-miscible solvents is advisable at the vesicle-generation stage in view of the subsequent in vivo application of the protein. Based on our own practical experience, even traces of denatured hemoglobin (which may well arise during treatment with organic solvents) may be enough to initiate significant prooxidant activity
A sixth approach, which has so far not been employed practically for blood substitutes purposes, involves supplementation with small-molecule antioxidants (e.g., ascorbate or selenium).
A seventh approach, which we hereby introduce for the first time, would involve encapsulation of hemoglobin in three-dimensional networks made of relatively stable biocompatible polymers, thereby generating particles compatible in size with an RBC but which have the added advantage of not involving chemical modification of the protein surface. In this respect, we will note that a product like DBBF, which is representative for the way clinical trials can fail in this field, features an increased autooxidation rate (and hence, enhanced prooxidant effect) following chemical modification.[1,13]
We now add a fourth class of blood substitutes, whose active ingredient is also a protein specialized in oxygen transport, but of non-heme nature: hemerythrin (Hr). Hr employs a binuclear non-heme diferrous center to bind oxygen; the resulting adduct is formally described as Fe(III)-Fe(III)-OOH, i.e. diferric peroxo.[14,15] This mode of binding oxygen has the remarkable advantage of (1) not featuring a superoxide ligand (unlike Hb), which drastically reduces the reactivity towards NO, and (2) exhibiting stability towards peroxide (unlike Hb). Related to Hr is rubrerythrin (Rbr), a non-heme diiron protein specializing which works as a very efficient peroxidase, with a very high affinity (low-micromolar range) towards hydrogen peroxide, som six orders of magnitude better than the catalase encountered in RBC. Another recently-discovered non-heme protein, superoxide reductase (SOR), functions as an efficient scavenger of superoxide.[17,18] The collection of Hr, Rbr, and SOR offers the possibility of designing a product with remarkable stability towards agents of oxidative and nitrosative stress.
The most recent concerted effort towards a viable blood substitute has been the project EUROBLOODSUBSTITUTES of FP6, within which all possible aspects of such a product were covered (ranging from chemistry, biology, medicine, sociology, and economics). In terms of results, the European project was the first to demonstrate the viability of using recombinant techniques for this field, and has demonstrated the relevance of oxidative and nitrosative reactivity for blood substitutes. The European project has also allowed for a platform to be set up with multidisciplinary competences, which may ensure collaboration beyond the framework of the FP6.
Our current project draws on the previous direct implication of the project leader (Silaghi-Dumitrescu) in the EUROBLOODUBSTITUTES initiative at the University of Essex; its working hypothesis is that the main requirements for alleviating blood substitute toxicity are a large apparent molecular weight and a reduced reactivity towards oxidative and nitrosative stress. The experiments that we propose line up along two main directions: (1) studies on the reactivity of blood substitute ingredients towards oxidative and nitrosative stress and ways to combat these reactivities, and (2) new techniques/approaches for preparation of blood substitutes that would be based on knowledge gathered under item (1) above and would take into account the possibility of employing proteins other than hemoglobin. Financing of our project has started in October 2007.
1. A. I. Alayash, Nat Rev Drug Discov, 2004, 3, 152-9.
2. D. H. Doherty, M. P. Doyle, S. R. Curry, R. J. Vali, T. J. Fattor, J. S. Olson, D. D. Lemon, Nat Biotechnol, 1998, 16, 672-6.
3. R. Silaghi-Dumitrescu, J. Mol. Struct. THEOCHEM, 2005, 722, 233-237.
4. R. Silaghi-Dumitrescu, J. Biol. Inorg. Chem., 2004, 9, 471-476.
5. R. Silaghi-Dumitrescu, C. E. Cooper, Dalton Trans., 2005, 3477-3482.
6. C. E. Cooper, M. Jurd, P. Nicholls, M. M. Wankasi, D. A. Svistunenko, B. J. Reeder, M. T. Wilson, Dalton Trans., 2005, 3483-3488.
7. N. B. Vollaard, B. J. Reeder, J. P. Shearman, P. Menu, M. T. Wilson, C. E. Cooper, Free Radic Biol Med, 2005, 39, 1216-28.
8. D. A. Svistunenko, B. J. Reeder, M. M. Wankasi, R. L. Silaghi-Dumitrescu, C. E. Cooper, S. Rinaldo, F. Cutruzzola, M. T. Wilson, Dalton Trans, 2007, 840-50.
9. R. Silaghi-Dumitrescu, B. J. Reeder, P. Nicholls, C. E. Cooper, M. T. Wilson, Biochem J, 2007, 403, 391-5.
10. B. J. Reeder, M. A. Sharpe, A. D. Kay, M. Kerr, K. Moore, M. T. Wilson, Biochem. Soc. Trans., 2002, 30, 745-748.
11. M. T. Green, J. H. Dawson, H. B. Gray, Science, 2004, 304, 1653-6.
12. A. I. Alayash, Free Rad. Biol. Med., 1994, 16, 137-138.
13. J. Dunne, A. Caron, P. Menu, A. I. Alayash, P. W. Buehler, M. T. Wilson, R. Silaghi-Dumitrescu, B. Faivre, C. E. Cooper, Biochem J, 2006, 399, 513-24.
14. D. M. Kurtz, Jr., Essays in Biochemistry, 1999, 55-80.
15. C. E. Isaza, R. Silaghi-Dumitrescu, R. B. Iyer, D. M. Kurtz, Jr., M. K. Chan, Biochemistry, 2006, 45, 9023-31.
16. R. Iyer, R. Silaghi-Dumitrescu, W. N. Lanzilotta, D. M. Kurtz, Jr., J. Biol. Inorg. Chem., 2005, 10, 407-416.
17. R. Silaghi-Dumitrescu, I. Silaghi-Dumitrescu, E. D. Coulter, D. M. Kurtz, Jr., Inorg. Chem., 2003, 42, 446-456.
18. D. M. Kurtz, Jr., Acc Chem Res, 2004, 37, 902-8.
Our working hypothesis is that the two main requirements for alleviating blood substitute toxicity are a large apparent molecular weight and a reduced reactivity towards oxidative and nitrosative stress. The experiments that we propose line up along two main directions: (1) studies on the reactivity of blood substitute ingredients towards oxidative and nitrosative stress and ways to combat these reactivities, and (2) new techniques/approaches for preparation of blood substitutes that would be based on knowledge gathered under item (1).
In terms of studying the reactivity of hemoglobin towards agents of oxidative stress, one of the main objectives will be to define a minimal, non-redundant set of tests that can be run on blood substitute candidates in order to assess their safety in terms of oxidative/nitrosative reactivities; in each case, a chemical focus will be maintained, seeking to understand the underlying causes and mechanisms of the phenomena studied. In vitro tests will be aimed at determining conditions that can reduce the effects of oxidative/nitrosative stress to non-detectable levels in a blood substitute. The knowledge gained this way is to be subsequently applied for tests on newly-developed materials.
In terms of preparing new blood substitutes, four directions of research will be followed.
First, a new class of blood substitutes will be generated, based non proteins other than hemoglobin.
Second, new techniques for derivatization of hemoglobin and other proteins will be sought, using biocompatible polymers with minimal effects on oxygen binding properties.
Third, new formulations will be proposed and tested, containing antioxidants as outlined above.
Fourth, genetic and/or chemical modifications will be sought, which may increase stability and/or reduce prooxidant activity of blood substitutes.
We aim to materialize our results into at least six ISI-indexed publications.
Novel blood substitute preparations have been obtained via chemical modifications of native proteins, recombinant protein overexpression, and site-directed mutagenesis. The reactivities of native proteins as well as of derivatized proteins towards agents of oxidative stress and nitrosative stress were examined; novel antioxidant pathways and preparations were studied, and contributions towards atomic-level mechanisms of oxidative stress were reported. A set of non-redundant tests for the functionality and possible toxicity of the blood substitutes has been developed. Some of the data has been published as detailed below, while other is still to be submitted for publication. Final tests are currently underway to verify that we have indeed fulfilled all objectives of the project.
Implication of young researchers:
The two PhD students involved in the project have so far been involved in 7 publications, with most of the data obtained by them still unpublished. There are also 10 publications benefiting from contribution from undergraduate students. This illustrates that the instructional goals of the project are being fulfilled.
Articles showing our recent contributions/benefiting from support of this grant (project members are shown in bold letters):
1. Silaghi-Dumitrescu, Radu; Uta, Matei-Maria; Makarov, Sergei. Nitrite linkage isomerism in hemes and related complexes: modulation by metal, oxidation state, macrocycle, and medium polarity Revue Roumaine de Chimie, 2010, in press.
2. Silaghi-Dumitrescu, Radu; Makarov, Sergei. A computational analysis of electromerism in hemoprotein Fe(I) models Journal of Biological Inorganic Chemistry, 2010, in press
3. Silaghi-Dumitrescu, Radu. A Density Functional Investigation of Hydrogen Peroxide Activation by High-Valent Heme Centers: Implications for the Catalase Catalytic Cycle Journal of Porphyrins and Phthalocyanines, 2010, in press.
4. Silaghi-Dumitrescu, Radu. Computational description of peptide architectures based on hydrogen bonds Studia Universitatis Babes-Bolyai, Chemia 2010, in press.
5. Kis, Zoltan; Makarov, Sergei V; Silaghi-Dumitrescu, Radu. Computational investigations on the electronic structure and reactivity of thiourea dioxide: sulfoxylate formation, tautomerism, dioxygen liberation Journal of Sulphur Chemistry, 2010, 31(1), 27-39.
6. Silaghi-Dumitrescu, Radu; Makarov, Sergei V., Hydrocarbon oxygenation by metal-nitrite adducts: a theoretical comparison with ferryl-based oxygenation agents European Journal of Inorganic Chemistry, 2010, 39(6):1464-6.
7. Mot, Augustin; Kis, Zoltan; Svistunenko, Dimitri A.; Damian, Grigore; Makarov, Sergei V.; Silaghi-Dumitrescu, Radu. Silaghi-Dumitrescu, Radu. ‘Super-reduced’ iron under physiologically-relevant conditions Dalton Transactions, 2010, 39(6):1464-6.
8. Deac, Florina-Violeta; Todea, Anamaria; Bolfa, Ana Maria; Podea, Paula; Petrar, Petronela; Silaghi-Dumitrescu, Radu. Ascorbate binding to globins Romanian Journal of Biochemistry, 2009, 46(2), 115–121.
9. Mot, Augustin Catalin; Damian, Grigore; Sarbu, Costel; Silaghi-Dumitrescu, Radu, Redox reactivity in propolis: direct detection of free radicals in basic medium and interaction with hemoglobin Redox Report, 2009, 14(6), 267-74.
10. Arkosi, Mariann-Kinga; Deac, Florina;Silaghi-Dumitrescu, Radu. Silaghi-Dumitrescu, Radu. Hemoglobin peroxidase activity: interaction with hydroquinone and anthracene Metal Elements in Environment, Medicine and Biology Tome IX, Radu Silaghi-Dumitrescu, Gabriela Garban, Eds., 2009, Cluj University Press, Cluj-Napoca, Romania, pp 99-110.
11. Mot, Augustin; Roman, Alina; Silaghi-Dumitrescu, Radu. Blood substitutes: can we do without hemoglobin? Metal Elements in Environment, Medicine and Biology Tome IX Radu Silaghi-Dumitrescu, Gabriela Garban, Eds., 2009, Cluj University Press, Cluj-Napoca, Romania, pp 122-125.
12. Taciuc, Vicentiu; Bischin, Cristina; Silaghi-Dumitrescu, Radu., A novel mechanism for platinum-based drugs: cisplatin and related compounds as pro-oxidants in blood Metal Elements in Environment, Medicine and Biology Tome IX, Radu Silaghi-Dumitrescu, Gabriela Garban, Eds., 2009, Cluj University Press, Cluj-Napoca, Romania, pp 130-134.
13. Deac, Florina; Todea, Anamaria; Silaghi-Dumitrescu, Radu., Glutaraldehyde derivatization of hemoglobin: a potential blood substitute Metal Elements in Environment, Medicine and Biology Tome IX, Radu Silaghi-Dumitrescu, Gabriela Garban, Eds., 2009, Cluj University Press, Cluj-Napoca, Romania, pp 165-173.
14. Silaghi-Dumitrescu, Radu; Bischin, Cristina; Deac, Florina; Kis, Zoltan; Mot, Augustin; Makarov, Sergei V., Unusual metal oxidation states in metalloproteins and related complexes: from degenerate orbitals to apoptosis Metal Elements in Environment, Medicine and Biology Tome IX, Radu Silaghi-Dumitrescu, Gabriela Garban, Eds., 2009, Cluj University Press, Cluj-Napoca, Romania, pp 174-182.
15. Silaghi-Dumitrescu, Radu., Superoxide interaction with nickel and iron superoxide dismutases Journal of Molecular Graphics and Modelling 2009, 28(2), 156-61.
16. Silaghi-Dumitrescu, Radu; Deac, Florina., The redox reactivity of globins: the chicken and egg paradox Metal Elements in Environment, Medicine and Biology Tome VIII, Corneliu Davidescu, Gabriela Garban, Iosif Gergen, Simona Dragan, Nicolae Vaszilcsin, Adina Avacovici, Eds., 2008, Eurobit Publishing House, Timisoara, Romania, pp 271-276.
17. Kis, Zoltan; Silaghi-Dumitrescu, Radu., The Electronic Structure of Biologically Relevant Fe(0) Systems International Journal of Quantum Chemistry 2010, in press.
18. Makarov,Sergei V.; Salnikov Denis S.; Pogorelova, Anna S.; Kis, Zoltan; Silaghi-Dumitrescu, Radu., A new route to carbon monoxide adducts of heme proteins Journal of Porphyrins and Phthalocyanines 2008, 12, 1096-1099.
19. Silaghi-Dumitrescu, Radu. Halide activation by heme peroxidases: theoretical predictions on putative adducts of halides with Compound I European Journal of Inorganic Chemistry, 2008, 5404-5407.
20. Silaghi-Dumitrescu, Radu. An alternative mechanism for catalase activity Studia Universitatis Babes-Bolyai, Chemia 2007, (4), 127-130.
21. Silaghi-Dumitrescu, Radu. Bonding in biologically-relevant high-valent iron centers International Journal of Chemical Modeling 2008, 1 (4).
22. Silaghi-Dumitrescu, Radu. Nitric oxide and nitrite reduction by metalloenzymes Revue Roumaine de Chimie, 2009, 54(6), 513–522.
23. Reeder, Brandon J.; Grey, Marie; Silaghi-Dumitrescu, Radu; Svistunenko, Dimitri A.; Bülow, L; Cooper, Chris E.; Wilson, Michael T. Tyrosine residues as redox cofactors in human hemoglobin: implications for engineering non toxic blood substitutes Journal of Biological Chemistry, 2008, 283, (45), 30780-30787.
24. Silaghi-Dumitrescu, Radu. The ferric-oxo moiety in porphyrin complexes – a ferryl in disguise? Macroheterocycles, 2008, 1, 79-81.
25. Silaghi-Dumitrescu, Radu; Uta, Matei-Maria. Nitrite linkage isomerism in bioinorganic chemistry – a case for mechanistic promiscuity Studia Universitatis Babes-Bolyai, Chemia 2008, (2), 61-65.
26. Cooper, Chris E.; Silaghi-Dumitrescu, Radu; Rukengwa, Martine; Alayash, Abdu I.; Buehler, Paul W. Peroxidase-activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of hemoglobin-based oxygen carriers (HBOC) Biochimica Biophysica Acta, 2008, 1784, 1415–1420.
27. Silaghi-Dumitrescu, Radu. The “push” effect of the thiolate axial ligand in superoxide reductase: a density functional study Revue Roumaine de Chimie, 2008, 53(12), 1149–1156.
28. Hillmann F, Riebe O, Fischer RJ, Mot Augustin, Caranto JD, Kurtz DM Jr, Bahl H, Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum FEBS Lett. 2009, 583(1), 241-5.
Relevant Romanian institutions:
Romanian chemistry/biochemistry journals