Redox Reactions of Cross-linked Haemoglobins with Oxygen and Nitrite

  • Celia Bonaventura
  • Robert Henkens
  • Katherine D. Weaver
  • Abdu I. Alayash
  • Alvin L. Crumbliss
Part of the Protein Reviews book series (PRON, volume 9)


Redox reactions of haemoglobin (Hb) with oxygen can initiate a cascade of oxidative reactions that appear to underlie the adverse side reactions observed when cell-free Hbs are introduced into the circulation to enhance oxygen delivery to respiring tissues. Redox reactions of cell-free Hbs with nitrite may also be of significance in vivo, as these reactions can lead to formation of nitrosylated Hb (NO-Hb) along with oxidised Hb (MetHb). To clarify the factors governing these redox reactions we measured the kinetics of nitrite-induced and oxygen-induced heme oxidation and obtained oxygen binding and oxidation curves for unmodified human Hb and four cross-linked Hbs. The four cross-linked Hbs studied were generated by cross-linking Hb with glutaraldehyde, dextran, O-raffinose or bis(3,5-dibromosalicyl)fumarate. Oxygen binding by the cross-linked Hbs occurred with reduced oxygen affinity, reduced cooperativity and reduced responses to organic phosphate effectors. The redox potentials of the cross-linked Hbs were shifted to higher potentials relative to unmodified Hb in the absence of allosteric effectors, indicating a reduced thermodynamic driving force for oxidation. In spite of this, these Hbs showed increased rates in oxidative reactions. Elevated rates of heme oxidation were observed for their oxy derivatives under aerobic conditions, and upon exposure to nitrite under both aerobic and anaerobic conditions. These results show that heme accessibility rather than heme redox potential is the major determinant of the kinetics of redox reactions of Hb with both oxygen and nitrite.


Oxygen Carrier Oxygen Affinity Oxygen Binding Blood Substitute Hill Plot 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abassi, Z., Kotob, S., Pieruzzi, F., Abouassali, M., Keiser, H. R., Fratantoni, J. C., and Alayash, A. I. 1997. Effects of polymerization on the hypertensive action of diaspirin cross-linked hemoglobin in rats. J. Lab. Clin. Med. 129:603–610.PubMedCrossRefGoogle Scholar
  2. Adamson, J. G., and Moore, C. 1997. Hemolink, an O-raffinose cross-linked hemoglobin-based oxygen carrier. In Blood Substitutes: Principals, Methods, Products and Clinical Trials, ed. T.M.S. Chang. pp. 62–79. Karger Landes Systems.Google Scholar
  3. Alayash, A. I. 2004. Oxygen therapeutics: Can we tame haemoglobin? Nat. Rev. Drug. Disc. 3:152–159.CrossRefGoogle Scholar
  4. Alayash, A. I., Fratantoni, J. C., Bonaventura, C., Bonaventura, J., and Bucci, E. 1992. Consequences of chemical modifications on the free radical reactions of human hemoglobin. Arch. Biochem. Biophys. 298:114–120.PubMedCrossRefGoogle Scholar
  5. Angelo, M., Singel, D. J., and Stamler, J.S. 2006. An S-nitrosothiol SNO. synthase function of hemoglobin that utilizes nitrite as a substrate. Proc. Nat. Acad. Sci. 103:8366–8371.PubMedCrossRefGoogle Scholar
  6. Baldwin, A. L., Wiley, E. B., and Alayash, A. I. 2002. Comparison of effects of two hemoglobin-based oxygen carriers on intestinal integrity and microvascular leakage. Am. J. Physiol. 283:H1292–1301.Google Scholar
  7. Baldwin, A. L., Wiley, E. B., and Alayash, A. I. 2004. Differential effects of sodium selenite in reducing tissue damage caused by three hemoglobin-based oxygen carriers. J. App. Physiol. 96:893–903.CrossRefGoogle Scholar
  8. Baldwin, A. L., Wiley, E. B., Summers, A. G., and Alayash, A. I. 2003. Sodium selenite reduces hemoglobin-induced venular leakage in the rat mesentery. Am. J. Physiol. 284:H81–91.Google Scholar
  9. Bonaventura, C., Cashon, R., Bonaventura, J., Perutz, M., Fermi, G., and Shih, D. T. B. 1991. Involvement of the distal histidine in the low affinity exhibited by Hb Chico Lys beta 66 to Thr. and its isolated beta chains. J. Biol. Chem. 266:23033–23040.PubMedGoogle Scholar
  10. Chevalier, A., Guillochon, D., Nadjar, N., Piot, J., Vijayakshmi, M. W., and Thomas, D. 1989. Effect of glutaraldehyde on haemoglobin: oxidation-reduction potentials and stability. Biochem. Cell. Biol. 68:813–818.Google Scholar
  11. Cosby, K., Partovi, K. S., Crawford, J. H., Patel, R. P., Reiter, C. D., Martyr, S., Yang, R. K., Waclawiw, M. A., Zalos, G., Xu, S. L., Huang, K. T., Shields, H., Kim-Shapiro, D. B., Schechter, A. N., Cannon, R. O., and Gladwin, M. T. 2003. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat. Med. 9:1498–1505.PubMedCrossRefGoogle Scholar
  12. D’Agnillo, F., and Alayash, A. I. 2000. Site-specific modifications and toxicity of blood substitutes. The case of diaspirin cross-linked hemoglobin. Adv. Drug Deliv. Rev. 28:199–212.CrossRefGoogle Scholar
  13. D’Agnillo, F. and Alayash, A. I. 2001. Redox cycling of diaspirin cross-linked hemoglobin induces G2/M arrest and apoptosis in cultured endothelial cells. Blood 98:3315–3323.PubMedCrossRefGoogle Scholar
  14. Guillochon, D., Vijayakshmi, M. W., Thion-Sow, A., and Thomas, D. 1989. Effect of glutaraldehyde on hemoglobin: functional aspects and Mossbauer parameters. Biochem. Cell. Biol. 64:29–37.CrossRefGoogle Scholar
  15. Jia, Y., Ramasamy, S., Wood, F., Alayash, A. I., and Rifkind, J. M. 2004a. Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation. Biochem. J. 384:367–375.PubMedCrossRefGoogle Scholar
  16. Jia, Y., Wood, F.., Menu, P., Faivre, B., Caron, A., and Alayash, A. I. 2004b. Oxygen binding and oxidation reactions of human hemoglobin conjugated to carboxylate dextran. Biochim. Biophys. Acta 1672:164–173.PubMedGoogle Scholar
  17. Kim-Shapiro, D. B., Gladwin, M. T., Patel, R. P., and Hogg, N. 2005. The reaction between nitrite and hemoglobin: the role of nitrite in hemoglobin-mediated hypoxic vasodilation. J. Inorg. Biochem. 99:237–246.PubMedCrossRefGoogle Scholar
  18. Kosaka, H., Imaizumi, K., Imai, K. and Tyuma, I. 1979. Stoichiometry of the reaction of oxyhemoglobin with nitrite. Biochim. Biophys. Acta 581:184–188.PubMedGoogle Scholar
  19. Lancaster, J. R. 1994. Simulation of the diffusion and reaction of endogeneously produced nitric oxide. Proc. Natl. Acad. Sci. U S A 91:8137–8141.PubMedCrossRefGoogle Scholar
  20. Nagababu, E., Ramasamy, S., Rifkind, J. M., Jia, Y., and Alayash, A. I. 2002. Site-specific cross-linking of human and bovine hemoglobins differentially alters oxygen binding and redox side reactions producing rhombic heme and heme degradation. Biochemistry 41:7407–7415.PubMedCrossRefGoogle Scholar
  21. Pearce, B. J. and Gawryl, M. S. 1998. The pharmacology of tissue oxygenation by Biopure’s hemoglobin-based oxygen carrier, Hemopure. In Blood Substitutes: Principles, Methods, Products and Clinical Trials, ed. T. M. S. Chang, pp. 82–100. Vol II. Basel, Switzerland: Karger Landes Systems.Google Scholar
  22. Perutz, M. F. 1970. Stereochemistry of cooperative effects in haemoglobin. Nature 228:726–734.PubMedCrossRefGoogle Scholar
  23. Perutz, M. F. 1978. Hemoglobin structure and respiratory transport. Sci. Am. 239:68–86.CrossRefGoogle Scholar
  24. Prouchayret, F., Fasan, G., Grandgeorge, M., Vigneron, C., Menu, P., and Dellacherie, E. 1992. A potential blood substitute from carboxylic dextran and oxyhemoglobin. I. Preparation, purification and characterization. Biomater. Artif. Cells Immobiliz. Biotechnol. 20:319–22.Google Scholar
  25. Reiss, J. G. 2001. Oxygen carriers “blood substitutes”-raison d’etre, chemistry, and some physiology. Chem. Rev. 101:2797–2919.CrossRefGoogle Scholar
  26. Riggs, A. F. and Wolbach, R. A. 1956. Sulfhydryl groups and the structure of hemoglobin. J. Gen. Physiol. 39:585–605.PubMedCrossRefGoogle Scholar
  27. Roche, C. J., Dantsker, D., Samuni, U., and Friedman, J. M. 2006. Nitrite reductase activity of sol-gel encapsulated deoxy hemoglobin: Influence of quaternary and tertiary structure. J. Biol. Chem. 281:36874–36882.PubMedCrossRefGoogle Scholar
  28. Spagnuolo, C., Rinelli, P., Coletta, M., Chiancone, E., and Ascoli, F. 1987. Oxidation reaction of human oxyhemoglobin with nitrite: a reexamination. Biochim. Biophys. Acta 911:59–65.PubMedGoogle Scholar
  29. Stellwagen, E. 1978. Haem exposure as the determinate of oxidation-reduction potential of haem proteins. Nature 275:73–74.PubMedCrossRefGoogle Scholar
  30. Taboy, C. H., Bonaventura, C., and Crumbliss, A. L. 2002. Anaerobic oxidations of myo-globin and hemoglobin by spectroelectrochemistry. In Methods in Enzymology, ed. M. I. Simon, pp. 187–209. New York: Academic Press.Google Scholar
  31. Taboy, C. H., Faulkner, K. M., Kraiter, D., Bonaventura, C., and Crumbliss, A. L. 2000. Concentration-dependent effects of anions on the anaerobic oxidation of hemoglobin and myoglobin. J. Biol. Chem. 275: 39048–39054.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Italia 2008

Authors and Affiliations

  • Celia Bonaventura
    • 1
  • Robert Henkens
    • 1
  • Katherine D. Weaver
    • 2
  • Abdu I. Alayash
    • 3
  • Alvin L. Crumbliss
    • 2
  1. 1.Nicholas School of the Environment and Earth SciencesDuke University Marine LaboratoryBeaufortUSA
  2. 2.Department of ChemistryDuke UniversityDurhamUSA
  3. 3.Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and ResearchFood and Drug AdministrationBethesdaUSA

Personalised recommendations