Share This Article:

Interaction of haptoglobin with hemoglobin octamers based on the mutation αAsn78Cys or βGly83Cys

Abstract Full-Text HTML Download Download as PDF (Size:1289KB) PP. 1-10
DOI: 10.4236/ajmb.2012.21001    3,644 Downloads   8,254 Views   Citations


Octameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function; in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized. Oxygen binding curves and CO kinetic studies indicate a correct allosteric transition of the tetramers within the octamer. Crystallographic studies of the two rHbs show two disulfide bonds per octamer. Reducing agents may provoke dissociation to tetramers, but the octamers are stable when mixed with fresh human plasma, indicating that the reduction by plasma is slower than the oxidation by the dissolved oxygen, consistent with an enhanced stability. The octameric rHbs were also mixed with a solution of haptoglobin (Hp), which binds the dimers of Hb: there was little interaction for incubation times of 15 min; however, on longer timescales a complex was formed. Dynamic light scattering was used to follow the interaction of Hp with the alpha Asn78Cys octamer during 24 hours; a transition from a simple complex of 15 nm to a final size of 60 nm was observed. The results indicate a specific orientation of the αβ dimers may be of importance for the binding to haptoglobin.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Brillet, T. , Marden, M. , Yeh, J. , Shen, T. , Ho, N. , Kettering, R. , Du, S. , Vasseur, C. , Domingues-Hamdi, E. , Ho, C. and Baudin-Creuza, V. (2012) Interaction of haptoglobin with hemoglobin octamers based on the mutation αAsn78Cys or βGly83Cys. American Journal of Molecular Biology, 2, 1-10. doi: 10.4236/ajmb.2012.21001.


[1] Dickerson, R.E. and Geis, I. (1983) Hemoglobin: Structure, function, evolution, and pathology. The Benjamin/Cummings Publishing Company, Menlo Park, CA.
[2] Sloan, E.P., Koenigsberg, M., Gens, D., Cipolle, M., Runge, J., Mallory, M.N. and Rodman, G. Jr. (1999) Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagic shock, a randomized controlled efficacy trial. Journal of the American Medical Association (JAMA), 282, 1857-1864. doi:10.1001/jama.282.19.1857
[3] Baudin-Creuza, V., Chauvierre, C., Domingues, E., Kiger, L., Leclerc, L., Vasseur, C., Célier C. and Marden, M.C. (2008) Octamers and nanoparticles as hemoglobin based blood substitutes. Biochimica et Biophysica Acta, 1784, 1448-1453.
[4] Fronticelli, C., Arosio, D., Bobofchak, K.M. and Vasquez, G.B. (2001) Molecular engineering of a polymer of tetrameric hemoglobins. Proteins, 44, 212-222. doi:10.1002/prot.1086
[5] Chauvierre, C., Manchanda, R., Labarre, D., Vauthier, C., Marden, M.C. and Leclerc, L. (2010) Artificial oxygen carrier based on polysaccharides-poly(alkylcyanoacrylates) nanoparticle templates. Biomaterials, 31, 6069-6074. doi:10.1016/j.biomaterials.2010.04.039
[6] Patrinos, G.P., Giardine, B., Riemer, C., Miller, W., Chui, D.H., Anagnou, N.P., Wajcman, H. and Hardison, R.C. (2004) Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. Nucleic Acids Research, 32, D537-541. doi:10.1093/nar/gkh006
[7] Blackwell, R.Q., Liu, C.S. and Wang, C.L. (1971) Hemoglobin Ta-Li, 83 Gly leads to Cys. Biochimica et Biophysica Acta, 243, 467-474.
[8] Fablet, C., Marden, M.C., Green, B.N., Ho, C., Pagnier, J. and Baudin-Creuza, V. (2003) Stable octameric structure of recombinant hemoglobin alpha(2)beta(2)83 Gly?Cys. Protein Science, 12, 690-695. doi:10.1110/ps.0234403
[9] Nagel, R.L. and Gibson Q.H. (1971) The binding of hemoglobin to haptoglobin and its relation to subunit dissociation of hemoglobin. Journal of Biological Chemistry, 246, 69-73.
[10] Kristiansen, M., Graversen, J.H., Jacobsen, C., Sonne, O., Hoffman, H.J., Law, S.K. and Moestrup, S.K. (2001) Identification of the haemoglobin scavenger receptor. Nature, 409, 198-201. doi:10.1038/35051594
[11] M?ller, H.J., Peterslund, N.A., Graversen, J.H. and Moes- trup, S.K. (2002) Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma. Blood, 99, 378-380. doi:10.1182/blood.V99.1.378
[12] Wejman, J.C., Hovsepian, D., Wall, J.S., Hainfeld, J.F. and Greer, J. (1984) Structure and assembly of haptoglobin polymers by electron microscopy. Journal of Molecular Biology, 174, 343-368. doi:10.1016/0022-2836(84)90342-5
[13] Shen, T.J., Ho, N.T., Simplaceanu, V., Zou, M., Green, B.N., Tam, M.F. and Ho, C. (1993) Production of unmodified human adult hemoglobin in Escherichia coli. Proceedings of the National Academy of Sciences USA, 90, 8108-8112. doi:10.1073/pnas.90.17.8108
[14] Shen, T.J., Ho, N.T., Zou, M., Sun, D.P., Cottam, P.F., Simplaceanu, V., Tam, M.F., Bell, D.A. and Ho, C. (1997) Production of human normal adult and fetal hemoglobins in Escherichia coli. Protein Engineering, 10, 1085-1097. doi:10.1093/protein/10.9.1085
[15] Marden, M.C., Kister, J., Bohn, B. and Poyart, C. (1988) T-state hemoglobin with four ligands bound. Biochemistry, 27, 1659-1664. doi:10.1021/bi00405a041
[16] Uzan, J., Dewilde, S., Burmester, T., Hankeln, T., Moens, L., Hamdane, D., Marden, M.C. and Kiger, L. (2004) Neuroglobin and other hexacoordinated hemoglobins show a weak temperature dependence of oxygen binding. Biophysical Journal, 87, 1196-1204. doi:10.1529/biophysj.104.042168
[17] Vasseur-Godbillon, C., Sahu, S.C., Domingues, E., Fablet, C., Giovannelli, J.L., Tam, T.C., Ho, N.T., Ho, C., Marden, M.C. and Baudin-Creuza, V. (2006) Recombinant hemoglobin betaG83C-F41Y. FEBS Journal, 273, 230- 241. doi:10.1111/j.1742-4658.2005.05063.x
[18] Pflugrath, J.W. (1999) The finer things in X-ray diffraction data collection. Acta Crystallographica Section D: Biological Crystallography, 55, 1718-1725. doi:10.1107/S090744499900935X
[19] Otwinowski, Z. and Minor, W. (1997) Processing of X-ray Diffraction Data Collected in Oscillation Method, Macromolecular Crystallography Part A. Methods Enzymology, 276, 307-326. doi:10.1016/S0076-6879(97)76066-X
[20] Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K. and Terwilliger, T.C. (2002) PHENIX, building new software for automated crystallographic structure determination. Acta Crystallographica Section D: Biological Crystallography, 58, 1948-1954. doi:10.1107/S0907444902016657
[21] Otwinowski, Z. and Minor, W. (1993) DENZO. A film processing program for macromolecular crystallography. Yale University, New Haven.
[22] Silva, M.M., Rogers, P.H. and Arnone, A. (1992) A third quaternary structure of human hemoglobin A at 1.7-A resolution. Journal of Biological Chemistry, 267, 17248- 17256.
[23] McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. and Read, R.J. (2005) Likelihood-enhanced fast translation functions. Acta Crystallographica Section D: Biological Crystallography, 61, 458-464. doi:10.1107/S0907444905001617
[24] Winn, M.D., Murshudov, G.N. and Papiz, M.Z. (2003) Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymology, 374, 300-321. doi:10.1016/S0076-6879(03)74014-2
[25] Brunger, A.T. (1992) Free R value, a novel statistical quantity for assessing the accuracy of crystal structures. Nature, 355, 472-475. doi:10.1038/355472a0
[26] Emsley, P. and Cowtan, K. (2004) Coot, model-building tools for molecular graphics. Acta Crystallographica Section D: Biological Crystallography, 60, 2126-2132. doi:10.1107/S0907444904019158
[27] Vaguine, A.A., Richelle, J. and Wodak, S.J. (1999) SFCHECK, a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallographica Section D: Biological Crystallography, 55, 191-205. doi:10.1107/S0907444998006684
[28] Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. (1993) PROCHECK, a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26, 283-291. doi:10.1107/S0021889892009944
[29] Collaborative Computational Project, Number 4. (1994) The CCP4 suite, programs for protein crystallography. Acta Crystallographica, D50, 760-763. doi:10.1107/S0907444994003112
[30] Nielsen, M.J. and Moestrup, S.K. (2009) Receptor targeting of hemoglobin mediated by the haptoglobins: roles beyond heme scavenging. Blood, 114, 764-771. doi:10.1182/blood-2009-01-198309

comments powered by Disqus

Copyright © 2019 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.