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Subunit Distribution of Calcium-Binding Sites in Lumbricus Terrestris Hemoglobin

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Abstract

The giant, ∼3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36–42 linkers L1–L4 (24–32 kDa). It is known to contain 50–80 Ca and 2–4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH ∼2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca2+ and all the Cu and Zn, the globin subassembly had 0.4 to ∼3 Ca2+, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH ∼2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH ∼2.2 and at neutral pH demonstrated avid binding of 1 Ca2+ and additional weaker binding of 7 Ca2+ in the presence of added Ca2+. Based on these and previous results which document the heterogeneous nature of the Ca2+-binding sites in Lumbricus Hb, we propose three classes of Ca2+-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) ∼30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) ∼25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1–100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.

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REFERENCES

  • Antonini, A., and Chiancone, E. (1977). Annu. Rev. Biophys. Bioeng. 6, 239–271.

    Article  CAS  PubMed  Google Scholar 

  • Blacklow, S., and Kim, P. S. (1996). Nature Struct. Biol. 3, 758–761.

    Article  CAS  PubMed  Google Scholar 

  • Chiancone, E., Bull, T. E., Norne, J. E., Forsen, S., and Antonini, E. (1976). J. Mol. Biol. 107, 25–34.

    Article  CAS  PubMed  Google Scholar 

  • Chiancone, E., Ferruzzi, G., Bonaventura, C., and Bonaventura, J. (1980). Biochim. Biophys. Acta 670, 84–92.

    Article  Google Scholar 

  • Chottard, G., El Ajouz, N., and Hervé, G (1992). Biochim. Biophys. Acta 1122, 113–117.

    Article  CAS  PubMed  Google Scholar 

  • Chung, M. C. C., and Ellerton, H. D. (1979). Progr. Biophys. Mol. Biol. 35, 51–102.

    Google Scholar 

  • De Haas, F., Taveau, J.-C., Boisset, N., Lambert, O., Vinogradov, S. N., and Lamy, J. N. (1996a). J. Mol. Biol. 255, 140–163.

    Article  CAS  PubMed  Google Scholar 

  • De Haas, F., Taveau, J.-C., Boisset, N., Lambert, O., Vinogradov, S. N., and Lamy, J. N. (1996b). Biophys. J. 70, 1973–1984.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • De Haas, F., Zal, F., Lallier, F. H., Toulmond, A., and Lamy, J. N. (1996c). Proteins Struct. Funct. Genet. 26, 241–256.

    Article  CAS  PubMed  Google Scholar 

  • De Haas, F., Zal, F., You, V., Lallier, F., Toulmond, A., and Lamy, J. N. (1996d). J. Mol. Biol. 264, 111–120.

    Article  CAS  PubMed  Google Scholar 

  • De Haas, F., Kuchumov, A., Taveau, J.-C., Boisset, N., Vinogradov, S. N., and Lamy, J. N. (1997). Biochemistry 36, 7330–7338.

    Article  CAS  PubMed  Google Scholar 

  • Fass, D., Blacklow, S., Kim, P. S., and Berger, J. M. (1997). Nature 388, 691–693.

    Article  CAS  PubMed  Google Scholar 

  • Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1980). Science 246, 64–71.

    Article  Google Scholar 

  • Fushitani, K., Higashiyama, K., Asao, M., and Hosokawa, K. (1996). Biochim. Biophys. Acta 1292, 273–280.

    Article  PubMed  Google Scholar 

  • Gotoh, T., and Suzuki, T. (1990). Zool. Sci. 7, 1–16.

    CAS  Google Scholar 

  • Green, B. G., Bordoli, R. S., Hanin, L. G., Lallier, F. H., Toulmond, A., and Vinogradov, S. N. (1999). J. Biol. Chem. 274, 28206–2812.

    Article  CAS  PubMed  Google Scholar 

  • Harrington, J. P. (1994). Comp. Biochem. Physiol. 109A, 799–802.

    Article  CAS  Google Scholar 

  • Krebs, A., Kuchumov, A. R., Shaarma, P. K., Braswell, E. H., Zipper, P., Weber, R. E., Chottard, G., and Vinogradov, S. N. (1996). J. Biol. Chem. 271, 18695–18704.

    Article  CAS  PubMed  Google Scholar 

  • Kuchumov, A. R., Taveau, J.-C., Lamy, J. N., Wall, J. S., Weber, R. E. and Vinogradov, S. N. (1999). J. Mol. Biol. 289, 1361–1374.

    Article  CAS  PubMed  Google Scholar 

  • Lamy, J. N., Green, B. N., Toulmond, A., Walls, J. S., Weber, R. E., and Vinogradov, S. N. (1996). Chem. Rev. 96, 3113–3124.

    Article  CAS  PubMed  Google Scholar 

  • Liochev, S. I., Kuchumov, A. R., Vinogradov, S. N., and Fridovich, I. (1996). Arch. Biochem. Biophys. 330, 281–284.

    Article  CAS  PubMed  Google Scholar 

  • Loo, J. A., and Pesch, R. (1994). Anal. Chem. 66, 3659–3663.

    Article  CAS  Google Scholar 

  • Loo, J. A., Edmunds, C. G., and Smith, R. D. (1993). Anal. Chem. 65, 4225–4238.

    Article  Google Scholar 

  • Mainwaring, M. G., Lugo, S. D., Fingal, R. A., Kapp, O. H., and Vinogradov, S. N. (1986). J. Biol. Chem. 261, 10899–10908.

    Article  CAS  PubMed  Google Scholar 

  • Martin, P. D., Eisele, K. L., Doyle, M. A., Kuchumov, A. R., Walz, D. A., Arutyunyan, E. G., Vinogradov, S. N., and Edwards, B. F. P. (1996). J. Mol. Biol. 255, 170–175.

    Article  CAS  PubMed  Google Scholar 

  • Ochiai, T. (1984). Arch. Biochem. Biophys. 231, 136–143.

    Article  CAS  PubMed  Google Scholar 

  • Ochiai, T., Hoshina, S., and Usuki, I. (1993). Biochim. Biophys. Acta 1203, 310–314.

    Article  CAS  PubMed  Google Scholar 

  • Ownby, D. W., Zhu, H., Schneider, K., Beavis, R. C., Chait, B. T., and Riggs, A. F. (1993). J. Biol. Chem. 268, 13539–13547.

    Article  CAS  PubMed  Google Scholar 

  • Perutz, M. (1990). Mechanisms of Cooperativity and Allosteric Regulation in Proteins, Cambridge University Press, Cambridge.

    Google Scholar 

  • Polidori, G., Maionwaring, M. G., Kosinski, T., Schwarz, C., Fingal, R. A., and Vinogradov, S. N. (1984). Arch. Biochem. Biophys. 233, 800–814.

    Article  CAS  PubMed  Google Scholar 

  • Polidori, G., Mainwaring, M. G., and Vinogradov, S. N. (1988). Comp. Biochem. Physiol. 89A, 541–545.

    Article  CAS  Google Scholar 

  • Rokosz, M. J., and Vinogradov, S. N. (1982). Biochim. Biophys. Acta 707, 291–293.

    Article  CAS  PubMed  Google Scholar 

  • Royer, W. E., Jr., Pardanani, A., Gibson, Q. E., Peterson, E. S., and Friedman, J. M. (1996). Proc. Natl. Acad. Sci. USA 93, 14526–14531.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schatz, M., Orlova, E. V., Dube, P., Jäger, J., and Van Heel, M. (1995). J. Struct. Biol. 114, 28–40.

    Article  CAS  PubMed  Google Scholar 

  • Senko, M. W., and McLafferty, F. W. (1994). Annu. Rev. Biophys. Biomol. Struct. 23, 763–785.

    Article  CAS  PubMed  Google Scholar 

  • Sharma, P. K., Kuchumov, A., Chottard, G., Martin, P. D., Wall, J. S., and Vinogradov, S. N. (1996). J. Biol. Chem. 271, 8754–8762.

    Article  CAS  PubMed  Google Scholar 

  • Smith, R. M., and Martell, A. E. (1989). Critical Stability Constants, Vol. 6, Plenum Press, New York, p. 96.

    Book  Google Scholar 

  • Standley, P. M., Mainwaring, M. G., Gotoh, T., and Vinogradov, S. N. (1988). Biochem. J. 249, 915–916.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suzuki, T., and Riggs, A. F. (1993). J. Biol. Chem. 268, 1354–1361.

    Google Scholar 

  • Suzuki, T., Takagi, T., and Ohta, S. (1990a). J. Biol. Chem. 265, 1551–1555.

    Article  CAS  PubMed  Google Scholar 

  • Suzuki, T., Takagi, T., and Gotoh, T. (1990b). J. Biol. Chem. 265, 12168–12177.

    Article  CAS  PubMed  Google Scholar 

  • Suzuki, T., Ohta, T., Yasa, H. J., and Takagi, T. (1994). Biochim. Biophys. Acta 1217, 291–296.

    Article  CAS  PubMed  Google Scholar 

  • Taveau, J.-C., Boisset, N, Vinogradov, S. N., and Lamy, J. N. (1999). J. Mol. Biol. 289, 1343–1349.

    Article  CAS  PubMed  Google Scholar 

  • Tsuneshige, A., Imai, K., Hori, H., Tyuma, I., and Gotoh, T. (1989). J. Biochem. 106, 406–417.

    Article  CAS  PubMed  Google Scholar 

  • Vinogradov, S. N., Shlom, J. M., Hall, B. C., Kapp, O. H., and Mizukami, H. (1977). Biochim. Biophys. Acta 492, 136–155.

    Article  CAS  PubMed  Google Scholar 

  • Vinogradov, S. N. (1985). In Respiratory Pigments in Animals (Lamy, J., Truchot, J. P., and Gilles, R., eds.), Springer-Verlag, Berlin. pp. 9–20.

    Chapter  Google Scholar 

  • Vinogradov, S. N., and Sharma, P. K. (1994). Meth. Enzmol. 231, 112–124.

    Article  CAS  Google Scholar 

  • Vinogradov, S. N., Kapp, O. H., and Ohtsuki, M. (1982). Electron Microscopy of Proteins, Vol. 3 (Harris, J., ed.), Academic Press, New York. p. 135.

    Google Scholar 

  • Vinogradov, S. N., Standley, P. R., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1985). Biochim. Biophys. Acta 828, 43–50.

    Article  CAS  PubMed  Google Scholar 

  • Vinogradov, S. N., Lugo, S. D., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986). Proc. Natl. Acad. Sci. USA 83, 8034–8038.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vinogradov, S. N., Sharma, P. K., Qabar, A. N., Wall, J. S., Westrick, J. A., Simmons, J. H., and Gill, S. J. (1991). J. Biol. Chem. 266, 13091–13096.

    Article  CAS  PubMed  Google Scholar 

  • Vinogradov, S. N., Walz, D. A., Pohajdak, B., Moens, L., Kapp, O. H., Suzuki, T., and Trotman, C. N. A. (1993). Comp. Biochem. Physiol. 106B, 1–26.

    CAS  Google Scholar 

  • Weber, R. E., and Vinogradov, S. N. (2000). Physiol. Rev. (in press).

  • Zhu, H., Hargrove, M., Xie, Q., Nozaki, Y., Linse, K., Smith, S. S., Olson, J. S., and Riggs, A. F. (1996a). J. Biol. Chem. 271, 29999–30006.

    Article  CAS  PubMed  Google Scholar 

  • Zhu, H., Ownby, D. W., Riggs, C. K., Nolasco, N. J., Stoops, J. K., and Riggs, A. F. (1996b). J. Biol. Chem. 271, 30007–30021.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan, 48201

    Askar R. Kuchumov

  2. Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan, 48105

    Joseph A. Loo

  3. Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan, 48201

    Serge N. Vinogradov

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  1. Askar R. Kuchumov
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  2. Joseph A. Loo
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  3. Serge N. Vinogradov
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Kuchumov, A.R., Loo, J.A. & Vinogradov, S.N. Subunit Distribution of Calcium-Binding Sites in Lumbricus Terrestris Hemoglobin. J Protein Chem 19, 139–149 (2000). https://doi.org/10.1023/A:1007086717412

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