Journal of comparative physiology

, Volume 128, Issue 2, pp 161–168 | Cite as

Hemocyanins in spiders

V. Fluorimetric recording of oxygen binding curves, and its application to the analysis of allosteric interactions inEurypelma californicum hemocyanin
  • Renate Loewe


  1. 1.

    Fluorescence (F) of the hemocyanin ofEurypelma californicum is strongly dependent on the degree of oxygenation (Fig. 2). Maximum excitation is found at 292 to 294 nm. There is only a small shift of maximum emission from 345 nm in oxygenated to 350 nm in deoxygenated hemocyanin, indicating that mainly tryptophan is responsible for oxygenation-dependent fluorescence (Fig. 3). Fluorescence enhancement depends linearly on the degree of deoxygenation (Fdeoxy/Foxy is about 16 at pH 7.4; Fig. 4).

  2. 2.

    Based on fluorescence quenching upon oxygenation, a fluorimetric-polarographic method for recording oxygen equilibrium curves was developed: With a favourable geometrical arrangement and low hemocyanin concentration, the error induced by reabsorption of emitted light is minimal (working range 0.02–0.2 mg/ml, corresponding to ca. 0.005–0.05 O.D. at 340 nm; Fig. 5). Data obtained by this method are in excellent agreement with data obtained by photometry (Figs. 6 and 7).

  3. 3.

    Oxygen affinity and cooperativity between oxygen binding sites ofEurypelma hemocyanin are strongly modified by protons: There is a very pronounced Bohr effect with a maximum between pH 8.0 and 8.4 (ΔlogP50/ΔpH=−1.2; Fig. 7). Cooperativity is maximal at about pH 8.0 (n50=7) and decreases towards low and high pH (Fig. 7). Oxygen affinity is independent of hemocyanin concentration, cooperativity, however, is slightly increased at high hemocyanin concentration.

  4. 4.

    Modification of oxygen affinity and cooperativity is interpreted in the framework of the Monod, Wyman and Changeux (1965) model. SinceK Tass andK Rass could not be estimated directly from the Hill plots, the intrinsic association constants of the first and the last oxygenation step,K1 andK24, were determined by means of a modified Scatchard plot (Edsall et al., 1954);K1=0.0036 mm Hg−1=0.0022×106 M−1;K24=2.69 mm Hg−1=1.636×106 M−1. With [T0]≫[R0],K1 representsK Tass , whereasK24 ([T0]≪[R0]) is equal toK Rass . From these constants, the MWC parameterc was calculated to be 0.00133 (c=K1/K24). The total free energy of interaction, ΔF1, is 3.9 kcal/site (25°C).



Tryptophan Association Constant Fluorescence Enhancement Scatchard Plot Total Free Energy 
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. Adair, G.S.: The hemoglobin system. VI. The oxygen dissociation curve of hemoglobin. J. Biol. Chem.63, 529–545 (1925)Google Scholar
  2. Antonini, E., Rossi-Fanelli, A., Caputo, A.: Studies on chlorocruorin I. The oxygen equilibrium ofSpirographis chlorocruorin. Arch. Biochem. Biophys.97, 336–342 (1962)Google Scholar
  3. Bannister, W.H., Wood, E.J.: Ultraviolet fluorescence ofMurex trunculus haemocyanin in relation to the binding of copper and oxygen. Comp. Biochem. Physiol.40B, 7–18 (1971)Google Scholar
  4. Brouwer, M., Bonaventura, C., Bonaventura, J.: Oxygen binding byLimulus polyphemus hemocyanin: Allosteric modulation by chloride ions. Biochemistry16, 3897–3902 (1977)Google Scholar
  5. Buc, H., Johannes, K.-J., Hess, B.: Appendix to: Allosteric kinetics of pyruvate kinase ofSaccharomyces carlsbergensis (by Johannes, K.-J., Hess, B.). J. Mol. Biol.76, 199–205 (1973)Google Scholar
  6. Colosimo, A., Brunori, M., Wyman, J.: Concerted changes in an allosteric macromolecule. Biophys. Chem.2, 338–344 (1974)Google Scholar
  7. Edsall, J.T., Felsenfeld, G., Goodman, D.S., Gurd, F.R.N.: The association of imidazole with the ions of zinc and cupric copper. J. Am. Chem. Soc.76, 3054–3058 (1954)Google Scholar
  8. Er-El, Z., Shaklai, N., Daniel, E.: Oxygen binding properties of haemocyanin fromLevantina hierosolina. J. Mol. Biol.64, 341–351 (1972)Google Scholar
  9. Imai, K., Morimoto, H., Kotani, M., Watari, H., Hirata, W., Kuroda, M.: Studies on the function of abnormal hemoglobins. I. An improved method for automatic measurement of the oxygen equilibrium curve of hemoglobin. Biochim. Biophys. Acta200, 189–196 (1970)Google Scholar
  10. Klarman, A., Shaklai, N., Daniel, E.: Tyrosyl fluorescence in hemocyanin from the scorpionLeiurus quinquestriatus. Biochim. Biophys. Acta490, 322–330 (1977)Google Scholar
  11. Kuiper, H.A., Antonini, E., Brunori, M.: Kinetic control of cooperativity in the oxygen binding ofPanulirus interruptus hemocyanin. J. Mol. Biol.115, 4–8 (1977)Google Scholar
  12. Loewe, R., Linzen, B.: Haemocyanins in spiders. I. Subunits and stability region ofDugesiella californica haemocyanin. Hoppe-Seyler's Z. Physiol. Chem.354, 182–188 (1973)Google Scholar
  13. Loewe, R., Linzen, B.: Haemocyanins in spiders. II. Automatic recording of oxygen binding curves, and the effect of Mg++ on oxygen affinity, cooperativity, and subunit association ofCupienius salei haemocyanin. J. comp. Physiol.98, 147–156 (1975)Google Scholar
  14. Long, C.: Biochemists' handbook, p. 33. London E. and F. Spon Ltd. 1961Google Scholar
  15. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem.193, 265–275 (1951)Google Scholar
  16. Markl, J., Schmid, R., Czichos-Tiedt, S., Linzen, B.: Haemocyanins in spiders, III. Chemical and physical properties of the proteins inDugesiella andCupienius blood. Hoppe-Seyler's Z. Physiol. Chem.354, 1713–1725 (1976)Google Scholar
  17. Miller, K., Van Holde, K.E.: Oxygen binding byCallianassa californiensis hemocyanin. Biochemistry13, 1668–1674 (1974)Google Scholar
  18. Monod, J., Wyman, J., Changeux, J.-P.: On the nature of allosteric transitions: A plausible model. J. Mol. Biol.12, 88–118 (1965)Google Scholar
  19. Roxby, R., Miller, K., Blair, D.P., Van Holde, K.E.: Subunits and association equilibria ofCallianassa californiensis hemocyanin. Biochemistry13, 1662–1668 (1974)Google Scholar
  20. Rubin, M.M., Changeux, J.-P.: On the nature of allosteric transitions: Implications of non-exclusive ligand binding. J. Mol. Biol.21, 265–274 (1966)Google Scholar
  21. Schneider, H.-J., Markl, J., Schartau, W., Linzen, B.: Subunit heterogeneity ofEurypelma (Dugesiella) hemocyanin, and separation of polypeptide chains. Hoppe-Seyler's Z. Physiol. Chem.358, 1133–1141 (1977)Google Scholar
  22. Shaklai, N., Daniel, E.: Fluorescence properties of hemocyanin fromLevantina hierosolima. Biochemistry13, 564–568 (1970)Google Scholar
  23. Van Driel, R.: Oxygen binding and subunit interactions inHelix pomatia hemocyanin. Biochemistry12, 2696–2698 (1973)Google Scholar
  24. Weber, G.: Enumeration of components in complex systems by fluorescence spectrophotometry. Nature (Lond.)190, 27–29 (1961)Google Scholar
  25. Wyman, J.: Reflections regarding hemoglobin. In: Oxygen affinity of hemoglobin and red cell acid base status. Proc. Alfred Benzon Symp. IV. Rorth, M., Astrup, P. (eds.), pp. 37–49. Copenhagen: Munksgaard 1972Google Scholar
  26. Zolla, L., Kuiper, H.A., Vecchini, P., Antonini, E., Brunori, M.: Dissociation and oxygen binding behaviour of β-hemocyanin fromHelix pomatia. (in press) (1978)Google Scholar

Copyright information

© Springer-Verlag 1978

Authors and Affiliations

  • Renate Loewe
    • 1
  1. 1.Zoologisches Institut der Universität MünchenMünchenFederal Republic of Germany

Personalised recommendations