Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1982 Nov 1;207(2):297–303. doi: 10.1042/bj2070297

Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

E Ilan, E Weisselberg, E Daniel
PMCID: PMC1153860  PMID: 7159384

Abstract

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.

Full text

PDF
300

Images in this article

300-1PLATE 1
on p.300-1
300Fig. 3.
on p.300

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. CASASSA E. F., EISENBERG H. THERMODYNAMIC ANALYSIS OF MULTICOMPONENT SOLUTIONS. Adv Protein Chem. 1964;19:287–395. doi: 10.1016/s0065-3233(08)60191-6. [DOI] [PubMed] [Google Scholar]
  2. FOX H. M. Haemoglobin in the Crustacea. Nature. 1957 Jan 19;179(4551):148–148. doi: 10.1038/179148a0. [DOI] [PubMed] [Google Scholar]
  3. Ilan E., Daniel E. Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki Characterization of the molecule and determination of the number of polypeptide chains. Biochem J. 1979 Nov 1;183(2):325–330. doi: 10.1042/bj1830325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ilan E., David M. M., Daniel E. Erythrocruorin from the crustacean Caenestheria inopinata. Quaternary structure and arrangement of subunits. Biochemistry. 1981 Oct 13;20(21):6190–6194. doi: 10.1021/bi00524a043. [DOI] [PubMed] [Google Scholar]
  5. Klotz I. M., Langerman N. R., Darnall D. W. Quaternary structure of proteins. Annu Rev Biochem. 1970;39:25–62. doi: 10.1146/annurev.bi.39.070170.000325. [DOI] [PubMed] [Google Scholar]
  6. Lee J. C., Timasheff S. N. The calculation of partial specific volumes of proteins in 6 M guanidine hydrochloride. Methods Enzymol. 1979;61:49–57. doi: 10.1016/0076-6879(79)61006-6. [DOI] [PubMed] [Google Scholar]
  7. MONOD J., WYMAN J., CHANGEUX J. P. ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. J Mol Biol. 1965 May;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
  8. Matthews B. W., Bernhard S. A. Structure and symmetry of oligomeric enzymes. Annu Rev Biophys Bioeng. 1973;4:257–317. doi: 10.1146/annurev.bb.02.060173.001353. [DOI] [PubMed] [Google Scholar]
  9. McMEEKIN T. L., MARSHALL K. Specific volumes of proteins and the relationship to their amino acid contents. Science. 1952 Aug 8;116(3006):142–143. doi: 10.1126/science.116.3006.142. [DOI] [PubMed] [Google Scholar]
  10. Shlom J. M., Vinogradov S. N. A study of the subunit structure of the extracellular hemoglobin of Lumbricus terrestris. J Biol Chem. 1973 Nov 25;248(22):7904–7912. [PubMed] [Google Scholar]
  11. Sugano H., Hoshi T. Purification and properties of blood hemoglobin from the fresh-water cladocera, Moina macrocopa and Daphnia magna. Biochim Biophys Acta. 1971 Feb 16;229(2):349–358. doi: 10.1016/0005-2795(71)90194-2. [DOI] [PubMed] [Google Scholar]
  12. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  13. Wood E. J., Mosby L. J. Physicochemical properties of Planorbis corneus erythrocruorin. Biochem J. 1975 Aug;149(2):437–445. doi: 10.1042/bj1490437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. YPHANTIS D. A. EQUILIBRIUM ULTRACENTRIFUGATION OF DILUTE SOLUTIONS. Biochemistry. 1964 Mar;3:297–317. doi: 10.1021/bi00891a003. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES