Skip to main content

Purification, Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

The Protein Journal volume 33, pages 377–385 (2014)Cite this article

Abstract

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P50) and 1.96 (n value), and a small Bohr effect (δH+ = −0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O2 affinity at P50 of 2.5 mmHg which may assure efficient utilization of the lung O2 reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl2·6H2O] solution at a pH of 7.0–8.5.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

Hb:

Hemoglobin

HCO3 − :

Bicarbonate ions

TEM:

Transmission electron microscopy

DTT:

Dithiothreitol

ATP:

Adenosine triphosphate

pO2 :

Partial pressure of oxygen

P50 :

Partial pressure of oxygen at half saturation

%Y:

Percent saturation of the hemoglobin with oxygen

References

  1. Srihongthong S, Pakdeesuwan A, Daduang S, Araki T, Dhiravisit A, Thammasirirak S (2012) Complete amino acid sequence of globin chains and biological activity of fragmented crocodile hemoglobin (Crocodylus siamensis). Protein J 31:466–476

    CAS  Article  Google Scholar 

  2. Bauer C, Forster M, Gros G, Mosca A, Perrella M, Rollema HS, Vogel D (1981) Analysis of bicarbonate binding to crocodilian hemoglobin. J Biol Chem 256:8429–8435

    CAS  Google Scholar 

  3. Deepthi S, Johnson A, Sathish R, Pattabhi V (2000) Purification, crystallisation and preliminary X-ray study of haemoglobin from Crocodilis palustris and Crocodilis porosus. Biochim Biophys Acta 1480:384–387

    CAS  Article  Google Scholar 

  4. Jelkmann W, Bauer C (1979) Oxygen binding properties of Caiman blood in the absence and presence of carbon dioxide. Comp Biochem Physiol 65:331–336

    Article  Google Scholar 

  5. Riggs A, Sullivan B, Agee JR (1964) Polymerization of frog and turtle hemoglobins. Proc Natl Acad Sci U S A 51:1127–1134

    CAS  Article  Google Scholar 

  6. Pata S, Yaraksa N, Daduang S, Temsiripong Y, Svasti J, Araki T, Thammasirirak S (2011) Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Dev Comp Immunol 35:545–553

    CAS  Article  Google Scholar 

  7. Jandaruang J, Siritapetawee J, Thumanu K, Songsiriritthigul C, Krittanai C, Daduang S, Dhiravisit A, Thammasirirak S (2012) The Effects of temperature and pH on secondary structure and antioxidant activity of Crocodylus siamensis hemoglobin. Protein J 31:43–50

    CAS  Article  Google Scholar 

  8. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  Article  Google Scholar 

  9. Asano T, Nishiuchi T (2011) Comparative analysis of phosphoprotein expression using 2D-DIGE. Methods Mol Biol 744:225–233

    CAS  Article  Google Scholar 

  10. Asakura T, Kawai Y, Yoneyama Y, Yoshikawa H (1964) Use of sodium borohydride in determination of oxygen dissociation curves of hemoglobin. Anal Biochem 7:393–400

    CAS  Article  Google Scholar 

  11. Falk J (1964) Porphyrins and metalloporphyrins. Elsevier, Amsterdam

    Google Scholar 

  12. Hill AV (1910) The possible effects of the aggregation of the molecules of hæmoglobin on its dissociation curves. J Physiol 40:4–7

    Google Scholar 

  13. Dasgupta J, Sen U, Choudhury D, Datta P, Chakrabarti A, Chakrabarty SB, Chakrabarty A, Dattagupta JK (2003) Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E, isolated from the blood samples of beta-thalassemic patients. Biochem Biophys Res Commun 303:619–623

    CAS  Article  Google Scholar 

  14. Bauer C, Jelkmann W (1977) Carbon dioxide governs the oxygen affinity of crocodile blood. Nature 269:825–827

    CAS  Article  Google Scholar 

  15. Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry. W H Freeman, New York

    Google Scholar 

  16. Arnone A (1972) X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhaemoglobin. Nature 237:146–149

    CAS  Article  Google Scholar 

  17. Perutz MF (1983) Species adaptation in a protein molecule. Mol Biol Evol 1:1–28

    CAS  Google Scholar 

  18. Weber Roy E, White FN (1994) Chloride-dependent organic phosphate sensitivity of the oxygenation reaction in crocodile hemoglobins. J Exp Biol 192:1–11

    CAS  Google Scholar 

  19. Komiyama NH, Miyazaki G, Tame J, Nagai K (1995) Transplanting a unique allosteric effect from crocodile into human haemoglobin. Nature 373:244–246

    CAS  Article  Google Scholar 

  20. Perutz M, Bauer C, Gros G, Leclercq F, Vandecasserie C, Schnek AG, Braunitzer G, Friday AE, Joysey KA (1981) Allosteric regulation of crocodilian haemoglobin. Nature 291:682–684

    CAS  Article  Google Scholar 

  21. Aki Y, Nakagawa T, Nagai M, Sasayama Y, Fukumori Y, Imai K (2007) Oxygenation properties of extracellular giant hemoglobin from Oligobrachia mashikoi. Biochem Biophys Res Commun 360:673–678

    CAS  Article  Google Scholar 

  22. Perutz MF, Fermi G, Poyart C, Pagnier J, Kister J (1993) A novel allosteric mechanism in haemoglobin. Structure of bovine deoxyhaemoglobin, absence of specific chloride-binding sites and origin of the chloride-linked Bohr effect in bovine and human haemoglobin. J Mol Biol 233:536–545

    CAS  Article  Google Scholar 

Download references

Acknowledgments

This investigation was supported by The Royal Golden Jubilee (RGJ) PhD Program of Thailand Research Fund; the Synchrotron Light Research Institute (Public Organization), Thailand; the Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Thailand; and the Biomolecular Physiology Laboratory, Department of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Japan. We would like to thank Dr. Yayoi Aki for her proof of the oxygen affinity calculation and Dr. Isao Nishiuchi for the tandem mass spectrometry analysis.

Author information

Affiliations

  1. Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand

    Jinda Jandaruang & Sompong Thammasirirak

  2. Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand

    Jinda Jandaruang & Sompong Thammasirirak

  3. School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand

    Jaruwan Siritapetawee

  4. Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima, 30000, Thailand

    Chomphunuch Songsiriritthigul

  5. Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand

    Sutthidech Preecharram

  6. Department of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Ishikawa, 920-1192, Japan

    Taoka Azuma & Yoshihiro Fukumori

  7. Faculty of Humanities and Social Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand

    Apisak Dhiravisit

Authors
  1. Jinda Jandaruang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jaruwan Siritapetawee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chomphunuch Songsiriritthigul
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sutthidech Preecharram
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Taoka Azuma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Apisak Dhiravisit
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yoshihiro Fukumori
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sompong Thammasirirak
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yoshihiro Fukumori or Sompong Thammasirirak.

Additional information

Yoshihiro Fukumori and Sompong Thammasirirak contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Jandaruang, J., Siritapetawee, J., Songsiriritthigul, C. et al. Purification, Characterization, and Crystallization of Crocodylus siamensis Hemoglobin. Protein J 33, 377–385 (2014). https://doi.org/10.1007/s10930-014-9569-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-014-9569-7

Keywords

  • Crocodylus siamensis
  • Hemoglobin
  • Crystal
  • Oxygen affinity