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The Protein Journal

, Volume 33, Issue 4, pp 377–385 | Cite as

Purification, Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

  • Jinda Jandaruang
  • Jaruwan Siritapetawee
  • Chomphunuch Songsiriritthigul
  • Sutthidech Preecharram
  • Taoka Azuma
  • Apisak Dhiravisit
  • Yoshihiro Fukumori
  • Sompong Thammasirirak
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.

Keywords

Crocodylus siamensis Hemoglobin Crystal Oxygen affinity 

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

Notes

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.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jinda Jandaruang
    • 1
    • 2
  • Jaruwan Siritapetawee
    • 3
  • Chomphunuch Songsiriritthigul
    • 4
  • Sutthidech Preecharram
    • 5
  • Taoka Azuma
    • 6
  • Apisak Dhiravisit
    • 7
  • Yoshihiro Fukumori
    • 6
  • Sompong Thammasirirak
    • 1
    • 2
  1. 1.Department of Biochemistry, Faculty of ScienceKhon Kaen UniversityKhon KaenThailand
  2. 2.Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI)Khon Kaen UniversityKhon KaenThailand
  3. 3.School of Biochemistry, Institute of ScienceSuranaree University of TechnologyNakhon RatchasimaThailand
  4. 4.Synchrotron Light Research Institute (Public Organization)Nakhon RatchasimaThailand
  5. 5.Department of Microbiology, Faculty of Liberal Arts and ScienceKasetsart UniversityNakhon PathomThailand
  6. 6.Department of Life Science, Graduate School of Natural Science and TechnologyKanazawa UniversityKanazawaJapan
  7. 7.Faculty of Humanities and Social SciencesKhon Kaen UniversityKhon KaenThailand

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