title: Purification, characterization, and crystallization of Crocodylus siamensis hemoglobin.
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 (P(50)) 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 O(2) affinity at P(50) of 2.5 mmHg which may assure efficient utilization of the lung O(2) 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 MgCl(2)·6H(2)O] solution at a pH of 7.0-8.5.
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.
authors.forename: Jinda
authors.initials: J
authors.lastname: Jandaruang
authors.forename: Jaruwan
authors.initials: J
authors.lastname: Siritapetawee
authors.forename: Chomphunuch
authors.initials: C
authors.lastname: Songsiriritthigul
authors.forename: Sutthidech
authors.initials: S
authors.lastname: Preecharram
authors.forename: Taoka
authors.initials: T
authors.lastname: Azuma
authors.forename: Apisak
authors.initials: A
authors.lastname: Dhiravisit
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Fukumori
authors.forename: Sompong
authors.initials: S
authors.lastname: Thammasirirak
mesh_terms: D000255:Adenosine Triphosphate; D000489:Alligators and Crocodiles / Q000097:blood*; D000595:Amino Acid Sequence; D000818:Animals; D001639:Bicarbonates; D003460:Crystallization; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D058105:Polymerization; D055550:Protein Stability; D016415:Sequence Alignment
chemical_list: D001639:Bicarbonates; D006454:Hemoglobins; D000255:Adenosine Triphosphate