Jandaruang Jinda, Siritapetawee Jaruwan, Songsiriritthigul Chomphunuch, Preecharram Sutthidech, Azuma Taoka, Dhiravisit Apisak, Fukumori Yoshihiro, Thammasirirak Sompong
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.
Protein J. 2014 Aug;33(4):377-85. doi: 10.1007/s10930-014-9569-7.
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.
暹罗鳄血红蛋白通过尺寸排阻色谱法（使用含二硫苏糖醇的缓冲液的Sephacryl S - 100）进行纯化。纯化后的血红蛋白解离为两种形式（α链和β链），通过SDS - PAGE观察到，这表明暹罗鳄血红蛋白是未聚合的形式。未聚合的血红蛋白（由两条α链和两条β链组成）在3.13 mmHg（P(50)）时显示出高氧亲和力，n值为1.96，在pH值为6.9 - 8.4时具有较小的玻尔效应（δH(+) = -0.29）。三磷酸腺苷不影响氧合特性，而碳酸氢根离子强烈降低氧亲和力。暹罗鳄粗血红蛋白溶液在P(50)为2.5 mmHg时显示出高O(2)亲和力，这可能确保在屏气和潜水期间有效利用肺部的O(2)储备。纯化后的血红蛋白在结晶前转变为高铁血红蛋白形式。通过坐滴气相扩散法，在5°C下，使用等体积的蛋白质溶液（37 mg/ml）和储液[10 - 13%（w/v）PEG 4000，在0.2 M MgCl₂·6H₂O存在下的0.1 M Tris缓冲液]溶液，在pH值为7.0 - 8.5时获得了棒状和平板状晶体。
