Moorthy Ponnuraj Sathya, Neelagandan Kamariah, Balasubramanian Moovarkumudalvan, Ponnuswamy Mondikalipudur Nanjappa Gounder
Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai - 600 025, India.
Protein Pept Lett. 2009;16(4):454-6. doi: 10.2174/092986609787847992.
Hemoglobin is a vital protein present in almost all higher species. It is a transport protein involved in carrying oxygen from lungs to tissues and carbon dioxide back to lungs by an intrinsically coordinated manner. Even though a good amount of work has been carried out in this direction there exists scarcity of structural insight on low oxygen affinity species. Attempts are being made to unravel the structural insight of this low oxygen affinity species. Goat blood plasma was collected, treated with EDTA to avoid blood clotting and purification was accomplished using DEAE-anion chromatographic column. The goat hemoglobin was crystallized using 50mM of phosphate buffer at pH 6.7 with 1M NaCl and PEG 3350 as precipitant by hanging drop vapor diffusion method. Crystals obtained are screened and suitable crystals are taken for data collection using mar345dtb as image plate detector system. Goat hemoglobin crystal diffracted up to 2.61 A resolution. Goat hemoglobin crystallizes in orthorhombic space group P212(1)2(1) as a whole biological molecule in the asymmetric unit with cell dimensions a=53.568A, b=67.365A, c=154.183A.
血红蛋白是几乎所有高等物种中都存在的一种重要蛋白质。它是一种运输蛋白，通过内在协调的方式参与将氧气从肺部输送到组织，并将二氧化碳带回肺部。尽管在这个方向上已经开展了大量工作，但对于低氧亲和力物种的结构认识仍然匮乏。目前正在尝试揭示这种低氧亲和力物种的结构奥秘。采集山羊血浆，用乙二胺四乙酸（EDTA）处理以避免血液凝固，然后使用二乙氨基乙基（DEAE）阴离子色谱柱进行纯化。通过悬滴气相扩散法，使用pH 6.7的50mM磷酸盐缓冲液、1M氯化钠和聚乙二醇3350（PEG 3350）作为沉淀剂，使山羊血红蛋白结晶。对获得的晶体进行筛选，选取合适的晶体使用mar345dtb作为图像板探测器系统进行数据收集。山羊血红蛋白晶体的衍射分辨率达到2.61埃。山羊血红蛋白在正交空间群P212(1)2(1)中结晶，在不对称单元中作为一个完整的生物分子，晶胞参数为a = 53.568埃，b = 67.365埃，c = 154.183埃。
