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NMR Studies on Molecular Mobility of Protein of Hen Egg White Lysome in Agarose Gels
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摘要: 运用脉冲梯度场测量自扩散系数及自旋自旋弛豫时间测量核磁共振技术对鸡蛋清溶菌酶(HEWL)在琼脂糖凝胶中的动力学进行了研究.试验结果表明, HEWL在琼脂糖凝胶中的自扩散系数及自旋自旋弛豫时间较其在纯乙酸钠溶液中变小, 说明琼脂糖凝胶的三维网状结构使HEWL分子整体运动及局部运动都受到阻碍.并且随着琼脂糖浓度的增大, 凝胶网孔尺寸不断减小, HEWL分子运动受限程度加剧, 从而蛋白质分子可以较长时间内停留在高浓度区, 分子间更容易互相碰撞, 发生反应, 晶核生长得以促进.同时琼脂糖凝胶较小流体力学网孔尺寸抑制聚晶或沉淀的出现, 晶体质量获得提高.Abstract: Changes in the molecular mobility of hen egg white lysome(HEWL)in aqueous solution and in agarose gel were measured by pulsed-field-gradient stimulated echo (PFG-STE) 1H NMR and spin-spin relaxation time(T2) 1H NMR methods in order to elucidate the effect of agarose gel on the crystallization of protein. Both the diffusion coefficient of HEWL(DHEWL) and T2 of HEWL showed a steep decrease with the increase of agarose concentration. These results suggested that both the local molecular motion and translational molecular motion were impeded by the three-dimensional polymeric network of agarose gel, with the result of a higher local concentration of HEWL in agarose gel. The low diffusion of HEWL molecules in agarose gel probably retained the higher local concentration for a longer time, which enhanced the nucleation. In addition, in the supersaturated solution, HEWL clusters diffused more slowly than monomers due to the large hydrodynamic interaction with the agarose matrix and their small hydrodynamic radius, which promoted the crystallization of the HEWL protein.
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Key words:
- agarose gel /
- NMR /
- HEWL /
- protein crystallization
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图 1 PFG-STE(a)和CPMG-SE(b)脉冲序列
Figure 1. PFG-STE(a)pulse sequence and CPMG-spin-echo(b)sequence with gradients
图 3 HEWL在乙酸钠溶液(a)、质量分数为1.6%琼脂糖凝胶(b)中的1H PFG-STENMR堆积谱
Figure 3. Stacked1H PFG-STE NMR spectra for HEWL solutions without (a) and with (b) 1.6w% agarose gel
图 4 HEWL的γ2g2δ2(Δ -δ/3)对积分强度的自然对数值作图
■ 0.1 mol/L乙酸钠溶液，△质量分数为0.4%琼脂糖凝胶，▲质量分数为1.2%琼脂糖凝胶
Figure 4. Diffusional spin-echo attenuations of HEWL in 0.1 mol/L sodium acetate (■), 0.4wt% agarose gel (△) and 1.2wt% agarose gel (▲) vs field gradient strength
图 5 HEWL在0.1 mol/L乙酸钠溶液及琼脂糖凝胶中存在形式示意图
Figure 5. Schematic representations of microscopic environments of HEWL in 0.1 mol/L sodium acetate and in agarose gel
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