Sol-gel trapping of functional intermediates of hemoglobin: Geminate and bimolecular recombination studies
Sol-gel trapping of functional intermediates of hemoglobin: Geminate and bimolecular recombination studies
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DOI：
10.1021/bi000536x
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发表时间：
2000-12-26
期刊：
影响因子：
2.9
通讯作者：
Friedman, JM
中科院分区：
文献类型：
--
作者：
Khan, I;Shannon, CF;Friedman, JM
将蛋白质封装在多孔溶胶-凝胶中是一种有前途的技术，用于生成、捕获和探测具有功能意义的非平衡蛋白质种类。实现这一目标所需的一个重要步骤是确定溶胶-凝胶在添加或去除底物时限制构象变化的程度。在本研究中，CO 与成人血红蛋白 (HbA) 的成对重组和溶剂相双分子重组被用作溶胶-凝胶内构象约束程度的敏感探针。产生两种形式的 CO 饱和封装的 HbA。在一种指定为[COHbA]的情况下，COHbA的平衡形式被直接封装。在第二种情况下，指定为[脱氧HbA] + CO，脱氧HbA的平衡形式被封装，只有在样品老化后，CO才会通过多孔溶胶-凝胶基质引入到HbA中。使用三种不同的制备方案来生成两种形式的封装 COHbA 中的每一种的溶胶-凝胶。从这些封装样品获得的动力学轨迹可以轻松评估溶胶-凝胶在初始三级/四级结构中的锁定程度。结果表明，溶胶-凝胶封装的样品可以与脉冲激光源一起使用，并且所测试的封装方案之一在构象锁定方面要优越得多。该方案用于捕获和探测非平衡形式，例如 HbA 的配体 T 态，这种物质的特性是充分探索 HbA 变构所必需的。
The encapsulation of proteins in porous sol-gels is a promising technique for generating, trapping, and probing functionally significant nonequilibrium protein species. An essential step needed in the pursuit of that goal is establishing the degree to which the sol-gel limits conformational change upon adding or removing substrates. In the present study, geminate recombination and solvent phase bimolecular recombination of CO to human adult hemoglobin (HbA) are used as sensitive probes of the degree of conformational constraint within the sol-gel. Two forms of CO saturated encapsulated HbA are generated. In one case, designated [COHbA], the equilibrium form of COHbA is directly encapsulated. In the second case, designated as [deoxyHbA] + CO, the equilibrium form of deoxyHbA is encapsulated and only after the sample has aged is CO introduced to the HbA through the porous sol-gel matrix. Three different preparative protocols are used to generate the sol-gels for each of the two forms of encapsulated COHbA. The kinetic traces obtained from these encapsulated samples allow for an easy evaluation of the extent to which the sol-gel is locking in the initial tertiary/quaternary structure. The results show that the sol-gel encapsulated samples can be used with pulsed laser sources and that one of the tested encapsulation protocols is far superior with respect to conformational locking. This protocol is used to trap and probe nonequilibrium forms such as the liganded T state of HbA, a species whose properties are needed to fully explore allostery in HbA.