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首页> 外文期刊>Biochemistry >Sol-gel trapping of functional intermediates of hemoglobin: geminate and bimolecular recombination studies.
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Sol-gel trapping of functional intermediates of hemoglobin: geminate and bimolecular recombination studies.

机译：血红蛋白功能中间体的溶胶-凝胶捕获：双链和双分子重组研究。

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摘要

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.
机译：蛋白质在多孔溶胶凝胶中的封装是一种有前途的技术，可用于产生，捕获和探测功能上重要的非平衡蛋白质种类。实现该目标所需的必要步骤是确定溶胶-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与人成年血红蛋白（HbA）的发芽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。生成两种形式的CO饱和封装的HbA。在一种情况下，命名为[COHbA]，可以直接封装平衡形式的COHbA。在第二种情况下，将其命名为[deoxyHbA] + CO，将平衡形式的deoxyHbA封装起来，只有在样品老化后，才通过多孔溶胶-凝胶基质将CO引入HbA。三种不同的制备方案用于生成两种形式的封装COHbA的溶胶凝胶。从这些封装样品中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，并且经过测试的封装方案之一在构象锁定方面要优越得多。该协议用于捕获和探测非平衡形式，例如HbA的配体T状态，该物种的特性需要充分探索HbA的变构作用。

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