Dissociation and denaturation equilibria and kinetics of a homogeneous human immunoglobulin Fab fragment.
Dissociation and denaturation equilibria and kinetics of a homogeneous human immunoglobulin Fab fragment.
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同质人免疫球蛋白 Fab 片段的解离和变性平衡以及动力学。
DOI：
10.1021/bi00649a028
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发表时间：
1976
期刊：
影响因子：
2.9
通讯作者：
E. Rowe
中科院分区：
文献类型：
--
作者：
E. Rowe
研究了IgG1骨髓瘤（Wes）Fab片段（SSFab）及其轻度还原和S-羧酰胺甲基化衍生物（RAFab）的构象平衡和接近平衡的动力学，作为盐酸胍浓度的函数。先前报道的SSFab的单分子变性、RAFab的双分子变性和Wes L链的变性（Rowe，E.美国，Tanford，C.，（1973），Biochemistry 12，4822）中所描述的方法，根据结构域结构进行解释，并根据蛋白质的热力学稳定性及其亚基之间的共价和非共价相互作用进行评价。发现Fd-L相互作用非常强，并且保持在足以使各个结构域变性的变性剂浓度下。结果表明，所有的数据是一致的两个区域的结构的Fab，一个由vL和vH结构域，和其他的cL和cH结构域组成，所以有两个网站的非共价Fd-L相互作用。其中一个区域被鉴定为C区，其稳定性是另一个区域的10（2）-10（4）倍;这种稳定性差异主要归因于该区域结构域之间更强和更广泛的相互作用。发现在过渡中心接近平衡的动力学极其缓慢，RAFab需要长达一周的平衡，SSFab需要数月。这种不寻常的动力学行为被证明是由于强大的Fd-L相互作用的条件下，单体域是不稳定的。
The conformational equilibria and the kinetics of the approach to equilibrium of an IgG1 myeloma (Wes) Fab fragment (SSFab) and its mildly reduced and S-carboxyamidomethylated derivative (RAFab) were studied as a function of guanidine hydrochloride concentration. The unimolecular denaturation of SSFab, the bimolecular denaturation of RAFab, and the denaturation of Wes L chain reported previously (Rowe, E. S., and Tanford, C., (1973), Biochemistry 12, 4822) are interpreted in terms of the domain structure and evaluated in terms of the thermodynamic stability of the protein and the covalent and noncovalent interactions among its subunits. The Fd-L interactions are found to be extremely strong and are maintained at concentrations of denaturant sufficient to denature the individual domains. It is shown that all of the data are consistent with a two region structure for Fab, one composed of the vL and vH domains, and the other composed of the cL and cH domains, so that there are two sites of noncovalent Fd-L interactions. One region, identified as the C region, is found to be 10(2)-10(4) times more stable than the other; this difference in stability is attributed largely to a stronger and more extensive interaction between the domains of this region. The kinetics of the approach to equilibrium are found to be extremely slow in the center of the transitions, requiring up to a week for equilibration for RAFab, and several months for SSFab. This unusual kinetic behavior is shown to be due to the strong Fd-L interaction under conditions where the monomeric domains are unstable.