Bonafe C F, Araujo J R, Silva J L
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Brazil.
Biochemistry. 1994 Mar 8;33(9):2651-60. doi: 10.1021/bi00175a038.
Under physiological conditions, the oxygen-transport protein from the gastropod Megalobulimulus ovatus, an extracellular hemocyanin, is composed of 20 identical subunits organized into a cylindrical structure (M(r) 9 x 10(6); 100 S). It dissociates in the pressure range of 0.4-2.5 kbar, as observed by spectroscopic methods (light scattering and intrinsic fluorescence) and gel filtration. In contrast to what is seen with smaller proteins, especially dimers, the pressure-dissociation curves for hemocyanin show little dependence on concentration, suggesting that native hemocyanin exists as a population of molecules with different free energies of association. The pressure-induced dissociation results from an equilibrium in which each aggregate responds to pressure independently of the others and, at any given pressure, is in one of two states, whole or dissociated, which persists for long times when compared with the duration of the experiment. The subunit-subunit affinity of dissociated hemocyanin is much lower than that of associated subunits, suggesting that a conformational drift of monomers occurs. When hemocyanin undergoes dissociation in the absence of calcium and at high pH (> 7.2), a large fraction of the dissociated products changes to a conformation that generates stable intermediate states of assembly, lacking the ability to fully reassemble into decamers and didecamers. These intermediates consist primarily of dimers (M(r) 900,000), and they bind oxygen reversibly with a higher affinity than the native hemocyanin. The binding of calcium or protons changes the conformation back to the "associable" state, which finally generates the assembled structure. The dissociation process is highly reversible at low pH (6.8-6.0) or in the presence of millimolar concentrations of calcium. At pH 5.7, dissociation is negligible at pressures up to 2.5 kbar. A decrease in pH from 7.6 to 6.6 increases the half-dissociation pressure (p 1/2) by 1.3 kbar, corresponding to a stabilization of 1.35 kcal per mole of subunit. The effects of Ca2+ and H+ may mean that, in vivo, special ionic conditions or other factors are required to be present at the assembly sites of oligomeric proteins such as hemocyanin.
在生理条件下，腹足纲动物卵形巨泡螺的氧运输蛋白——一种细胞外血蓝蛋白，由20个相同的亚基组成，这些亚基组装成圆柱形结构（相对分子质量9×10⁶；100 S）。通过光谱方法（光散射和固有荧光）和凝胶过滤观察到，它在0.4 - 2.5 kbar的压力范围内会解离。与较小的蛋白质，尤其是二聚体不同，血蓝蛋白的压力解离曲线对浓度的依赖性很小，这表明天然血蓝蛋白以具有不同结合自由能的分子群体形式存在。压力诱导的解离源于一种平衡，在该平衡中，每个聚集体独立于其他聚集体对压力做出反应，并且在任何给定压力下，处于两种状态之一，即完整或解离状态，与实验持续时间相比，这种状态会持续很长时间。解离后的血蓝蛋白亚基间亲和力远低于结合状态的亚基，这表明单体发生了构象漂移。当血蓝蛋白在无钙且高pH（> 7.2）条件下发生解离时，很大一部分解离产物转变为一种构象，该构象产生稳定的组装中间状态，缺乏完全重新组装成十聚体和双十聚体的能力。这些中间体主要由二聚体（相对分子质量900,000）组成，它们以比天然血蓝蛋白更高的亲和力可逆地结合氧气。钙或质子的结合会使构象变回“可结合”状态，最终形成组装结构。在低pH（6.8 - 6.0）或存在毫摩尔浓度钙的情况下，解离过程是高度可逆的。在pH 5.7时，在高达2.5 kbar的压力下解离可忽略不计。pH从7.6降至6.6会使半解离压力（p 1/2）增加1.3 kbar，相当于每摩尔亚基稳定1.35千卡。Ca²⁺和H⁺的作用可能意味着，在体内，寡聚蛋白如血蓝蛋白的组装位点需要存在特殊的离子条件或其他因素。
