Bismuto E, Sirangelo I, Irace G, Gratton E
Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Italy.
Biochemistry. 1996 Jan 30;35(4):1173-8. doi: 10.1021/bi951163g.
The nature of the structural changes that apomyoglobin undergoes when subjected to hydrostatic pressure, ranging from atmospheric pressure to 2.4 kbar, has been investigated by steady-state fluorescence and frequency domain fluorometry. In particular, we have examined the intrinsic tryptophanyl emission and that of the extrinsic probe 1-anilino-8-naphthalenesulfonate (ANS) bound to apomyoglobin at neutral pH, as well as at strongly acidic high-salt conditions. Apomyoglobin at neutral pH undergoes a pressure-induced structural transition, which causes the disorganization of the heme binding region with a consequent ANS dissociation; a concomitant increase in solvent accessibility to the N-terminus of the macromolecule in which tryptophans are located is also observed. At 2.4 kbar, the tryptophanyl emission is not coincident with that of a fully solvent exposed residue, thus suggesting that the N-terminal region of the apomyoglobin molecule retains elements of organized structure. The spectroscopic properties of the structural state attained at 2.4 kbar and neutral pH are different from those of the acidic compact state. The acidic compact state of apomyoglobin undergoes a pressure-induced structural change that brings the tryptophanyl residues in contact with the solvent, but does not affect the ability to bind ANS.
利用稳态荧光和频域荧光法研究了脱辅基肌红蛋白在从大气压到2.4千巴的静水压力下所经历的结构变化的性质。具体而言，我们研究了中性pH以及强酸性高盐条件下，脱辅基肌红蛋白自身色氨酸的荧光发射，以及与脱辅基肌红蛋白结合的外在探针1-苯胺基-8-萘磺酸盐（ANS）的荧光发射。中性pH条件下的脱辅基肌红蛋白会发生压力诱导的结构转变，这会导致血红素结合区域的无序化，进而导致ANS解离；同时还观察到，大分子中色氨酸所在的N端对溶剂的可及性增加。在2.4千巴时，色氨酸的荧光发射与完全暴露于溶剂中的残基的荧光发射不一致，因此表明脱辅基肌红蛋白分子的N端区域保留了有序结构的元素。在2.4千巴和中性pH条件下达到的结构状态的光谱性质与酸性致密状态的光谱性质不同。脱辅基肌红蛋白的酸性致密状态会发生压力诱导的结构变化，使色氨酸残基与溶剂接触，但不影响其结合ANS的能力。
