Pressure-Volume Properties of Molten Globule

关于牛α-乳清蛋白典型和经典熔融球的体积行为，我们已经获得了以下结果：虽然α-乳清蛋白的体积在熔融球天然构象变化期间减小（ΔV[约等于] - 60 cm^3/mol），但从熔融球到完全展开状态的体积接近0 cm^3/mol（Kobashigawa et al.，蛋白质科学，8，2765-2772（1999））。蛋清溶菌酶不具有稳定的熔融球，其解折叠和再折叠均为双态机制，在100 Mpa压力下的解折叠行为不能用双态机制来解释，尤其是在较低温度（0 ~ 20℃）下。这表明，体积行为是有点复杂。鸡蛋清溶菌酶的熔融球在压力下是稳定的。犬乳溶菌酶显示非常稳定的熔融球。用差示扫描量热法研究了犬乳溶菌酶的热行为，结果表明，从天然状态到熔融球状态和熔融球状态到未折叠状态的构象变化是明显分开的。这是因为与α-乳白蛋白和常规溶菌酶相比，犬乳溶菌酶的熔融球状态非常稳定。突变研究表明，这种稳定性来源于His 21、Ile 56、Ala 93和Val 109在熔融球状态下形成的疏水簇。由于稳定性的来源是疏水相互作用，因此预期在从熔融小球到未折叠状态的构象变化期间体积增加。然而，由于仪器在更高温度下缺乏稳定性，我们还没有在实验上证明这一点。

We had already obtained the following result concerning to the volumetric behavior of the typical and classical molten globule of bovine α-lactalbumin : although the volume of α-lactalbumin decreased during the conformational change ftom native to molten-globule (ΔV【approximately equal】 - 60 cm^3/mol)s, that from molten-globule to fully-unfolded state was nearly 0 cm^3/mol (Kobashigawa et al., Protein Sci., 8, 2765-2772(1999)). In the case of egg-white lysozyme, which does not show stable molten-globule and unfolds and refolds with two-state mechanism, the unfolding behavior under the pressure up to 100 Mpa cannot be explained with two-state mechanism, especially at lower temperature (0 〜 20℃). It suggests that the volumetric behavior is somewhat complicated. The molten globule of hen egg-white lysozyme is stabilized under pressure. Canine milk lysozyme shows extremely stable molten globule. The thermal behavior of canine milk lysozyme was investigated with differential scanning calorimetry and it is shown that the conformational changes from native to molten-globule and molten-globule to unfolded state are clearly separated to each other. This is because that the molten-globule state of canine milk lysozyme is extremely stable as compared to those of α-lactalbumins and conventional lysozymes. From mutational investigation, this stability is originated from the formation of hydrophobic cluster of His21, Ile56, Ala93 and Val109 in molten-globule state. As the origin of the stability is hydrophobic interaction, the volume is anticipated to increase during the conformational change from molten-globule to unfolded state. However, we have not yet proved it experimentally due to the lack of stability of the instrument under higher temperatures.