STRATEGY FOR DEPRESSION OF IRREVERSIBLE REACTIONS OF PEPTIDES OR PROTEINS AS MEDICINE

在溶液中，蛋白质处于折叠和未折叠状态之间的平衡。不可逆的化学反应通常与蛋白质的未折叠状态相结合。因此，我提出了两个策略：1，抑制蛋白质从折叠态到去折叠态的转变，2，抑制蛋白质去折叠态的不可逆反应。本研究以鸡溶菌酶为模型蛋白，得到以下结果：1.在pH = 3的条件下，用差示扫描量热法测定了溶菌酶在甘油、葡萄糖、半乳糖、甘露糖、海藻糖、蔗糖和肌氨酸等添加剂存在下的变性温度（Tm）。在这些添加剂的存在下，溶菌酶的Tm增加。特别是，加入1.5 M海藻糖使溶菌酶的Tm增加了12 μ C。因此，我发现这些添加剂的加入抑制了溶菌酶从折叠状态到未折叠状态的转变。在1000 ℃下，即使在这些添加剂的存在下也观察到溶菌酶的失活，但程度小于不存在添加剂的情况。在这些添加剂的存在下，溶菌酶对加热的失活被抑制的原因被发现取决于这些添加剂诱导溶菌酶的未折叠状态是紧凑的，从而导致溶菌酶分子之间的不利的分子间相互作用的抑制。此外，这些添加剂也被发现抑制不可逆的化学反应，如氨基酸残基的脱酰胺或消旋溶菌酶。因此，我证明了这些添加剂也对蛋白质中不可逆化学反应的抑制起作用。

In solution, a protein is in an equilibrate between the folded and the unfolded state. Irreversible chemical reactions are usually coupled with the unfolded state of a protein. Therfore, I proposed two strategy : 1, depression of the shift from the folded state to the unfolded state in a protein, 2, depression of irreversible reactions in the unfolded state of a protein. In this study, we used hen lysozyme as a model protein and obtained the following results.As for 1, the denaturation temperatures (Tm) of lysozyme were measured using differential scanning calorimetry at pH 3 in the presence of several additives such as glycerol, glucose, galactose, mannose, trehalose, sucrose and sarcosine. In the presence of these additives, the Tm of lysozyme increased. Especially, addition of 1.5 M trehalose increased the Tm of lysozyme by 12ﾟC.Therefore, I found that the addition of these additives depressed the shift from the folded state to the unfolded state in lysozyme.As for 2, inactivation experiments of lysozyme against heating in the presence of sucrose, trehalose and sarcosine were carried out. Inactivations of lysozyme at 1000ﾟC were observed even in the presence of these additives but the extents were less than those in the absence of additives. The reason why inactivations of lysozyme against heating were depressed in the presence of these additives was found to depend that these additives induced the unfolded state of lysozyme to be compact leading to the depression of unfavorable intermolecular interactions between lysozyme molecules. Moreover, these additives were also found to depress the irreversible chemical reactions such as deamidations or racemizations of amino acid residues in lysozyme. Therefore, I showed that these additives also play a role on the depression of the irreversible chemical reactions in a protein.