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状态。不可逆的化学反应通常与蛋白质的展开状态相结合。因此，我提出了两个策略：1，抑制蛋白质从折叠状态到未折叠状态的转变；2，抑制蛋白质在未折叠状态下的不可逆反应。本研究以鸡溶菌酶为模型蛋白，采用差示扫描量热法测定了鸡溶菌酶在甘油、葡萄糖、半乳糖、甘露糖、海藻糖、蔗糖和肌氨酸等多种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。特别是添加1.5M的海藻糖，使溶菌酶的Tm提高了12ﾟ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ﾟC下，即使有这些添加剂也能观察到溶菌酶的失活，但失活程度比没有添加剂时要小。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是这些添加剂诱导了溶菌酶的未折叠状态致密，从而抑制了溶菌酶分子间不利的分子间相互作用。此外，这些添加剂还可以抑制溶菌酶中氨基酸残基的脱酰胺或消旋等不可逆的化学反应。因此，我证明了这些添加剂也起到了抑制蛋白质中不可逆化学反应的作用。

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.