| 摘要: | 摘要 吸附於氣/液界面上的蛋白質分子層可以降低界面的表面張力 g（surface tension），而降低的部分則稱之表面壓力 P（surface pressure）。於是研究人員便發展出一套系統模式來結合表面壓力與蛋白質分子吸附層之吸附密度（adsorption density, G）、分子展開程度（the degree of unfolding）的關係等[van Aken, 1995；van Aken and Merks, 1996]。 蛋白質分子在界面上結構展開的程度與所需的時間和蛋白質分子的自然結構及它結構中所具的極性-非極性之結構特性（polar-nonpolar character）有很大的關聯性[Tanford, 1973]。而蛋白質分子的結構特性主要是由胺基酸的組成所決定的。因此，蛋白質單分子層對於了解蛋白質的自然結構與其穩定性是佔有重要地位的[Andrade, 1992]。 在本研究當中我們嘗試著將5種具有不同結構特性的蛋白質經變性劑溶液變性，利用蛋白質於氣/液界面上的行為來探討變性劑對於蛋白質結構的影響。本研究將蛋白質溶液置放於氣/液界面使其形成蛋白質的單分子層（protein monolayer）並使用Langmuir-Blodgett（LB）Nima-trough系統來量測蛋白質溶液經變性後在氣/液界面上的表面壓與時間的關係圖與表面壓和分子所佔據面積的關係圖。而且我們也試著改變變性劑的濃度與種類以便於我們可以探討變性劑對各種蛋白質的影響。 結果顯示，較具結構性的球型蛋白質溶菌酶（Lysozyme），它在表面壓-時間、表面壓-每分子在氣/液界面佔據的面積之結果中：經32mM DTT+6M Gu-HCl變性後，平衡表面壓達至最高，且分子佔據面積明顯比自然態的蛋白質高出一倍；但經8M Urea變性時，表面壓並無上升且每分子所佔據的面積維持和自然態時相同。因此，不同的變性劑會造成蛋白質有不同程度的結構破壞與展開；這樣的行為可經由簡單的表面壓力的量測即可得知。另一方面，較不具結構性的蛋白質如酪蛋白（β-Casein），由於其本身結構特性的關係，經不同的變性劑將它變性後，其在界面上的界面行為表面並看不出其結構展開變化。 因此，蛋白質經不同變性劑變性後，因其結構展開程度的不同所以在氣/液界面上的界面行為亦不同。不同的界面行為表示不同的變性效果即蛋白質經變性後在氣/液界面上的構型狀態並不相同[周, 2002]。當然，蛋白質的界面行為也和本身的自然結構特質有相當的關係。 不同的蛋白質構型狀態即可提供給一些藉由界面方法如固體或觸手來幫助蛋白質復性的研究一些關於復性時結構狀態的資訊。 Abstract The presence of an adsorbed protein layer in the interface leads to decreased surface tension and its decrease is called the surface pressureΠ.A model was developed that relates the surface pressure to adsorption density, Γ, and the degree of unfolding , α, of the molecules in the adsorbed layer. The conformational changes of the adsorbed protein molecules proceed relatively slowly and lead to the viscoelastic behavior of protein monolayers. Proteins at air/liquid interfaces are usually denatured or unfolded because interfacial interactions are generally stronger than cohesive interactions within protein molecules. The degree of unfolding and time taken to unfold at the interface depend on protein structure and on its polar-nonpolar character, which determined by the amino acid composition. Therefore, behaviors of protein monolayer at the interface play an important role in understanding the native conformation and stability of protein. In this study, we discuss the interfacial behaviors of five native and denatured proteins with different conformational character. The protein solution is layered and spread along the air/solution interfaces to form protein monolayer. Measurements of equilibrium surface pressure–time (Π-t) and surface pressure-area per molecule (Π-A) by using the Langmuir-Blodgett (LB) balance consisted of a NIMA-trough system. Besides, we also change the kind and concentration of denaturants to discuss the influences of denaturants. The data suggests that Π-t and Π-A isotherm of proteins with more structural stability (eg: Lysozyme) denatured by 32mM DTT+6M GuHCl are different from those of native protein and become high and large. But, Π-t and Π-A isotherm of proteins with more structural stability denatured by 8M Urea are similar to those of native proteins. Both disulfide and noncovalent bonds apparently restrict the unfolding of globular proteins at the interfaces. Protein denatured by denaturants can relax those restrictions, resulting in more complete interfacial unfolding. The interfacial behaviors (activity) of β-Casein, a random-coil type protein, is not influenced by denaturants, as it readily and completely unfolds at the interfaces. Different types of denaturants cause different unfolding degree of protein structure. The variations of equilibrium surface pressure and the area/per molecule of denatured proteins indicate different degree of protein structure changed. The structural unfolding degree of proteins depends on their natural conformational stability and the kinds of denaturants. The collective interfacial behaviors of the protein in the study to provide protein structure stability and fundamental information for protein refolding process by solid or ligand surface. |
