Determination of structural parameters of protein-containing reverse micellar solution by near-infrared absorption spectroscopy

近赤外吸収分光に基づいた蛋白質逆ミセル溶液の構造パラメータの決定

村上 洋; 西 孝樹*; 豊田 祐司*

Murakami, Hiroshi; Nishi, Takaki*; Toyota, Yuji*

逆ミセルは、界面活性剤分子が会合してできたナノメートルサイズの球状の空洞であり、その中に、水溶性の生体高分子を保持できる。逆ミセルは細胞の一つのモデル系となり、逆ミセルを用いて、生体高分子の機能・物性の空洞サイズや水和水環境依存性を調べるなど、水溶液ではできない研究が可能である。そのような研究のためには、生体高分子を含んだ逆ミセルの構造情報が必要である。空洞に水のみを含む逆ミセルでは、X線小角散乱測定など広範な研究により、[水濃度]/[界面活性剤濃度](=)が構造パラメータを決める主因子であり、空洞半径や会合界面活性剤分子数などの依存性が明らかにされている。それに対して、生体高分子逆ミセルの構造パラメータはよくわかっていない。本研究では、近赤外分光の結果を基礎に、生体高分子逆ミセルの構造パラメータのW依存性を決定するモデルを提案する。

We present a method based on near-infrared absorption spectroscopy of the OH stretching vibration band of water around 3400 cm to examine if the aqueous cavity size of a protein-unfilled reverse micelle is affected by solubilization of protein, and it has been found for AOT (= bis (2-ethylhexyl) sulfosuccinate) reverse micellar solution with myoglobin that it does not change before and after solubilization of the protein in the water-to-surfactant molar ratio () from 2 to 18, that is, the values of the protein-filled and unfilled reverse micelles are the same as that of the reverse micellar solution regardless of size relation between the aqueous cavity of the unfilled reverse micelle and the protein. On the basis of this experimental fact, we propose a model to determine the structural parameters of protein-filled reverse micelles, such as the aqueous cavity radius and the aggregation number of surfactant molecules constituting the micelle, and the molar concentration of the unfilled reverse micelle that exists with the protein-filled reverse micelle in the reverse micellar solution, and derive their values for AOT reverse micellar solution with myoglobin in the range from 2 to 24. On the other hand, circular dichroism measurements and UV-visible absorption spectroscopy of myoglobin/AOT reverse micellar solution and myoglobin/AOT aqueous solution were carried out in order to examine the conformational state of myoglobin in the reverse micellar solution. These experimental results lead to the conclusion that myoglobin is located in the aqueous cavity of the reverse micelle, although the conformational state of the protein is to some extent distorted because of the interaction with AOT compared with that of native myoglobin. Finally, it is suggested that the proposed model is appropriate for reverse micellar solution with a hydrophilic protein molecule that is located in the aqueous cavity of the reverse micelle.