Determination of Structural Parameters of Protein-Containing Reverse Micellar Solution by Near-Infrared Absorption Spectroscopy

Abstract

We present a method based on near-infrared absorption spectroscopy of the OH stretching vibration band of water around 3400 cm(-1) to examine whether 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 the cavity size does not change before and after solubilization of the protein at water-to-surfactant molar ratios (w(0)) from 2 to ∼18; that is, the w(0) values of the protein-filled and unfilled reverse micelles are the same as that of the reverse micellar solution, regardless of the size relationship 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 w(0) range from 2 to 24. On the other hand, circular dichroic 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.