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.

Terahertz dynamics of water before and after water shedding from reverse micelles

“…A reverse micellar solution was prepared using the injection method [23]. AOT (Sigma-Aldrich), isooctane (Wako), and sterile Milliporefiltered water (Yamato) were used as the surfactant, oil, and water, respectively.…”

“…Accordingly, the structure and dynamics of water in reverse micelles have been extensively studied through various experimental methods, and these studies have revealed that the water's features are quite different from those of liquid water and depend on the size of the reverse micelles [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. On the other hand, reverse micelles are considered good models of cells, because biomolecules, such as proteins and DNA, can be dissolved in them [2,[20][21][22][23]. Water plays an important role in the function of biomolecules, and the condition of water in cells is different from that in a dilute aqueous solution of biomolecules.…”

Infrared spectroscopy of proteins in reverse micelles

“…A concern at the outset might be that enzymes tend to be much larger than reverse micelles. However, reverse micelles readily fuse as necessary to encapsulate enzymes, and they retain their w 0 in so doing [36]. …”

Nanometer-scale water droplet free from the constraint of reverse micelles at low temperatures

“…A reverse micelle is a nanometer-scale cage filled with water [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. The aqueous cavity radius of the reverse micelle is proportional to the water-surfactant molar ratio w 0 (=[water]/[surfactant]), and can be controlled experimentally [11,12].…”