J . Phys. Chem. 1988, 92, 6680-6685
6680
Thermodynamic Study of Globular Protein Stability in Microemulsions Ezio Battistel,+ Pier Luigi Luisi, Institut fur Polymere. ETH, Zurich, Switzerland
and Giovanni Rialdi* Centro Studi Chimico Fisici Macromolecole Sintetiche e Naturali. CNR, Corso Europa 30, 16132 Genova, Italy (Received: November 30, 1987; I n Final Form: April 28, 1988)
Aqueous solutions can be solubilized in organic solvents with the help of reverse micelles formed by bis(2-ethylhexyl) sodium sulfosuccinate (AOT), a negatively charged surfactant. The physicochemical properties of the proteins solubilized in the reverse micelles can be modulated by varying the amount of water present in the system. We studied the unfolding transition of globular proteins (ribonuclease, cytochrome c, lysozyme) in the AOT/isooctane micellar system by differential scanning calorimetry (DSC). The thermodynamic parameters associated with the transition were studied as a function of the micellar critical parameters and system composition. The DSC thermogram of a protein micellar solution showed a single sharp transition that can be attributed to a cooperative unfolding process of the protein structure. The T,, AH, M, and AG of the unfolding process calculated from the thermograms were strongly dependent on the amount of water present in the system. Protein micellar solutions at low water content were remarkably stable as a function of time.
Introduction Reverse micelles are spheroidal aggregates formed by certain surfactants in organic media. The polar head groups of the surfactant molecules are directed toward the interior of the aggregate, whereas the hydrocarbon tails are in contact with the bulk organic solvent. The properties of the water present in the micellar core differ from those of the bulk water, depending on the mole ratio of water and s~rfactant,'-~ usually indicated as Wo (= [water]/[surfactant]). For instance, at low W,, water has the characteristics of the water associated with in vivo interfaces4 A typical surfactant is bis( 2-ethylhexyl) sodium sulfosuccinate (AOT), a negatively charged amphiphilic molecule that forms stable reverse micelles in several hydrocarbons. The AOT micellar system in isooctane is able to solubilize water up to 6-7% v/v, forming spheroidal aggregates with the radius of the water pool ranging from 18 to 150 A, which strictly depends on W, (= [water]/[AOT]). Inside the micellar water pool, large biopolymers such as enzymes5-' and nucleic acids8 may be hosted without denaturation. The physicochemical properties of these macromolecules (such as the enzymatic activity, substrate affinity, stability, etc.) can be modulated by varying the amount of water present in the system and, therefore, the hydration of the macromolecules, the properties of water, and the electrostatic interactions. On the basis of spectroscopic evidence (UV, circular dichroism (CD), and fluorescence), globular proteins are assumed to be located inside the water pool. This situation is best described by the so-called the water-shell model5 according to which the protein molecule is surrounded by one or more layers of water molecules that protect the protein from the denatur-ting effects of isooctane and AOT molecules. In fact, enzymes such as a-chymotrypsin and lysozyme are still active in the AOT micelles. For low amounts of water, the volume of the micellar water pool is comparable to the hydration shell of a small globular protein, and under such conditions novel properties of these and other enzymes may be found. At low Wothe specific activity of a-chymotrypsin9 and peroxidase'O increases several times with respect to that in aqueous solution (for a review see ref 11). Significant increases in the operational stability are also observed.8 This study presents a thermodynamic approach to the peculiar properties of the proteins inside microemulsions and more in general to the protein functions. This is also, in view of the particular relevance of the enzymatic catalysis in microemulsions, a potential tool in applied enzymology and biote~hnology.'~-'~ In particular, we have studied the unfolding transition of a few 'Present address: Istituto G. Donegani. Novara, Italy
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globular proteins (lysozyme, ribonuclease, and cytochrome c) by differential scanning calorimetry (DSC). The thermodynamic parameters associated with the transition (the temperature of the the enthalpy, entropy, middle point of the melting process (Tm), and free energy changes AH, AS, and AG,respectively) have been AOT studied as a function of the critical micellar parameters (Wo, concentration, and composition of the system) in the temperature range in which the micellar system is stable.
Materials and Methods Bis(2-ethylhexyl) sodium sulfosuccinate (AOT) was purchased from Sigma (West Germany) and used without further purification. According to standard tests of purity,I5 the batch used was found to contain minimal amounts of acid and UV-absorbing impurities (
