Proc. Nati. Acad. Sci. USA Vol. 85, pp. 2979-2983, May 1988 Biophysics Cholesterol aggregation and interaction with cholesterol oxidase in supercritical carbon dioxide (electron paramagnetic resonance spectroscopy/Krafft behavior/enzymes in nonaqueous solvents) T. W. RANDOLPH, D. S. CLARK, H. W. BLANCH*, AND J. M. PRAUSNITZ Department of Chemical Engineering, University of California, Berkeley, CA 94720 Contributed by J. M. Prausnitz, December 7, 1987 ABSTRACT High-pressure EPR spectroscopy indicates concerned only with cholesterol oxidase from G. chryso- that cholesterol forms aggregates in supercritical carbon diox- creas. As has been shown elsewhere (5), addition of small ide. In pure carbon dioxide, changes in cholesterol aggregate amounts of cosolvents to supercritical carbon dioxide may size or packing structure are observed with changing pressure. cause substantial increases in the rate of enzymatic reaction. Near the critical point of carbon dioxide, cholesterol solubility These changes are not easily explained in terms of choles- is too low to permit sigificant aggregation, and monomeric terol-solubility increases caused by cosolvent addition; al- cholesterol is observed. Addition of small amounts of dopants though methanol and acetone cause the largest increases in to supercritical carbon dioxide strongly affects cholesterol cholesterol solubility, these cosolvents yield smaller in- aggregation. Branched butanols (2-methyl-1-propanol and 2- creases in reaction rate than do 2-methyl-1-propanol (iso- methyl-2-propanol) and ethanol (to a lesser degree) promote butyl alcohol) and 2-methyl-2-propanol (tert-butyl alcohol), cholesterol aggregation, while methanol, acetone, and 1- which cause the smallest solubility increases. butanol do not. Cosolvents that promote aggregation also High-pressure EPR spectroscopy was used to study the increase the rate at which cholesterol oxidase from Gloeocysti- effect of cosolvent addition. Nitroxide-labeled cholesterol cum chrysocreas catalyzes the oxidation of cholesterol. In oxidase from G. chrysocreas was used in addition to a supercritical carbon dioxide solutions, the EPR spectroscopy nitroxide-labeled derivative of cholesterol, 3-doxyl-5-a cho- reveals little or no conformational change in cholesterol oxi- lestane, to study (i) the conformation of the enzyme as a dase as 2-methyl-2-propanol or methanol is added. Damp function of cosolvent addition, (ii) the self-association of cholesterol oxidase binds multiple cholesterol molecules; dry cholesterol in supercritical carbon dioxide and supercritical enzyme loses the ability to bind cholesterol. When molecular carbon dioxide/cosolvent mixtures, and (iii) the interaction oxygen is the oxidizing agent, the rate of enzymatic cholesterol of cholesterol and cholesterol oxidase under supercritical oxidation is greatly reduced in bone-dry carbon dioxide com- conditions. pared to that in water-saturated carbon dioxide. MATERIALS AND METHODS Supercritical carbon dioxide and supercritical carbon diox- ide cosolvent mixtures have been shown (1, 2) to provide a EPR spectra were recorded on an IBM ER 200 D-SRC EPR medium wherein enzymes maintain catalytic activity (also spectrometer with a custom-made quartz high-pressure cell. unpublished results of T.W.R., D. A. Miller, H.W.B., and Three spectra were recorded at each pressure with a sweep- J.M.P.). This supercritical-fluid medium is advantageous for time of 150 sec, and the spectra were averaged. Pressures enzyme-catalyzed reactions for a number of reasons: (i) high were recorded with an Omega pressure transducer (model diffusivities and low viscosities (relative to liquid solvents); PX 420-2KGI) calibrated with a dead-weight pressure gauge. (ii) simplified downstream separations of products, unre- Cholesterol oxidase from G. chrysocreas (Chemical acted substrates, and catalysts; and (iii) large changes in Dynamics, South Plainfield, NJ) was spin-labeled by incu- solvent power and dielectric constant caused by small bating the enzyme in a 100-fold molar excess of 2,2,5,5- changes in pressure and temperature. tetramethyl-l-pyrrolin-3-oxyl-carboxylic acid N-hydroxy The modification of steroids constitutes a class ofenzyme- succinimide (Kodak Chemicals; used as received) for 24 hr catalyzed reactions in which supercritical-fluid processing at room temperature. Unreacted spin label was removed by may be of particular interest. Since aqueous solubilities of dialysis for 48 hr at 0°C against a 50 mM phosphate buffer many steroids are low, reaction rates are also low in aqueous (pH 7.0). media. Substantial increases in steroid solubility may be The interaction of cholesterol and cholesterol oxidase obtained by using a supercritical fluid as a solvent. For from G. chrysocreas was examined by EPR spectroscopy by example, cholesterol is about 50 times more soluble in using 3-doxyl-5-a-cholestane, a cholesterol analogue spin- supercritical carbon dioxide at 123 bars (1 bar = 100 kPa) labeled with a nitroxide group (Aldrich; used as received). and 308 K (3) than in water at 298 K (4). Addition of small Cholesterol oxidase [Chemical Dynamics; dialyzed for 48 hr amounts of cosolvents to supercritical carbon dioxide (e.g., against 50 mM phosphate buffer (pH 7.0)] was first immobi- 3.5 mol % of methanol) may increase solubility by an lized on porous aminosilanized glass beads (Sigma). One additional order of magnitude (3). gram of beads was activated by treating the surface amine We have examined the enzyme-catalyzed kinetics of cho- groups with a 2.5% glutaraldehyde solution in 50 mM phos- lesterol oxidation by molecular oxygen in carbon dioxide. phate buffer (pH 7.0). The mixture was allowed to react for Although cholesterol oxidases from Streptomyces sp., Pseu- 1 hr at room temperature. Unreacted glutaraldehyde was domonas sp., Norcardia sp, and Gloeocysticum chrysocreas removed by washing on a Buchner funnel with 50 mM are active in supercritical carbon dioxide, this study is phosphate buffer (pH 7.0). Five milliliters of cholesterol oxidase solution [10 mg/ml of 50 mM phosphate buffer (pH The publication costs of this article were defrayed in part by page charge 7.0)] was added to the glass beads. After 3 hr of reaction at payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. Downloaded by guest on September 27, 2021 2979 2980 Biophysics: Randolph et al. Proc. Natl. Acad. Sci. USA 85 (1988) room temperature, uncoupled enzyme was washed away on When a solution of 3.9 mM 3-doxyl-5-a-cholestane in a Buchner funnel with the phosphate buffer. The final wash carbon dioxide at 112 bars and 308 K was examined by EPR contained 0.01% sodium azide as a preservative. spectroscopy, the expected sharp three-peak signal was not Spin-labeled cholesterol was added to the high-pressure observed. Instead, the spectrum (Fig. 2) is composed of EPR cell in a methylene chloride solution. The methylene three broad peaks on a sharply sloping baseline. Reducing chloride was evaporated under vacuum leaving the spin- the 3-doxyl-5-a-cholestane concentration to 65 ILM did not labeled cholesterol derivative deposited on the walls of the give a typical "dilute solution" EPR signal; the spectrum in EPR cell. Spin-labeled enzyme was added as a solid immo- Fig. 2 resembles a "solid-like" form typical of much higher bilized on glass beads. spin-label concentrations. Although the overall concentra- tion of slin-labeled cholesterol had been greatly reduced, the RESULTS local concentration remained sufficiently high for spin-spin interactions to cause merging of the three peaks. Therefore, Conformation of Cholesterol Oxidase from G. chrysocreas in it appears that the nitroxide-labeled cholesterol molecules Supercritical Carbon Dioxide. Cholesterol oxidase from G. exist as aggregates in supercritical carbon dioxide under chrysocreas was derivatized by using 2,2,5,5-tetramethyl- these conditions. pyrrolin-1-oxyl-3-carboxylic acid N-hydroxy succinimide es- Krafft Pressure Behavior in Supercritical Carbon Dioxide. ter, a spin label that is reactive towards lysine residues (6). Further evidence of cholesterol aggregation was provided by Nitroxide groups were attached to the enzyme with an the EPR spectrum of 3-doxyl-5-a-cholestane in carbon diox- average stoichiometry of eight per enzyme. Fig. 1 shows ide at lower pressures, where carbon dioxide's solvent power EPR spectra of spin-labeled cholesterol oxidase at atmo- spheric conditions and under supercritical conditions in is low. In liquid-micellar systems, there often exists a carbon dioxide with addition of 3% methanol or 2-methyl-2- "Krafft" temperature, where the solubility ofthe surfactant is propanol. Surprisingly little difference in the spectra is seen equal to the critical micelle concentration. Below the Krafft as conditions change from atmospheric to supercritical or temperature, surfactant molecules are not sufficiently con- upon addition of2-methyl-2-propanol. Some distortion ofthe centrated to form micellar aggregates. Similarly, in a super- spectrum occurs upon addition of methanol; the peak split- critical fluid, a Krafft pressure may be defined as the pressure ting decreases somewhat and the shoulder on the first peak (at a given temperature) at which the solubility of a surfactant is more pronounced. While these spectra cannot rule out a is equal to the critical micelle concentration. small conformational change in the enzyme caused by co- For