Cholesterol aggregation and interaction with

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 that cholesterol forms aggregates in supercritical carbon dioxide. In pure carbon dioxide, changes in cholesterol aggregate size or packing structure are observed with changing pressure. Near the critical point of carbon dioxide, cholesterol solubility is too low to permit sigifi cant aggregation, and monomeric cholesterol is observed. Addition of small amounts of dopants to supercritical carbon dioxide strongly affects cholesterol aggregation. Branched butanols (2-methyl-1-propanol and 2methyl-2-propanol) and ethanol (to a lesser degree) promote cholesterol aggregation, while methanol, acetone, and 1butanol do not. Cosolvents that promote aggregation also increase the rate at which cholesterol oxidase from Gloeocysticum chrysocreas catalyzes the oxidation of cholesterol. In supercritical carbon dioxide solutions, the EPR spectroscopy reveals little or no conformational change in cholesterol oxidase as 2-methyl-2-propanol or methanol is added. Damp cholesterol oxidase binds multiple cholesterol molecules; dry enzyme loses the ability to bind cholesterol. When molecular oxygen is the oxidizing agent, the rate of enzymatic cholesterol oxidation is greatly reduced in bone-dry carbon dioxide compared to that in water-saturated carbon dioxide.

concerned only with cholesterol oxidase from G. chrysocreas. As has been shown elsewhere (5), addition of small amounts of cosolvents to supercritical carbon dioxide may cause substantial increases in the rate of enzymatic reaction. These changes are not easily explained in terms of cholesterol-solubility increases caused by cosolvent addition; although methanol and acetone cause the largest increases in cholesterol solubility, these cosolvents yield smaller increases in reaction rate than do 2-methyl-1-propanol (isobutyl alcohol) and 2-methyl-2-propanol (tert-butyl alcohol), which cause the smallest solubility increases. High-pressure EPR spectroscopy was used to study the effect of cosolvent addition. Nitroxide-labeled cholesterol oxidase from G. chrysocreas was used in addition to a nitroxide-labeled derivative of cholesterol, 3-doxyl-5-a cholestane, to study (i) the conformation of the enzyme as a function of cosolvent addition, (ii) the self-association of cholesterol in supercritical carbon dioxide and supercritical carbon dioxide/cosolvent mixtures, and (iii) the interaction of cholesterol and cholesterol oxidase under supercritical conditions.

MATERIALS AND METHODS

Supercritical carbon dioxide and supercritical carbon dioxide cosolvent mixtures have been shown (1, 2) to provide a medium wherein enzymes maintain catalytic activity (also unpublished results of T.W.R., D. A. Miller, H.W.B., and J.M.P.). This supercritical-fluid medium is advantageous for enzyme-catalyzed reactions for a number of reasons: (i) high diffusivities and low viscosities (relative to liquid solvents); (ii) simplified downstream separations of products, unreacted substrates, and catalysts; and (iii) large changes in solvent power and dielectric constant caused by small changes in pressure and temperature. The modification of steroids constitutes a class of enzymecatalyzed reactions in which supercritical-fluid processing may be of particular interest. Since aqueous solubilities of many steroids are low, reaction rates are also low in aqueous media. Substantial increases in steroid solubility may be obtained by using a supercritical fluid as a solvent. For example, cholesterol is about 50 times more soluble in supercritical carbon dioxide at 123 bars (1 bar = 100 kPa) and 308 K (3) than in water at 298 K (4). Addition of small amounts of cosolvents to supercritical carbon dioxide (e.g., 3.5 mol % of methanol) may increase solubility by an additional order of magnitude (3).

EPR spectra were recorded on an IBM ER 200 D-SRC EPR spectrometer with a custom-made quartz high-pressure cell. Three spectra were recorded at each pressure with a sweeptime of 150 sec, and the spectra were averaged. Pressures were recorded with an Omega pressure transducer (model PX 420-2KGI) calibrated with a dead-weight pressure gauge. Cholesterol oxidase from G. chrysocreas (Chemical Dynamics, South Plainfield, NJ) was spin-labeled by incubating the enzyme in a 100-fold molar excess of 2,2,5,5tetramethyl-l-pyrrolin-3-oxyl-carboxylic acid N-hydroxy succinimide (Kodak Chemicals; used as received) for 24 hr at room temperature. Unreacted spin label was removed by dialysis for 48 hr at 0°C against a 50 mM phosphate buffer

(pH 7.0).

The interaction of cholesterol and cholesterol oxidase from G. chrysocreas was examined by EPR spectroscopy by using 3-doxyl-5-a-cholestane, a cholesterol analogue spinlabeled with a nitroxide group (Aldrich; used as received). Cholesterol oxidase [Chemical Dynamics; dialyzed for 48 hr against 50 mM phosphate buffer (pH 7.0)] was first immobilized on porous aminosilanized glass beads (Sigma). One gram of beads was activated by treating the surface amine groups with a 2.5% glutaraldehyde solution in 50 mM phosphate buffer (pH 7.0). The mixture was allowed to react for 1 hr at room temperature. Unreacted glutaraldehyde was removed by washing on a Buchner funnel with 50 mM phosphate buffer (pH 7.0). Five milliliters of cholesterol oxidase solution [10 mg/ml of 50 mM phosphate buffer (pH 7.0)] was added to the glass beads. After 3 hr of reaction at

We have examined the enzyme-catalyzed kinetics of cholesterol oxidation by molecular oxygen in carbon dioxide. Although cholesterol oxidases from Streptomyces sp., Pseudomonas sp., Norcardia sp, and Gloeocysticum chrysocreas are active in supercritical carbon dioxide, this study is The publication costs of this article were defrayed in part by page charge

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.

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room temperature, uncoupled enzyme was washed away on a Buchner funnel with the phosphate buffer. The final wash contained 0.01% sodium azide as a preservative. Spin-labeled cholesterol was added to the high-pressure EPR cell in a methylene chloride solution. The methylene chloride was evaporated under vacuum leaving the spinlabeled cholesterol derivative deposited on the walls of the EPR cell. Spin-labeled enzyme was added as a solid immobilized on glass beads.

RESULTS Conformation of Cholesterol Oxidase from G. chrysocreas in Supercritical Carbon Dioxide. Cholesterol oxidase from G. chrysocreas was derivatized by using 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-carboxylic acid N-hydroxy succinimide ester, a spin label that is reactive towards lysine residues (6). Nitroxide groups were attached to the enzyme with an average stoichiometry of eight per enzyme. Fig. 1 shows EPR spectra of spin-labeled cholesterol oxidase at atmospheric conditions and under supercritical conditions in carbon dioxide with addition of 3% methanol or 2-methyl-2propanol. Surprisingly little difference in the spectra is seen as conditions change from atmospheric to supercritical or upon addition of 2-methyl-2-propanol. Some distortion of the spectrum occurs upon addition of methanol; the peak splitting decreases somewhat and the shoulder on the first peak is more pronounced. While these spectra cannot rule out a small conformational change in the enzyme caused by cosolvent addition, such a change seems unlikely, considering that the magnitude of the observed changes in the EPR spectra was small and that the enzyme had been labeled at eight different sites. Changes in the EPR spectrum with addition of methanol do not necessarily reflect enzymeconformation changes; methanol addition changes the supercritical solvent's properties (e.g., dielectric constant and viscosity), which in turn can affect the EPR spectrum without causing conformational changes. Self-Association of Cholesterol in Supercritical Carbon Dioxide. A rapidly tumbling nitroxide group in dilute solution (of the order of 1 mM or less) gives an EPR signal composed of three sharp peaks of approximately equal height separated by a flat baseline. Under more concentrated conditions, the peaks widen until, at concentrations of approximately 0.1 M or more, the three peaks merge into a single broad peak (7, 8).

a

b

C

When a solution of 3.9 mM 3-doxyl-5-a-cholestane in carbon dioxide at 112 bars and 308 K was examined by EPR spectroscopy, the expected sharp three-peak signal was not observed. Instead, the spectrum (Fig. 2) is composed of three broad peaks on a sharply sloping baseline. Reducing the 3-doxyl-5-a-cholestane concentration to 65 ILM did not give a typical "dilute solution" EPR signal; the spectrum in Fig. 2 resembles a "solid-like" form typical of much higher spin-label concentrations. Although the overall concentration of slin-labeled cholesterol had been greatly reduced, the local concentration remained sufficiently high for spin-spin interactions to cause merging of the three peaks. Therefore, it appears that the nitroxide-labeled cholesterol molecules exist as aggregates in supercritical carbon dioxide under these conditions. Krafft Pressure Behavior in Supercritical Carbon Dioxide. Further evidence of cholesterol aggregation was provided by the EPR spectrum of 3-doxyl-5-a-cholestane in carbon dioxide at lower pressures, where carbon dioxide's solvent power is low. In liquid-micellar systems, there often exists a "Krafft" temperature, where the solubility of the surfactant is equal to the critical micelle concentration. Below the Krafft temperature, surfactant molecules are not sufficiently concentrated to form micellar aggregates. Similarly, in a supercritical fluid, a Krafft pressure may be defined as the pressure (at a given temperature) at which the solubility of a surfactant is equal to the critical micelle concentration. For a solution containing 0.27 mM 3-doxyl-5-a-cholestane and 1.4 mM cholesterol, the EPR spectrum changed dramatically as the pressure was reduced from 112 bars. The three nitroxide peaks are clearly distinguishable in Fig. 3 above 89 bars, although they are quite broad and exhibit a steeply sloping baseline. In this region, the local concentration of unpaired electrons is significantly higher than the overall bulk-phase concentration. The local nitroxide concentration increases further as the pressure is lowered from 89 to 85 bars. In this region in Fig. 3, the peaks are so broad that three peaks can scarcely be defined. This unexpected increase in local concentration may be due to the formation of an aggregate that is different (from those observed elsewhere) in size or packing structure. The region between 83.2 and 81 bars may be defined as the Krafft-pressure region. Here an abrupt change occurs in Fig. 3 from a spectrum corresponding to high aggregation to a spectrum typical of rapidly tumbling "monomeric" spin label. Further evidence that at higher pressure cholesterol is indeed found in aggregate form was provided by the appearance of this sharp signal corresponding to nonaggregation at lower pressures. Below 81 bars (not shown), the signal remained sharp but decreased with decreasing 3-doxyl-5-acholestane solubility until, below about 75 bars, the 3-doxyl5-a-cholestane concentration was too low for detection. Transient EPR Spectra of 3-Doxyl-5-a-cholestane After Pressure Jumps in Supercritical Carbon Dioxide. The EPR spectra described in the previous section represent steady-

d

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FIG. 1. EPR spectra of spin-labeled cholesterol oxidase from G. chrysocreas under atmospheric and supercritical conditions. Spectra: a, atmospheric pressure (308 K); b, in carbon dioxide at 104 bars (308 K); c, in carbon dioxide and 3% (vol/vol) 2-methyl-2-propanol at 104 bars (308 K); d, in carbon dioxide and 3% (vol/vol) methanol at 104 bars (308 K).

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FIG. 2. EPR spectrum of 65 ,uM 3-doxyl-5-a-cholestane in supercritical carbon dioxide at 308 K.

Biophysics: Randolph et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 3. EPR spectra of 0.27 mM 3-doxyl-5-a-cholestane with 1.4 mM cholesterol in carbon dioxide at 308 K. Pressure in bars is indicated.

state measurements. Fig. 4 shows the EPR spectrum of 3-doxyl-5-a-cholestane as a function of time after the pressure was rapidly decreased (during a period of about 2 sec) from 84.1 to 82.7 bars and from 82.7 to 82.4 bars, respectively. The spectra show large changes for approximately 2 min until steady state was reached. Such changes may represent transient aggregate packing structures or aggregate sizes. The large magnitude of such spectral changes is surprising, considering the small pressure changes that produced them. These results indicate the extreme sensitivity of density-dependent solvent characteristics to pressure changes in the region near the solvent's critical point. Cosolvent Effects on Cholesterol Aggregation in Supercritical Carbon Dioxide. Addition of cosolvents to supercritical

carbon dioxide solutions of 3-doxyl-5-a-cholestane caused large changes in the extent of aggregation. Fig. 5 shows the EPR spectrum of 3-doxyl-5-a-cholestane in supercritical carbon dioxide at 104 bars (308 K) with 3% (vol/vol of various cosolvents added. Addition of the branched butanols-2-methyl-1-propanol and 2-methyl-propanol-pro moted cholesterol aggregation; only one broad peak is evident in the EPR spectrum in Fig. 5. Ethanol promoted aggregation less effectively, as indicated by the appearance of three poorly resolved peaks in the EPR spectrum. Addition of cosolvents 1-butanol acetone, and methanol caused progressively less aggregation. Although still not as sharp as would be typical of nonassociating nitroxide groups at this concentration, the spectral line shapes were considerably narrower than those observed when either of the branched butanols was added. Addition of cosolvents also changed the Krafft-pressure behavior for nitroxide-labeled cholesterol. Krafft-pressure behavior was observed near the critical pressure of carbon dioxide for the cosolvents investigated, but the Krafftpressure transitions were less sharp in cosolvent-containing mixtures. Transitions were sharper when aggregatepromoting cosolvents (e.g., 2-methyl-2-propanol) were added than when cosolvents like methanol were added. Fig. 6 shows the Krafft-pressure transition for carbon dioxide solutions containing 3% (vol/vol) 2-methyl-2-propanol and methanol, respectively. The broadened Krafft-pressure region may be the result of a broadened distribution of aggregation number. The degree of aggregation of cholesterol in carbon dioxide cosolvent mixtures correlates well with the observed rate of enzymatic cholesterol oxidation (5). The cosolvents that promote cholesterol aggregation also provide the largest increases in the rate of enzymatic oxidation in supercritical carbon dioxide.

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FIG. 4. Transient EPR spectra of 3-doxyl-5-a-cholestane after a rapid pressure drop. (Upper) Pressure drop from 84.1 to 82.7 bars. Spectra were recorded after 12 sec (spectrum A), 24 sec (spectrum B), 36 sec (spectrum C), 48 sec (spectrum D) and 60 sec (spectrum E). (Lower) Pressure drop from 82.7 to 82.4 bars. Spectra were recorded after 12 sec (spectrum A), 24 sec (spectrum B), 36 sec (spectrum C), 48 sec (spectrum D), 60 sec (spectrum E), 72 sec (spectrum F), and 9 min (spectrum G).

Interaction of Spin-Labeled Cholesterol with Cholesterol Oxidase from G. chrysocreas. The interaction of cholesterol with cholesterol oxidase from G. chrysocreas was examined

FIG. 5. EPR spectra of 0.31 mM 3-doxyl-5-a-cholestane in supercritical carbon dioxide at 103 bars and 308 K with 3% (vol/vol) of various cosolvents added. Spectra: a, methanol, b, acetone; c, 1-butanol; d, ethanol; e, (2-methyl-2-propanol); f, 2-methyl-ipropanol.

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FIG. 6. Krafft-pressure behavior of 3-doxyl-5-a-cholestane in supercritical carbon dioxide with 3% cosolvent. (Left) With 2methyl-2-propanol. Pressure in bars as indicated. (Right) With methanol. Pressure in atmospheres (1 atm = 101.3 kPa) is indicated.

by EPR spectroscopy by using 3-doxyl-5-a-cholestane and enzyme covalently attached to 50-gum glass beads. EPR spectra were recorded at 308 K and various pressures. The spectra were very broad, with only one peak present (Fig. 7). As pressure was reduced through the critical region, the signal did not sharpen to three peaks, in contrast to previous experiments with no enzyme present (see Fig. 3). Since there was no evidence of monomeric spin-label signal, it appears that more than one spin-labeled cholesterol molecule adsorbs onto the enzyme surface at a time, and the polar nitroxide groups remain in sufficiently close proximity to cause spin-spin broadening of the EPR spectrum. The spin-labeled cholesterol remains on the enzyme surface even in pressure regions where spin-labeled cholesterol does not form aggregates in supercritical carbon dioxide. No freely tumbling, monomeric spin-label signal could be detected, even at 81.7 bars (Fig. 7). Earlier experiments in a continuous enzyme reactor showed that reducing the water content of a supercritical carbon dioxide solution has a substantially negative effect on the rate of enzymatic oxidation: a reversible decrease by a factor of 10 in enzymatic activity was found when the carbon dioxide/cholesterol solution was dried over a molecular sieve (T.W.R., H.W.B., and J.M.P., unpublished results). A corresponding situation was investigated with EPR spectroscopy. A 0.29 mM solution of 3-doxyl-5-a-cholestane in methylene chloride was evaporated onto the walls of a quartz high-pressure EPR cell. One hundred milligrams of damp enzyme on glass beads was added, and the entire cell was dried for 5 hr at 3.3 x 10-5 bar and room temperature.

The EPR cell was then pressurized at 308 K with bone-dry carbon dioxide. The sample was allowed to equilibrate for 1 hr at a pressure of 104 bars. EPR spectra were then recorded at various pressures. The spectrum recorded at 104 bars is shown in Fig. 8. The spectrum appears to be a composite comprising a broad, exchange-broadened spectrum with the maximum peak splitting AN of about 35 G and a sharper spectrum with an AN of about 17.5 G. The sharper spectrum appears to be asymmetric, with the third peak smaller than the first two, indicating restricted motion of the spin label. In Fig. 8, as the pressure decreases to 90.6 bars, both spectra narrow slightly. The broad signal decreases in intensity relative to the sharper signal, and the peaks of the sharper spectrum become more even. These trends continue with decreasing pressure in Fig. 8 until at 76.9 bars the broad spectrum is almost undetectable. The sharp spectrum displays a splitting AN of about 15 G, and the third peak has grown to about 80%o of the size of the first peak. This lower-pressure spectrum is due to monomeric, freely tumbling 3-doxyl-5-a-cholestane. There was a sharp contrast between the EPR spectra of spin-labeled cholesterol with immobilized cholesterol oxidase in dry supercritical carbon dioxide and the corresponding spectra in water-saturated carbon dioxide. Although 3-doxyl-5-a-cholestane adsorbed to the enzyme at 104 bars in both cases, the adsorption apparently was much stronger when water was present. As the pressure was lowered below 104 bars in the case of dry supercritical carbon dioxide, there was significant (almost complete) desorption of 3-doxyl-5-acholestane from the enzyme; on the other hand, the spinlabeled cholesterol remained on the enzyme surface, even very near the critical point, when the carbon dioxide was saturated with water. In a water-restricted environment, the diminished capability for the enzyme to bind to multiple substrate molecules correlates well with the observed loss of oxidative activity of the enzyme in bone-dry carbon dioxide. Enzyme binding to a cholesterol-containing membrane may be a necessary first step for enzymatic activity, and this binding appears to be hampered when the enzyme is dry.

CONCLUSIONS Damp cholesterol oxidase binds more than one cholesterol molecule. Dry cholesterol oxidase loses the ability to bind multiple substrate molecules. This loss of binding ability is accompanied by a large reduction in cholesterol oxidase

activity.

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FIG. 7. EPR spectra of 3-doxyl-5-a-cholestane (0.29 mM) in carbon dioxide at 308 K in the presence of damp cholesterol oxidase from G. chrysocreas. Pressure in bars is indicated.

(thousands)

FIG. 8. EPR spectra of 3-doxyl-5-a-cholestane (0.29 mM) in bone-dry carbon dioxide at 308 K in the presence of dry cholesterol oxidase from G. chrysocreas. Pressure in bars is indicated.

Biophysics: Randolph et al. Cholesterol aggregation provides an explanation for the varied effects of cosolvents on cholesterol oxidase activity in supercritical carbon dioxide. EPR spectroscopy shows that the cosolvents that cause large reaction-rate increases also promote cholesterol aggregation. An alternative explanation, cosolvent-induced enzyme-conformation changes, may be discounted. Only small changes in the EPR spectrum of spin-labeled cholesterol oxidase are seen in supercritical carbon dioxide with cosolvent addition. Cholesterol aggregation in supercritical carbon dioxide is affected by cosolvent addition and the solution pressure. Changes in the EPR spectrum of spin-labeled cholesterol may be a result of changes in cholesterol-aggregate packing or size. At the "Krafft pressure," cholesterol exists in monomeric form in supercritical carbon dioxide; cosolvent addition broadens the Krafft pressure region. We are grateful to Mr. Paul S. Skerker for assistance with the EPR measurements and for aid in interpreting EPR spectra. For

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financial support, we are grateful to the National Science Foundation (Grant CBT8513642) and to the Center for Biotechnology Research, San Francisco, CA. 1. Randolph, T. W., Blanch, H. W., Prausnitz, J. M. & Wilke, C. R. (1985) Biotech. Lett. 7, 325-328. 2. Hammond, D. A., Karel, M., Klibanov, A. & Krukonis, V. J. (1985) Appl. Biochem. Biotechnol. 11, 393-400. 3. Wong, J. M. & Johnston, K. P. (1986) Biotechnol. Prog. 2, 29-39. 4. Haberland, M. E. & Reynolds, J. A. (1973) Proc. Natl. Acad. Sci. USA 70, 2313-2316. 5. Randolph, T. W., Clark, D. S., Blanch, H. W. & Prausnitz, J. M. (1988) Science 239, 387-390. 6. Twinning, S. S., Sealy, R. C. & Glick, D. M. (1981) Biochemistry 20, 1267-1272. 7. Likhtenshtein, G. I. (1976) Spin Labelling Methods in Molecular Biology (Wiley, New York), pp. 40-45. 8. Jost, P. & Griffith, 0. H. (1976) in Spin Labeling: Theory and Applications, ed., Berliner, L. J. (Academic, New York), pp. 251-272.