Blotechnol. Prog. 1002, 8,421-423
421
In Situ Studies of Protein Conformation in Supercritical Fluids: Trypsin in Carbon Dioxide JoAnn Zagrobelny and Frank V. Bright* Department of Chemistry, Acheson Hall, State University of New York at Buffalo, Buffalo, New York 14214
The conformation of the monomeric enzyme trypsin has been studied in supercritical carbon dioxide. Steady-state fluorescence spectroscopy is used t o follow the conformation of trypsin in situ as a function of C02 density. Our results show for the first time that protein denaturation can occur during the fluid compression step and that the native trypsin is only slightly more stable (1.2 kcal/mol) than the unfolded form. These results demonstrate the power of fluorescence spectroscopy as a tool for studying protein conformation and dynamics in supercritical fluids.
1. Introduction Supercritical fluids have received a great deal of attention because of the ease with which their chemical potential can be adjusted with pressure (Eckert et al., 1986a,b). That is, their physical properties (e.g., dielectric constant, refractive index, and density) can be tuned while the inherent molecular structure of the solvent is maintained. As a consequence, supercritical solvents have been used extensively for extractions (Brennecke and Eckert, 1989; McHugh and Krukonis, 19861, chromatography (Smith, 19881, chemical reaction processes (Eckert et al., 1986a),enhanced oil recovery (Squires and Paulaitis, 1987), and most recently as a medium for enzyme-catalyzed reactions (Aaltonen and Rantakyla, 1991). In 1985, two groups reported on enzymatic reactions in supercritical solvents (Randolph et al., 1985; Hammond et al., 1985). Followingthese reports, there has been steady work in this area (Randolph et al., 1988a,b) and the stateof-the-art has been reviewed recently (Aaltonen and Rantakyla, 1991). Although efforts in this area have increased, to the best of our knowledge, only a single report has appeared that compares the conformation of an enzyme dissolved in water and supercritical solvents (Randolph, 1987; Randolph et al., 1988a). In these experiments, several spin-labeled variants of cholesterol oxidase were studied in supercritical CO2 using electron paramagnetic resonance spectroscopy. The conclusion was that the conformation of these particular proteins was not influenced by the supercritical fluid. In contrast, Kasche and co-workers (Kasche et al., 1988) concluded that the achymotrypsin, trypsin, and penicillin amidase were partially denatured by supercritical C02. They proposed that decompression led to denaturation; however, in situ measurements were not carried out to substantiate this proposition. The enzymes were only studied after being subjected to supercritical C02. In this paper, we report on the in situ conformation of trypsin as it is maintained in supercritical CO2. Steadystate fluorescence spectroscopy is used as our analytical tool (Bright, 1988; Lakowicz, 1983). COz was chosen because it is the most widely used supercritical fluid, it is readily available in high purity, it has mild critical conditions (T,= 304.1 K; P, = 73.8 bar) (Johnston and Penninger, 1989;Reid et al., 1987;Williams, 1981;Schneider, 19781, it is nontoxic, there are extensive tabulations on the phase equilibria of CO2 with many cosolvents, including water (Jennings et al., 1991; Suzuki et al., 1990; 8756-7938/92/3008-0421$03.00/0
Chrastil, 19821, and its density is adjustable over a fairly broad range (e.g., at 310 K density can be adjusted from 0.116 to 0.857 g/cm3 with pressures from 50 to 200 bar) (Angus et al., 1976). Trypsin was studied because it was reported previously (Kasche et al., 1988) that (1) it is partially denatured in supercritical C02 and (2) denaturation presumably occurred during fluid decompression.
2. Experimental Procedures Bovine trypsin (EC 3.4.21.4) and C02 (SFC grade) were from Sigma and Scott, respectively, and were used as received. Distilled-deionized water was used throughout. The high-pressure pumping apparatus, optical cells, and fluorescence spectrometer have been described in detail elsewhere (Betta and Bright, 1990). In a typical experiment, a 145 mg/mL stock solution of aqueous trypsin is prepared. Ten microliters of this solution is added directly into the high-pressure optical cell (5 mL volume) resulting in a final concentration of 0.29 mg of trypsin/mL. The cell is then connected to the pumping apparatus, and the temperature is allowed to equilibrate a t 35 O C for 30-40 min. A vacuum is then maintained ( ~ 5 pmHg) 0 on the system for 10-20 min. This step was taken to remove residual 0 2 which may quench the protein fluorescence (Lakowicz, 1983). The cell is then slowly charged with C02 as described previously (Betta and Bright, 1990). The cell pressure is raised to the desired starting point, and the experiments are performed in order of increasing CO2 density while the contents of the cell are continuously stirred. The concentration of trypsin, in the cell, remains constant throughout our experiments. The experiments were carried out at densities where the amount of aqueous solution added would be solubilized in supercritical C02. The solubility of water is calculated using the following semiempirical formula (Chrastil, 1982): c = p'.55 exp(-2826/T
- 0.81)
(1) where c is the concentration of water in grams per liter, p is the density in grams per liter, and T is the temperature in Kelvin. The tryptophan amino acid residues within trypsin were selectively excited at 290 nm (Lakowicz, 1983). The subsequent tryptophan emission was studied because of its sensitivity to the local environment and its yield (Lakowicz, 1983). In this way, we can use the trypsin emission
0 1992 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1992, Vol. 8, No. 5 1.0,
-1.04 50 Pressure (bar)
Figure 1. Emission center of gravity for trypsin in supercritical COz (with 110 mM added HzO) as a function of pressure at 35 "C. Lower and upper dashed horizontal lines denote resulta for trypsin in pure liquid water and 8 M urea, respectively,at 35 O C . &,= = 290 nm (band-pass = 4 nm).
to follow the effects of the supercritical solvent on the enzyme conformation.
3. Results and Discussion When a protein unfolds, its unique three-dimensional structure is affected drastically and the protein proceeds ultimately to a random-coiled conformation (Pace et al., 1991;Van Holde, 1971). Because of the magnitude of these conformational changes, one can often follow the process with standard optical techniques (UV-vis, IR, and fluorescence) (Bright, 1988; Lakowicz, 1983). Figure 1shows the effects of CO2 pressure on the tryptophan residue fluorescence of trypsin. These results are shown as the center of gravity of the emission spectrum (Lakowiczand Hogan, 1981). Under these particular conditions, all water would be dissolved at about 84 bar (Chrastil, 19821, and a small portion of the protein becomes suspended in the supercritical fluid. For comparison, we show also fluorescence results (horizontal, dashed lines) for trypsin in liquid water and 8 M urea. Several interesting aspects of these results merit additional discussion. First, the emission spectra observed in water and urea are quite different from one another. Specifically, the spectrum for trypsin in water is blue shifted and indicates that the average tryptophan residue encounters a moderately hydrophobic environment (Lakowicz, 1983; Teale and Weber, 1957). This is to be contrasted with the urea results in which the protein is denatured significantly (Bohinski, 1979)and the emission contour is red shifted nearly 13 nm. The indication here is that trypsin is unfolded such that the tryptophan residues become accessible to water (a less hydrophobic environment) (Lakowicz, 1983). Second, at low C02 pressures we recover an emission spectrum similar to trypsin in water, but as CO2 pressure is increased, the emission contour steadily red shifts and approaches that of trypsin in urea. These results are consistent with increased C02 pressure leading to conformational changes in trypsin. Third, following a pressure increase the emission spectrum shifted rapidly (within 2-3 min) and remained constant over time. This indicates that there is not any slow, long-term drift in conformation over time. Finally, as can be seen in Figure 1, the conformational changes in trypsin do not, as reported previously (Kasche et al., 19881, occur exclusively during decompression. To prove this point, we performed a set of simple decompression experiments from 200 to 85 bar, and the emission contours were found to be identical a t the two pressures (results not shown). From these results, we conclude that decompression has very little effect on the conformation of trypsin.
I
100
150
200
I
250
Pressure (bar)
Figure 2. Recovered free energy of unfolding for trypsin as a function of COz pressure. Experimentalconditions are the same as in Figure 1.
To further analyze these C02 pressure-dependent denaturation data, we assume a two-state model of the form (Pace et al., 1991) folded a unfolded (2) where folded and unfolded represent the native and conformationally altered forms of trypsin, respectively. From this we can calculate the fraction of unfolded trypsin, F,, using
F,(P) = (CG(P) - CGf)/(CG, - CGf) (3) where CG(P) is the pressure-dependent emission center of gravity (Figure 11, which is used to follow changes in conformation, and CGf and CG, are the centers of gravity for the folded (water) and unfolded (urea) trypsin conformations, respectively. F,(P)is related to the pressuredependent equilibrium constant, K,,(P), by K,,(P) = F,(P)/(l- F,(P)) (4) which is in turn related to the pressure-dependent free energy of unfolding, AG(P), by
AG(P) = -RT In [K,,(P)] (5) where R and T are the gas constant and absolute temperature, respectively. For trypsin in supercritical Cog, AG is found to vary linearly with pressure (Figure 2). These results are reminiscent of results for protein denaturation by urea and temperature (Thomson et al., 1989). Extrapolation of these experimental results to zero pressure yields AG(H20),an estimate of the trypsin conformational stability. From thisparticular set of experiments,AG(H20) is 1.2 f 0.1 kcal/mol, which indicates that native trypsin is only marginally more stable than the unfolded form.
4. Conclusions We present the first in situ fluorescence measurements on protein conformation in supercritical fluid media. These results show clearlythat (1)there can be significant changes in protein conformation induced by supercritical solvents, (2) most of the conformational change occurs during compression, and (3) the native trypsin conformation is only slightly more stable compared to the unfolded form. These results also indicate that enzyme segments that undergo significant changes in environment may not be associated with the active site. In an effort to more accurately study the influence of supercritical fluids on protein conformation, we are investigating systems which contain a single tryptophan residue. We are also applying dynamic fluorescence techniques to these same systems (Bright et al., 1990).We shall report on the progress of these experiments in due course.
Biotechnol. hog., 1992, Vol. 8, No. 5
Acknowledgment This work was supported in part by the United States Department of Energy (DE-FG02-90ER14143).
Literature Cited Aaltonen, 0.;Rantakyla, M. Biocatalysis in Supercritical C02. CHEMTECH 1991,21,240-248. Angus, S.; Armstrong, B.; de Reuck, K. M. Carbon Dioxide: International Thermodynamic Tables of the Fluid State; Pergamon: New York, 1976; Vol. 3. Betta, T. A.; Bright, F. V. Instrumentation for Steady-Stateand Dynamic Fluorescence Studies in Supercritical Media. Appl. Spectrosc. 1990,44, 1196-1202. Brennecke, J. F.; Eckert, C. A. Phase Equilibria for Supercritical Fluid Process Design. MChE J. 1989, 35, 1409-1427. Bohinski, R. C. Modern Concepts in Biochemistry; Allyn and Bacon: Boston, 1979. Bright, F. V. Bioanalytical Applications of Fluorescence Spectroscopy. Anal. Chem. 1988, 60, 1031A-1039A. Bright, F. V.; Betta, T. A.; Litwiler, K. S. Advances in Multifrequency Phase and Modulation Fluorimetric Analysis. CRC Crit. Rev. Anal. Chem. 1990,21, 389-405. Chrastil, J. Solubilityof Solidsand Liquids in Supercritical Gases. J,Phys. Chem. 1982,86,3016-3021. Eckert, C. A.; Van Alsten, J. G.; Stoicos, T. Supercritical Fluid Processing. Environ. Sei. Technol. 1986a, 20, 319-325. Eckert, C. A.; Ziger, D. H.; Johnston, K. P.; Kim, S. Solute Partial Molal Volumes in Supercritical Fluids. J.Phys. Chem. 1986b, 90,27362745. Hammond, D. A.; Karel, M.; Klibanov, A. M.; Krukonis, V. J. Enzymatic Reactions in Supercritical Gases. Appl. Biochem. Biotechnol, 1985,11, 393-400. Jennings, D. W.; Lee, R.-J.; Teja, A. S. Vapor-Liquid Equilibria in the Carbon Dioxide Ethanol and Carbon Dioxide + 1Butanol System. J. Chem. Eng. Data 1991,36, 303-307. Johnston, K. P.; Penninger, J. M. L. Supercritical Fluid Science and Technology; ACS Symposium Series 406; ACS: Washington, DC, 1989. Kasche, V.; Schlothauer, R.; Brunner, G. Enzyme Denaturation in Supercritical COz: Stabilizing Effects of S-S Bonds During the Depressurization Step. Biotechnol. Lett. 1988, 10, 569574. Lakowicz, J. R. Principles ofFluorescence Spectroscopy;Plenum: New York, 1983. Lakowicz, J. R.; Hogan, D. Dynamic Properties of the LipidWater Interface of Model Membranes as Revealed by LifetimeResolved FluorescenceEmission Spectra. Biochemistry 1981, 20, 1366-1377.
+
423
McHugh, M. A.; Krukonis, V. J. Supercritical Fluid Extraction-Principles and Practice; Butterworths: Boston, 1986. Pace, C. N.; Heinemann, U.; Hahn, U.; Saenger, W. Ribonuclease T1: Structure, Function and Stability. Angew. Chem., Int. Ed. Engl. 1991, 30, 343-360. Squires, T. G.; Paulaitis, M. E. Supercritical Fluids-Chemical Engineering Principles and Applications; ACS Symposium Series 329; ACS: Washington, 1987. Randolph, T. W.; Clark, D. S.; Blanch, H. W.; Prausnitz, J. M. Enzymatic Oxidation of Cholesterol Aggregates in Supercritical Carbon Dioxide. Science 1988a, 238,387-390. Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M. Enzyme Catalyzed Oxidation of Cholesterol in Supercritical Carbon Dioxide. AIChE J. 1988b, 34,1354-1360. Randolph, T. W. Enzymatic Catalysis in Supercritical Fluids. Ph.D. Thesis, University of California-Berkley, 1987. Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M.; Wilke, C. R. Enzymatic Catalysis in Supercritical Fluids. BiotechnoLLett. 1985, 7, 325-328. Reid, R. C.; Prausnitz, J. M.; Poling, B. E. The Properties of Gases & Liquids, 4th ed.; McGraw-Hill: New York, 1987. Schneider, G. M. Physicochemical Principles of Extraction with Supercritical Gases. Angew. Chem., Int. Ed. Engl. 1978,17, 716-727. Smith, R. M. Supercritical Fluid Chromatography; Smith, R. M., Ed.; Royal Society of Chemistry Monograph London, 1985. Suzuki, K.; Sue, H.; Itou, M.; Smith, R. L.; Inomata, H.; Arai, K.; Saito, S. Isothermal Vapor-Liquid Equilibria Data for Binary Systems at High Pressure: Carbon Dioxide-Methanol, Carbon Dioxide-Ethanol, Carbon Dioxide-1-Propanol, Methane-Ethmol, Methane-1-Propanol, Ethane-Ethanol, and E t h a n e l Propanol Systems. J. Chem. Eng. Data 1990,35, 63-66. Teale, F. W. J.; Weber, G. Ultraviolet Fluorescence of the Aromatic Amino Acids. Biochem. J. 1957,65, 476-482. Thomson, J. A.; Shirley, B. A.; Grimsley, G. R.; Pace, C. N. Conformational Stability and Mechanism of Folding of Ribonuclease T1. J. Biol. Chem. 1989,264, 11614-11620. Van Holde, K. E. Physical Biochemistry; Prentice-Hall: New Jersey, 1971. Williams, D. F. Extraction with Supercritical Gases. Chem.Eng. Sci. 1981,36, 1769-1788. Accepted March 24, 1992. Registry No. C02, 124-38-9;trypsin, 9002-07-7.
421
In Situ Studies of Protein Conformation in Supercritical Fluids: Trypsin in Carbon Dioxide JoAnn Zagrobelny and Frank V. Bright* Department of Chemistry, Acheson Hall, State University of New York at Buffalo, Buffalo, New York 14214
The conformation of the monomeric enzyme trypsin has been studied in supercritical carbon dioxide. Steady-state fluorescence spectroscopy is used t o follow the conformation of trypsin in situ as a function of C02 density. Our results show for the first time that protein denaturation can occur during the fluid compression step and that the native trypsin is only slightly more stable (1.2 kcal/mol) than the unfolded form. These results demonstrate the power of fluorescence spectroscopy as a tool for studying protein conformation and dynamics in supercritical fluids.
1. Introduction Supercritical fluids have received a great deal of attention because of the ease with which their chemical potential can be adjusted with pressure (Eckert et al., 1986a,b). That is, their physical properties (e.g., dielectric constant, refractive index, and density) can be tuned while the inherent molecular structure of the solvent is maintained. As a consequence, supercritical solvents have been used extensively for extractions (Brennecke and Eckert, 1989; McHugh and Krukonis, 19861, chromatography (Smith, 19881, chemical reaction processes (Eckert et al., 1986a),enhanced oil recovery (Squires and Paulaitis, 1987), and most recently as a medium for enzyme-catalyzed reactions (Aaltonen and Rantakyla, 1991). In 1985, two groups reported on enzymatic reactions in supercritical solvents (Randolph et al., 1985; Hammond et al., 1985). Followingthese reports, there has been steady work in this area (Randolph et al., 1988a,b) and the stateof-the-art has been reviewed recently (Aaltonen and Rantakyla, 1991). Although efforts in this area have increased, to the best of our knowledge, only a single report has appeared that compares the conformation of an enzyme dissolved in water and supercritical solvents (Randolph, 1987; Randolph et al., 1988a). In these experiments, several spin-labeled variants of cholesterol oxidase were studied in supercritical CO2 using electron paramagnetic resonance spectroscopy. The conclusion was that the conformation of these particular proteins was not influenced by the supercritical fluid. In contrast, Kasche and co-workers (Kasche et al., 1988) concluded that the achymotrypsin, trypsin, and penicillin amidase were partially denatured by supercritical C02. They proposed that decompression led to denaturation; however, in situ measurements were not carried out to substantiate this proposition. The enzymes were only studied after being subjected to supercritical C02. In this paper, we report on the in situ conformation of trypsin as it is maintained in supercritical CO2. Steadystate fluorescence spectroscopy is used as our analytical tool (Bright, 1988; Lakowicz, 1983). COz was chosen because it is the most widely used supercritical fluid, it is readily available in high purity, it has mild critical conditions (T,= 304.1 K; P, = 73.8 bar) (Johnston and Penninger, 1989;Reid et al., 1987;Williams, 1981;Schneider, 19781, it is nontoxic, there are extensive tabulations on the phase equilibria of CO2 with many cosolvents, including water (Jennings et al., 1991; Suzuki et al., 1990; 8756-7938/92/3008-0421$03.00/0
Chrastil, 19821, and its density is adjustable over a fairly broad range (e.g., at 310 K density can be adjusted from 0.116 to 0.857 g/cm3 with pressures from 50 to 200 bar) (Angus et al., 1976). Trypsin was studied because it was reported previously (Kasche et al., 1988) that (1) it is partially denatured in supercritical C02 and (2) denaturation presumably occurred during fluid decompression.
2. Experimental Procedures Bovine trypsin (EC 3.4.21.4) and C02 (SFC grade) were from Sigma and Scott, respectively, and were used as received. Distilled-deionized water was used throughout. The high-pressure pumping apparatus, optical cells, and fluorescence spectrometer have been described in detail elsewhere (Betta and Bright, 1990). In a typical experiment, a 145 mg/mL stock solution of aqueous trypsin is prepared. Ten microliters of this solution is added directly into the high-pressure optical cell (5 mL volume) resulting in a final concentration of 0.29 mg of trypsin/mL. The cell is then connected to the pumping apparatus, and the temperature is allowed to equilibrate a t 35 O C for 30-40 min. A vacuum is then maintained ( ~ 5 pmHg) 0 on the system for 10-20 min. This step was taken to remove residual 0 2 which may quench the protein fluorescence (Lakowicz, 1983). The cell is then slowly charged with C02 as described previously (Betta and Bright, 1990). The cell pressure is raised to the desired starting point, and the experiments are performed in order of increasing CO2 density while the contents of the cell are continuously stirred. The concentration of trypsin, in the cell, remains constant throughout our experiments. The experiments were carried out at densities where the amount of aqueous solution added would be solubilized in supercritical C02. The solubility of water is calculated using the following semiempirical formula (Chrastil, 1982): c = p'.55 exp(-2826/T
- 0.81)
(1) where c is the concentration of water in grams per liter, p is the density in grams per liter, and T is the temperature in Kelvin. The tryptophan amino acid residues within trypsin were selectively excited at 290 nm (Lakowicz, 1983). The subsequent tryptophan emission was studied because of its sensitivity to the local environment and its yield (Lakowicz, 1983). In this way, we can use the trypsin emission
0 1992 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1992, Vol. 8, No. 5 1.0,
-1.04 50 Pressure (bar)
Figure 1. Emission center of gravity for trypsin in supercritical COz (with 110 mM added HzO) as a function of pressure at 35 "C. Lower and upper dashed horizontal lines denote resulta for trypsin in pure liquid water and 8 M urea, respectively,at 35 O C . &,= = 290 nm (band-pass = 4 nm).
to follow the effects of the supercritical solvent on the enzyme conformation.
3. Results and Discussion When a protein unfolds, its unique three-dimensional structure is affected drastically and the protein proceeds ultimately to a random-coiled conformation (Pace et al., 1991;Van Holde, 1971). Because of the magnitude of these conformational changes, one can often follow the process with standard optical techniques (UV-vis, IR, and fluorescence) (Bright, 1988; Lakowicz, 1983). Figure 1shows the effects of CO2 pressure on the tryptophan residue fluorescence of trypsin. These results are shown as the center of gravity of the emission spectrum (Lakowiczand Hogan, 1981). Under these particular conditions, all water would be dissolved at about 84 bar (Chrastil, 19821, and a small portion of the protein becomes suspended in the supercritical fluid. For comparison, we show also fluorescence results (horizontal, dashed lines) for trypsin in liquid water and 8 M urea. Several interesting aspects of these results merit additional discussion. First, the emission spectra observed in water and urea are quite different from one another. Specifically, the spectrum for trypsin in water is blue shifted and indicates that the average tryptophan residue encounters a moderately hydrophobic environment (Lakowicz, 1983; Teale and Weber, 1957). This is to be contrasted with the urea results in which the protein is denatured significantly (Bohinski, 1979)and the emission contour is red shifted nearly 13 nm. The indication here is that trypsin is unfolded such that the tryptophan residues become accessible to water (a less hydrophobic environment) (Lakowicz, 1983). Second, at low C02 pressures we recover an emission spectrum similar to trypsin in water, but as CO2 pressure is increased, the emission contour steadily red shifts and approaches that of trypsin in urea. These results are consistent with increased C02 pressure leading to conformational changes in trypsin. Third, following a pressure increase the emission spectrum shifted rapidly (within 2-3 min) and remained constant over time. This indicates that there is not any slow, long-term drift in conformation over time. Finally, as can be seen in Figure 1, the conformational changes in trypsin do not, as reported previously (Kasche et al., 19881, occur exclusively during decompression. To prove this point, we performed a set of simple decompression experiments from 200 to 85 bar, and the emission contours were found to be identical a t the two pressures (results not shown). From these results, we conclude that decompression has very little effect on the conformation of trypsin.
I
100
150
200
I
250
Pressure (bar)
Figure 2. Recovered free energy of unfolding for trypsin as a function of COz pressure. Experimentalconditions are the same as in Figure 1.
To further analyze these C02 pressure-dependent denaturation data, we assume a two-state model of the form (Pace et al., 1991) folded a unfolded (2) where folded and unfolded represent the native and conformationally altered forms of trypsin, respectively. From this we can calculate the fraction of unfolded trypsin, F,, using
F,(P) = (CG(P) - CGf)/(CG, - CGf) (3) where CG(P) is the pressure-dependent emission center of gravity (Figure 11, which is used to follow changes in conformation, and CGf and CG, are the centers of gravity for the folded (water) and unfolded (urea) trypsin conformations, respectively. F,(P)is related to the pressuredependent equilibrium constant, K,,(P), by K,,(P) = F,(P)/(l- F,(P)) (4) which is in turn related to the pressure-dependent free energy of unfolding, AG(P), by
AG(P) = -RT In [K,,(P)] (5) where R and T are the gas constant and absolute temperature, respectively. For trypsin in supercritical Cog, AG is found to vary linearly with pressure (Figure 2). These results are reminiscent of results for protein denaturation by urea and temperature (Thomson et al., 1989). Extrapolation of these experimental results to zero pressure yields AG(H20),an estimate of the trypsin conformational stability. From thisparticular set of experiments,AG(H20) is 1.2 f 0.1 kcal/mol, which indicates that native trypsin is only marginally more stable than the unfolded form.
4. Conclusions We present the first in situ fluorescence measurements on protein conformation in supercritical fluid media. These results show clearlythat (1)there can be significant changes in protein conformation induced by supercritical solvents, (2) most of the conformational change occurs during compression, and (3) the native trypsin conformation is only slightly more stable compared to the unfolded form. These results also indicate that enzyme segments that undergo significant changes in environment may not be associated with the active site. In an effort to more accurately study the influence of supercritical fluids on protein conformation, we are investigating systems which contain a single tryptophan residue. We are also applying dynamic fluorescence techniques to these same systems (Bright et al., 1990).We shall report on the progress of these experiments in due course.
Biotechnol. hog., 1992, Vol. 8, No. 5
Acknowledgment This work was supported in part by the United States Department of Energy (DE-FG02-90ER14143).
Literature Cited Aaltonen, 0.;Rantakyla, M. Biocatalysis in Supercritical C02. CHEMTECH 1991,21,240-248. Angus, S.; Armstrong, B.; de Reuck, K. M. Carbon Dioxide: International Thermodynamic Tables of the Fluid State; Pergamon: New York, 1976; Vol. 3. Betta, T. A.; Bright, F. V. Instrumentation for Steady-Stateand Dynamic Fluorescence Studies in Supercritical Media. Appl. Spectrosc. 1990,44, 1196-1202. Brennecke, J. F.; Eckert, C. A. Phase Equilibria for Supercritical Fluid Process Design. MChE J. 1989, 35, 1409-1427. Bohinski, R. C. Modern Concepts in Biochemistry; Allyn and Bacon: Boston, 1979. Bright, F. V. Bioanalytical Applications of Fluorescence Spectroscopy. Anal. Chem. 1988, 60, 1031A-1039A. Bright, F. V.; Betta, T. A.; Litwiler, K. S. Advances in Multifrequency Phase and Modulation Fluorimetric Analysis. CRC Crit. Rev. Anal. Chem. 1990,21, 389-405. Chrastil, J. Solubilityof Solidsand Liquids in Supercritical Gases. J,Phys. Chem. 1982,86,3016-3021. Eckert, C. A.; Van Alsten, J. G.; Stoicos, T. Supercritical Fluid Processing. Environ. Sei. Technol. 1986a, 20, 319-325. Eckert, C. A.; Ziger, D. H.; Johnston, K. P.; Kim, S. Solute Partial Molal Volumes in Supercritical Fluids. J.Phys. Chem. 1986b, 90,27362745. Hammond, D. A.; Karel, M.; Klibanov, A. M.; Krukonis, V. J. Enzymatic Reactions in Supercritical Gases. Appl. Biochem. Biotechnol, 1985,11, 393-400. Jennings, D. W.; Lee, R.-J.; Teja, A. S. Vapor-Liquid Equilibria in the Carbon Dioxide Ethanol and Carbon Dioxide + 1Butanol System. J. Chem. Eng. Data 1991,36, 303-307. Johnston, K. P.; Penninger, J. M. L. Supercritical Fluid Science and Technology; ACS Symposium Series 406; ACS: Washington, DC, 1989. Kasche, V.; Schlothauer, R.; Brunner, G. Enzyme Denaturation in Supercritical COz: Stabilizing Effects of S-S Bonds During the Depressurization Step. Biotechnol. Lett. 1988, 10, 569574. Lakowicz, J. R. Principles ofFluorescence Spectroscopy;Plenum: New York, 1983. Lakowicz, J. R.; Hogan, D. Dynamic Properties of the LipidWater Interface of Model Membranes as Revealed by LifetimeResolved FluorescenceEmission Spectra. Biochemistry 1981, 20, 1366-1377.
+
423
McHugh, M. A.; Krukonis, V. J. Supercritical Fluid Extraction-Principles and Practice; Butterworths: Boston, 1986. Pace, C. N.; Heinemann, U.; Hahn, U.; Saenger, W. Ribonuclease T1: Structure, Function and Stability. Angew. Chem., Int. Ed. Engl. 1991, 30, 343-360. Squires, T. G.; Paulaitis, M. E. Supercritical Fluids-Chemical Engineering Principles and Applications; ACS Symposium Series 329; ACS: Washington, 1987. Randolph, T. W.; Clark, D. S.; Blanch, H. W.; Prausnitz, J. M. Enzymatic Oxidation of Cholesterol Aggregates in Supercritical Carbon Dioxide. Science 1988a, 238,387-390. Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M. Enzyme Catalyzed Oxidation of Cholesterol in Supercritical Carbon Dioxide. AIChE J. 1988b, 34,1354-1360. Randolph, T. W. Enzymatic Catalysis in Supercritical Fluids. Ph.D. Thesis, University of California-Berkley, 1987. Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M.; Wilke, C. R. Enzymatic Catalysis in Supercritical Fluids. BiotechnoLLett. 1985, 7, 325-328. Reid, R. C.; Prausnitz, J. M.; Poling, B. E. The Properties of Gases & Liquids, 4th ed.; McGraw-Hill: New York, 1987. Schneider, G. M. Physicochemical Principles of Extraction with Supercritical Gases. Angew. Chem., Int. Ed. Engl. 1978,17, 716-727. Smith, R. M. Supercritical Fluid Chromatography; Smith, R. M., Ed.; Royal Society of Chemistry Monograph London, 1985. Suzuki, K.; Sue, H.; Itou, M.; Smith, R. L.; Inomata, H.; Arai, K.; Saito, S. Isothermal Vapor-Liquid Equilibria Data for Binary Systems at High Pressure: Carbon Dioxide-Methanol, Carbon Dioxide-Ethanol, Carbon Dioxide-1-Propanol, Methane-Ethmol, Methane-1-Propanol, Ethane-Ethanol, and E t h a n e l Propanol Systems. J. Chem. Eng. Data 1990,35, 63-66. Teale, F. W. J.; Weber, G. Ultraviolet Fluorescence of the Aromatic Amino Acids. Biochem. J. 1957,65, 476-482. Thomson, J. A.; Shirley, B. A.; Grimsley, G. R.; Pace, C. N. Conformational Stability and Mechanism of Folding of Ribonuclease T1. J. Biol. Chem. 1989,264, 11614-11620. Van Holde, K. E. Physical Biochemistry; Prentice-Hall: New Jersey, 1971. Williams, D. F. Extraction with Supercritical Gases. Chem.Eng. Sci. 1981,36, 1769-1788. Accepted March 24, 1992. Registry No. C02, 124-38-9;trypsin, 9002-07-7.
