ANALYTICAL
BIOCHEMISTRY
73,
?6?-36%
(1976)
A High-Pressure Sample Cell for Circular Dichroism Studies R. D. HARRIS, M. JACOBS, M. M. LONG, Laboratory
of Molecular
Biophysics, Birmingham,
University Alabama
AND D. W. URRY
of Alabama 35294
in Birmingham.
Received November 10, 1975; accepted March 4. 1976 The design and application of a high-pressure sample cell is described for circular dichroism studies of molecular systems that undergo conformational change with application of high pressure. Mechanical and optical problems in highpressure cells, safety considerations in cell design, and the pressurizing system are discussed. Operating procedures developed to yield reproducible data and the results of a trifluoroethanol solution of a derivative of gramicidin A used as an example are outlined.
Circular dichroism is a sensitive means of following conformational changes of biomolecules. For those cases where conformational changes involve changes in volume of a molecule, it would be useful to be able to identify a given circular dichroism pattern with a conformation of greater 01 lesser volume and to calculate the volume change in going from one conformation to another. A particular area of application is the channel-forming polypeptides. It has been proposed that new polypeptide conformations occur wherein a channel is formed which is coincident with the helix axis, and the concept was introduced that these structures span the lipid layer of a cell membrane and provide a channel through which ion-selective permeation of membranes would occur (I). If such structures occur in homogeneous solutions then, depending on the relative size of solvent and channel diameter, the volume required for the molecule in a channel containing conformation would be significantly different in a solvent that can fill the channel than in a solvent that cannot fill the channel. Especially when multiple conformations are possible for a channel-forming molecular system, an increase in pressure should be able to cause a shift in the equilibria between conformations, and the change in volume would be calculable from the change in the equilibrium constant (2), i.e.; TV = 2.3RT log(KP,IKP,) p2 - PI
[II
In the present effort we report the design of a high-pressure cell suitable for circular dichroism instruments which use an electro-optic 363 Copyright All rights
0 1976 by Academic hess, Inc. of reproduction in any form rew’ved.
364
HARRIS
ET AL.
modulator for quarter wavelength retardation, and we demonstrate the efficacy of the high-pressure cell by showing the dramatic conformational change which occurs when a pressure of 2500 psi is applied to a trifluoroethanol solution of a derivative of gramicidin A, a channel-forming polypeptide antibiotic. METHODS Cell Design
A cell body suitable for high-pressure study was obtained from Precision Cells, Inc. The cell body, 316 stainless steel, is constructed as described by the manufacturer’s sketch No. 505. Assembly of sample chamber, fused silica windows, and retaining rings with requisite seals and adequate beam path (see Fig. 1) presented three major problems in obtaining a satisfactory high-pressure cell for circular dichroism studies: (i) distortion of CD baseline resulting from pressure-induced stress on the windows; (ii) shift in window position on pressurization; and (iii) obtaining O-ring seals which could withstand the solvents and not yield unacceptable amounts of absorbing materials into the solvents. The retaining rings were designed with a lo-mm aperture and for cylindrical windows 16 mm in diameter and 10 mm thick. In this configuration, testing to 400 atm indicated that the fused silica material was capable of withstanding a shear force of 105 kg/cm2. The windows are positioned within the cell body by threaded stainlesssteel retaining rings, and a pressure-tight O-ring seal is placed between the window and cell body. The retaining-ring configuration is such that it provides window support as well as forming one surface of the O-ring gland. The retaining ring being threaded provides the capability of varying the optical pathlength, but it also presents some window alignment difficulty. To ensure safe use of the pressure cell, the possibility of shearing the threads on the window retaining rings was considered. From the thread-
DCEA
FIG. 1. Exploded view of a high-pressure CD sample cell. A: Retaining rings; B: O-rings; C: fused silica windows; D: O-ring and window guides; E: window spacer; F: cell body; G: flare fitting for cell access: H: bottom plug. All the dimensions are in millimeters.
A HIGH-PRESSURE
CD SAMPLE
CELL
365
loaded relationship (3) T = 2FhDL, the shear stress T can be calculated, where F is the applied force, D is the thread root diameter, and L is the length of the threads. The root diameter is the overall diameter of the male threads less twice the thread depth. The applied force is the test pressure divided by the area defined by the root diameter of the retainingring threads. This expression, while normally applied to square threads, gives a good indication for safety considerations. A safety factor of two is inherent when this expression is applied to unified fine threads, making a rigorous treatment of thread fit unnecessary. For the pressure cell being described, the thread root diameter is 20.2 mm. The shear strength of 304 stainless steel, of which the retaining rings were made, is 1050 kg/cm*. The shear force that the threaded area would be exposed to at 136 atm was calculated to be 554 kg/cm2. Another important consideration that presented difficulty was the selection of O-rings of suitable composition. With the fixed inside diameter of the cell body and the window diameter required for the desired aperture an O-ring of 50 or 60 durometer was required. For the solvents being used, an inert material such as Teflon would probably have been the best. The noncompressibility of Teflon O-rings, however, prevented their use without modification of the cell body. O-rings of buna rubber and polyvinyl chloride produced pronounced effects on the optical absorbance in the wavelengths of interest using TFE, methanol, and ethanol as solvents. An O-ring with a special composition, material specification No. 6865, was obtained from American Seals, Inc., which had a minimal optical effect on the solvents being used. Pressurization
System
The pressurization system consists of a gaseous nitrogen tank, regulator, Haskel Engineering Model AG75C pressure intensifier, dial gauge, and two hand valves. The nitrogen supply is regulated to 1000 psi which feeds the high-pressure inlet to the intensifier. The line from the intensifier outlet to the cell has a bleed valve, a shut-off vlave. and a dial gauge. The shut-off valve permits closing off the cell at the desired pressure, and the bleed valve is necessary for system depressurization. The low-pressure drive to the intensifier is from the laboratory air supply or another gaseous nitrogen tank if pressures above 240 atm are required. The pressurant is coupled to the cell through a flare tube fitting. A flare fitting is used so that the cell can be connected and disconnected repeatedly without degradation of the seal. All other connections are Parker-Hannifin CPI tube fittings. Temperature Control A temperature jacket enables temperature studies using a circulating controlled temperature fluid. The jacket is cylindrical and of stainless-
366
HARRIS
ETAL.
steel construction with the top and bottom cut away for access to the cell ports. A special base plate was fabricated that positions the temperature jacket and cell such that the light path is perpendicular to the windows and passes through the center of the cell. Experimental
Procedure
The following procedure and sample handling techniques were established: Prior to pressurization, baseline distortions arising from nonparallel alignment of the windows were minimized by rotating the retaining rings with solvent in the cell. In this manner a baseline could be achieved in the wavelength range of interest, 300 to 200 nm, which varied no more than 11 millidegrees from the air baseline. Once optimal window alignment was achieved, the stainless-steel plugs (parts G and H of Fig. 1) were removed, and the cell was cleaned with repeated rinses of deionized water and ethanol and dried with vacuum. Before the plugs were replaced, they were wrapped with Teflon pipe thread tape to ensure a tight, leakproof seal. The cell was filled with solvent using the following technique to eliminate trapped bubbles of air in the bottom of the cell, i.e., the needle of aglass syringe was inserted through the top hole to the bottom plug and the solvent was injected. A check for air bubbles involved pressurizing the cell to 2500 psi, visually inspecting the windows, depressurizing, and then checking again. The pressure cell was placed in its temperature jacket in the CD with the same end always positioned close to the phototube. The pressure connection and clamps which anchor the temperature jacket to the base of the CD chamber were then secured. Next, baselines were run with solvent wavelength scans at 14.7 and 2500 psi; the latter pressure being attained by going to 4000 psi and then backing off to 2500 psi or any other pressure of interest. These baseline scans were duplicated by cycling through the series 14.7,4000, and 2500 psi. Sample wavelength scans were done in the identical way as the solvent scans and the effect of pressure is judged by the magnitude of the difference between the solvent baseline and sample with and without pressure. Sample concentration is adjusted to correspond with cell pathlength which can be changed by varying the thickness and number of stainless steel inserts (part E in Fig. 1). The advantage of the above procedure is that it provides reproducible data, and the inherent pressure effects on the solvent baseline can be minimized allowing detection of real differences in sample scans. RESULTS
The effect of pressure on the circular dichroism spectrum of hydrogenated gramicidin A is dramatic (Fig. 2). In trifluoroethanol, the conformation of gramicidin A changes from one of negative to positive
A HIGH-PRESSURE
CD SAMPLE
CELL
367
0.05 P bX 32 -0.05 -0. IO
I
1 220
I 240
1 260
X (nm)
FIG. 2. Circular dichroism scan of hydrogenated gramicidin A in TFE at 1 mgiml and 25°C with 14.7 and 2500 psi applied to the sample. This had been prepared 5 days prior to the experiment and remained at room temperature throughout this period. Traces of acetic acid from the hydrogenation reaction were present.
ellipticity, indicating a volume change with application of pressure. Studies in which the potentiometer baseline corrections are not made indicate that application of 2500 psi can alter the solvent baseline by 26 millidegrees and that the baseline at 2500 psi is reproducible to within 11 millidegrees at 225 nm. The total pressure effect for the sample at 225 nm was greater than 2000 millidegrees. The effect of pressure on sample molar ellipticity is 20 times greater than the effect of pressure on the baseline alone. For the sample conditions listed in Fig. 2, the change in ellipticity due to the change in pressure is 214 millidegrees with an uncertainty of the order of +ll millidegrees. DISCUSSION
Gramicidin A provides a model system for illustrating the applicability of this high-pressure cell to circular dichroism measurements. Pressure exerted a significant conformational change which correlates with the channel forming ability of this antibiotic. In trifluoroethanol, with the outlined experimental conditions, the structure would not have a pore large enough for solvent, thereby leaving a void in the molecule’s interior. Pressure causes a shift in the equilibrium between conformations to a con-
368
HARRISET
AL.
formational state of lesser volume. If a solvent system could be developed in which solvent entered the gramicidin A channel and thereby eliminated the void, this system would be an appropriate control for the trifluoroethanol experiment. There should be no pressure effect in such a system. Ethanol and ethanol:water (9: 1) provide an interesting control solvent system. Pressure significantly changed the CD spectrum of gramicidin A in ethanol; it had no effect when the solvent system was ethanol:water (9: 1). In this case, the channel can be thought of as large enough for water to fill the void but too small for ethanol to enter. A parallel to this work is the study made by Derechin ef al. (4) on the effect of concentration on the partial specific volume of gramicidin A. Above 1 mg/ml in ethanol, the partial specific volume was constant, below 1 mg/ml, it varied nonlinearly. In ethanol:water (9: 1) the change in partial specific volume with changes in concentration was not observed. An explanation of the result would be that only water entered the channel; ethanol was excluded. Gramicidin A exists in two or more conformational states. A temperature study of gramicidin A followed by plotting the single wavelength molar ellipticity as a function of temperature defines two conformational states for gramicidin A. A high temperature and a low temperature plateau are found which enable the equilibrium constant to be calculated (5). The temperature jacket of the pressure cell allows states to be defined at different pressures, and the ellipticity values which characterize high and low temeprature states allow equilibrium constants to be calculated at two different pressures. With values for KP, and KP, the change in volume can be calculated by Eq. [ 11. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grant HL-I 1310. The authors wish to thank Dr. Kouji Okamoto for the hydrogenation of the gramicidin A and David W. Mason for technical assistance.
REFERENCES 1. Urry, D. W. (1971) Proc. Nat. Acad. Sci. USA 68, 672-676. 2. Johnson, F. H., Eyring, H.. and Polissar, M. J. (1974) in The Theory of Rate Processes in Biology and Medicine, p. 295, Wiley. New York. 3. Shigley, J. E. (1972) in Mechanical Engineering Design, 2nd ed., p. 300, McGraw-Hill, New York. 4. Derechin, M., Hayashi, D. M., and Jordan, B. E. (1974) Life Sci. 15, 403-413. 5. Urry. D. W., Long, M. M.. Jacobs. M., and Harris, R. D. (1976)Ann. N.Y. Acad. Sci., 264,203-220. 6. Veatch. W. R., Fossel, E. T.. and Blout, E. R. (1974) Biochemistry 13,5249-5256.
BIOCHEMISTRY
73,
?6?-36%
(1976)
A High-Pressure Sample Cell for Circular Dichroism Studies R. D. HARRIS, M. JACOBS, M. M. LONG, Laboratory
of Molecular
Biophysics, Birmingham,
University Alabama
AND D. W. URRY
of Alabama 35294
in Birmingham.
Received November 10, 1975; accepted March 4. 1976 The design and application of a high-pressure sample cell is described for circular dichroism studies of molecular systems that undergo conformational change with application of high pressure. Mechanical and optical problems in highpressure cells, safety considerations in cell design, and the pressurizing system are discussed. Operating procedures developed to yield reproducible data and the results of a trifluoroethanol solution of a derivative of gramicidin A used as an example are outlined.
Circular dichroism is a sensitive means of following conformational changes of biomolecules. For those cases where conformational changes involve changes in volume of a molecule, it would be useful to be able to identify a given circular dichroism pattern with a conformation of greater 01 lesser volume and to calculate the volume change in going from one conformation to another. A particular area of application is the channel-forming polypeptides. It has been proposed that new polypeptide conformations occur wherein a channel is formed which is coincident with the helix axis, and the concept was introduced that these structures span the lipid layer of a cell membrane and provide a channel through which ion-selective permeation of membranes would occur (I). If such structures occur in homogeneous solutions then, depending on the relative size of solvent and channel diameter, the volume required for the molecule in a channel containing conformation would be significantly different in a solvent that can fill the channel than in a solvent that cannot fill the channel. Especially when multiple conformations are possible for a channel-forming molecular system, an increase in pressure should be able to cause a shift in the equilibria between conformations, and the change in volume would be calculable from the change in the equilibrium constant (2), i.e.; TV = 2.3RT log(KP,IKP,) p2 - PI
[II
In the present effort we report the design of a high-pressure cell suitable for circular dichroism instruments which use an electro-optic 363 Copyright All rights
0 1976 by Academic hess, Inc. of reproduction in any form rew’ved.
364
HARRIS
ET AL.
modulator for quarter wavelength retardation, and we demonstrate the efficacy of the high-pressure cell by showing the dramatic conformational change which occurs when a pressure of 2500 psi is applied to a trifluoroethanol solution of a derivative of gramicidin A, a channel-forming polypeptide antibiotic. METHODS Cell Design
A cell body suitable for high-pressure study was obtained from Precision Cells, Inc. The cell body, 316 stainless steel, is constructed as described by the manufacturer’s sketch No. 505. Assembly of sample chamber, fused silica windows, and retaining rings with requisite seals and adequate beam path (see Fig. 1) presented three major problems in obtaining a satisfactory high-pressure cell for circular dichroism studies: (i) distortion of CD baseline resulting from pressure-induced stress on the windows; (ii) shift in window position on pressurization; and (iii) obtaining O-ring seals which could withstand the solvents and not yield unacceptable amounts of absorbing materials into the solvents. The retaining rings were designed with a lo-mm aperture and for cylindrical windows 16 mm in diameter and 10 mm thick. In this configuration, testing to 400 atm indicated that the fused silica material was capable of withstanding a shear force of 105 kg/cm2. The windows are positioned within the cell body by threaded stainlesssteel retaining rings, and a pressure-tight O-ring seal is placed between the window and cell body. The retaining-ring configuration is such that it provides window support as well as forming one surface of the O-ring gland. The retaining ring being threaded provides the capability of varying the optical pathlength, but it also presents some window alignment difficulty. To ensure safe use of the pressure cell, the possibility of shearing the threads on the window retaining rings was considered. From the thread-
DCEA
FIG. 1. Exploded view of a high-pressure CD sample cell. A: Retaining rings; B: O-rings; C: fused silica windows; D: O-ring and window guides; E: window spacer; F: cell body; G: flare fitting for cell access: H: bottom plug. All the dimensions are in millimeters.
A HIGH-PRESSURE
CD SAMPLE
CELL
365
loaded relationship (3) T = 2FhDL, the shear stress T can be calculated, where F is the applied force, D is the thread root diameter, and L is the length of the threads. The root diameter is the overall diameter of the male threads less twice the thread depth. The applied force is the test pressure divided by the area defined by the root diameter of the retainingring threads. This expression, while normally applied to square threads, gives a good indication for safety considerations. A safety factor of two is inherent when this expression is applied to unified fine threads, making a rigorous treatment of thread fit unnecessary. For the pressure cell being described, the thread root diameter is 20.2 mm. The shear strength of 304 stainless steel, of which the retaining rings were made, is 1050 kg/cm*. The shear force that the threaded area would be exposed to at 136 atm was calculated to be 554 kg/cm2. Another important consideration that presented difficulty was the selection of O-rings of suitable composition. With the fixed inside diameter of the cell body and the window diameter required for the desired aperture an O-ring of 50 or 60 durometer was required. For the solvents being used, an inert material such as Teflon would probably have been the best. The noncompressibility of Teflon O-rings, however, prevented their use without modification of the cell body. O-rings of buna rubber and polyvinyl chloride produced pronounced effects on the optical absorbance in the wavelengths of interest using TFE, methanol, and ethanol as solvents. An O-ring with a special composition, material specification No. 6865, was obtained from American Seals, Inc., which had a minimal optical effect on the solvents being used. Pressurization
System
The pressurization system consists of a gaseous nitrogen tank, regulator, Haskel Engineering Model AG75C pressure intensifier, dial gauge, and two hand valves. The nitrogen supply is regulated to 1000 psi which feeds the high-pressure inlet to the intensifier. The line from the intensifier outlet to the cell has a bleed valve, a shut-off vlave. and a dial gauge. The shut-off valve permits closing off the cell at the desired pressure, and the bleed valve is necessary for system depressurization. The low-pressure drive to the intensifier is from the laboratory air supply or another gaseous nitrogen tank if pressures above 240 atm are required. The pressurant is coupled to the cell through a flare tube fitting. A flare fitting is used so that the cell can be connected and disconnected repeatedly without degradation of the seal. All other connections are Parker-Hannifin CPI tube fittings. Temperature Control A temperature jacket enables temperature studies using a circulating controlled temperature fluid. The jacket is cylindrical and of stainless-
366
HARRIS
ETAL.
steel construction with the top and bottom cut away for access to the cell ports. A special base plate was fabricated that positions the temperature jacket and cell such that the light path is perpendicular to the windows and passes through the center of the cell. Experimental
Procedure
The following procedure and sample handling techniques were established: Prior to pressurization, baseline distortions arising from nonparallel alignment of the windows were minimized by rotating the retaining rings with solvent in the cell. In this manner a baseline could be achieved in the wavelength range of interest, 300 to 200 nm, which varied no more than 11 millidegrees from the air baseline. Once optimal window alignment was achieved, the stainless-steel plugs (parts G and H of Fig. 1) were removed, and the cell was cleaned with repeated rinses of deionized water and ethanol and dried with vacuum. Before the plugs were replaced, they were wrapped with Teflon pipe thread tape to ensure a tight, leakproof seal. The cell was filled with solvent using the following technique to eliminate trapped bubbles of air in the bottom of the cell, i.e., the needle of aglass syringe was inserted through the top hole to the bottom plug and the solvent was injected. A check for air bubbles involved pressurizing the cell to 2500 psi, visually inspecting the windows, depressurizing, and then checking again. The pressure cell was placed in its temperature jacket in the CD with the same end always positioned close to the phototube. The pressure connection and clamps which anchor the temperature jacket to the base of the CD chamber were then secured. Next, baselines were run with solvent wavelength scans at 14.7 and 2500 psi; the latter pressure being attained by going to 4000 psi and then backing off to 2500 psi or any other pressure of interest. These baseline scans were duplicated by cycling through the series 14.7,4000, and 2500 psi. Sample wavelength scans were done in the identical way as the solvent scans and the effect of pressure is judged by the magnitude of the difference between the solvent baseline and sample with and without pressure. Sample concentration is adjusted to correspond with cell pathlength which can be changed by varying the thickness and number of stainless steel inserts (part E in Fig. 1). The advantage of the above procedure is that it provides reproducible data, and the inherent pressure effects on the solvent baseline can be minimized allowing detection of real differences in sample scans. RESULTS
The effect of pressure on the circular dichroism spectrum of hydrogenated gramicidin A is dramatic (Fig. 2). In trifluoroethanol, the conformation of gramicidin A changes from one of negative to positive
A HIGH-PRESSURE
CD SAMPLE
CELL
367
0.05 P bX 32 -0.05 -0. IO
I
1 220
I 240
1 260
X (nm)
FIG. 2. Circular dichroism scan of hydrogenated gramicidin A in TFE at 1 mgiml and 25°C with 14.7 and 2500 psi applied to the sample. This had been prepared 5 days prior to the experiment and remained at room temperature throughout this period. Traces of acetic acid from the hydrogenation reaction were present.
ellipticity, indicating a volume change with application of pressure. Studies in which the potentiometer baseline corrections are not made indicate that application of 2500 psi can alter the solvent baseline by 26 millidegrees and that the baseline at 2500 psi is reproducible to within 11 millidegrees at 225 nm. The total pressure effect for the sample at 225 nm was greater than 2000 millidegrees. The effect of pressure on sample molar ellipticity is 20 times greater than the effect of pressure on the baseline alone. For the sample conditions listed in Fig. 2, the change in ellipticity due to the change in pressure is 214 millidegrees with an uncertainty of the order of +ll millidegrees. DISCUSSION
Gramicidin A provides a model system for illustrating the applicability of this high-pressure cell to circular dichroism measurements. Pressure exerted a significant conformational change which correlates with the channel forming ability of this antibiotic. In trifluoroethanol, with the outlined experimental conditions, the structure would not have a pore large enough for solvent, thereby leaving a void in the molecule’s interior. Pressure causes a shift in the equilibrium between conformations to a con-
368
HARRISET
AL.
formational state of lesser volume. If a solvent system could be developed in which solvent entered the gramicidin A channel and thereby eliminated the void, this system would be an appropriate control for the trifluoroethanol experiment. There should be no pressure effect in such a system. Ethanol and ethanol:water (9: 1) provide an interesting control solvent system. Pressure significantly changed the CD spectrum of gramicidin A in ethanol; it had no effect when the solvent system was ethanol:water (9: 1). In this case, the channel can be thought of as large enough for water to fill the void but too small for ethanol to enter. A parallel to this work is the study made by Derechin ef al. (4) on the effect of concentration on the partial specific volume of gramicidin A. Above 1 mg/ml in ethanol, the partial specific volume was constant, below 1 mg/ml, it varied nonlinearly. In ethanol:water (9: 1) the change in partial specific volume with changes in concentration was not observed. An explanation of the result would be that only water entered the channel; ethanol was excluded. Gramicidin A exists in two or more conformational states. A temperature study of gramicidin A followed by plotting the single wavelength molar ellipticity as a function of temperature defines two conformational states for gramicidin A. A high temperature and a low temperature plateau are found which enable the equilibrium constant to be calculated (5). The temperature jacket of the pressure cell allows states to be defined at different pressures, and the ellipticity values which characterize high and low temeprature states allow equilibrium constants to be calculated at two different pressures. With values for KP, and KP, the change in volume can be calculated by Eq. [ 11. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grant HL-I 1310. The authors wish to thank Dr. Kouji Okamoto for the hydrogenation of the gramicidin A and David W. Mason for technical assistance.
REFERENCES 1. Urry, D. W. (1971) Proc. Nat. Acad. Sci. USA 68, 672-676. 2. Johnson, F. H., Eyring, H.. and Polissar, M. J. (1974) in The Theory of Rate Processes in Biology and Medicine, p. 295, Wiley. New York. 3. Shigley, J. E. (1972) in Mechanical Engineering Design, 2nd ed., p. 300, McGraw-Hill, New York. 4. Derechin, M., Hayashi, D. M., and Jordan, B. E. (1974) Life Sci. 15, 403-413. 5. Urry. D. W., Long, M. M.. Jacobs. M., and Harris, R. D. (1976)Ann. N.Y. Acad. Sci., 264,203-220. 6. Veatch. W. R., Fossel, E. T.. and Blout, E. R. (1974) Biochemistry 13,5249-5256.
