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vis biochemical spectroscopy under high pressure
High Pressure Chemical Engineering Ph. Rudolf von Rohr and Ch. Trepp (Editors) 1996 Elsevier Science B.V.
553
UV/vis Biochemical Spectroscopy under High Pressure C. Balny a, J.-L. Saldana a, R. Lange a, M. J. Kornblatt b and J. A. Kornblatt b aINSERM, Unit~ 128, BP 5051, 34033 Montpellier, Cedex 1, France bConcordia University, 1455 de Maisonneuve Blvd W, PQ H3G 1M8 Canada Abstract
High hydrostatic pressure induces changes in protein conformation, solvation and enzyme activities via reversible and non-reversible effects on intra- and inter-molecular interactions (noncovalent bonds) [1]. To have access to these structural modifications, spectroscopic investigations are required which necessitate special spectroscopic adaptations. Two improvements are presented : first for enzyme reactions and second for structural determination. 1. I N T R O D U C T I O N
A complete study of catalytic reactions involving a succession of very rapid different steps, consists of the exploration of the properties of these steps, including thermodynamic parameters obtained by the action of temperature and pressure. One difficulty in measuring kinetics using spectroscopic detection method is the relatively long dead-time of the high-pressure techniques. Different devices have been proposed to eliminate sources of errors in time and temperature. These techniques include in situ initiation of the reaction after the heat of compression has been dissipated. To reduce the deadtime with respect to the reactions studied, we have combined low temperature which decreases reaction velocities according to the Arrhenius expression, with the stopped-flow method which provides the rapid mixing of two compounds. The second recent spectroscopic improvement is second and fourth derivative spectroscopies in the ultraviolet region of proteins. Derivative spectroscopy is a new tool for analyzing the effects of pressure on proteins. It permits one to enhance selectively spectral changes due to the UV absorbance of phenylalanine, tyrosine and tryptophan. The solvent polarity affects the amplitude, the position and the shape of the second and fourth derivative spectral bands. 2. THE HIGH PRESSURE STOPPED-FLOW APPARATUS The relatively long dead-time of high-pressure techniques using spectroscopic detection is the first important limitation to exploiting enzyme kinetics. If the system under study can be characterized via optical detection
554 (absorbancy, fluorescence), the stopped-flow method is easily used. The second limitation deals with the t e m p e r a t u r e control which m u s t be efficient to compensate the heat of compression. To solve these problems, devices were designed to mix samples under high pressure, at controlled temperatures. The first a p p a r a t u s was described by Grieger and Eckert and modified by Sasaki et al.. Both systems were thermostated and used the breakage of a foil d i a p h r a g m for the mixing of two components. After different improvements, the Heremans' group described a stopped-flow a p p a r a t u s designed for spectroscopic detections of fast reactions at pressures up to 120 MPa by m e a n s of i m m e r s i n g a stopped-flow unit in a high-pressure bomb. Since the, other high-pressure stopped-flow devices have been described [2]. To reduce the dead-time with respect to the reactions studied, we have developped an a p p a r a t u s which permits rapid mixing. The high p r e s s u r e stopped-flow system functions at low t e m p e r a t u r e thereby slowing reaction velocities according to the A r r h e n i u s equation. The design of our i n s t r u m e n t incorporates certain features of previous stopped-flow systems described for cryoenzymological studies and for investigations under high-pressure. The general design, already published [3,4], consists of a powerful p n e u m a t i c system driving the syringe mechanism, two vertical drive syringes c o n t a i n i n g the samples to be mixed, a mixing chamber, an observation chamber with quartz windows, and a waste syringe. Both t e m p e r a t u r e and p r e s s u r e homogeneities are m a i n t a i n e d by housing the whole a p p a r a t u s in a highpressure thermostated bomb. The stopped-flow a p p a r a t u s can operate in absorbance or fluorescence mode over temperature and pressure ranges of + 40 to - 35 ~ C and of 1 to 300 MPa, respectively. The system is mounted either on a n Aminco DW2 spectrophotometer or on a spectrofluorometer specially designed in the laboratory (wavelength limits : 230 - 650 nm). The dead-time, nearly independent of pressure, is less than 5 ms using aqueous solutions at room temperature. Using this device, different biological systems have been examined allowing the development of cryobaroenzymatic studies [1, 5]. 3. DERIVATIVE S P E C T R O S C O P Y METHODS
The main problems for a protein chemist or enzymologist who wishes to study the effects of pressure on a protein are a) to observe and quantitate the effects and b) to interpret them. The majority of proteins do not contain easily studied chromophores but they do contain substantial amounts of the a r o m a t i c amino acids, tryptophan and tyrosine. These amino acids are characterized by strong but overlapping absorption bands in the ultraviolet. A protein that contains five to ten of each of the aromatics presents a spectrum that is a composite of the contributions of each. A change in a protein, such as a conformational change, dissociation, or denaturation, may result in a c h a n g e in the environment of one or more of these residues. However, the UV spectrum of a protein is broad and featureless; the changes of interest are m a s k e d by the contributions from all the other aromatics whose e n v i r o n m e n t has not changed.
555 3.1 Second derivative spectroscopy Second derivative spectroscopy is a means to extract information from the spectrum. In contrast to the relatively featureless UV spectra, the second derivatives of UV spectra are characterized by 2 sharp peaks and troughs [6,7]. Figure 1 shows the ultraviolet spectra of yeast enolase at 0.28 and 200 MPa and Figure 2 shows the second derivative of those spectra.
Figure 1. UVspectra of yeast enolase at 0.28 and 200 MPa
Figure 2. Second derivative of the UV spectra
Ragone [7] has determined the effects of solvent polarity on the second derivative spectra of model compounds. For both tyrosine and tryptophan, as the solvent polarity goes from non-polar (as when the residue is buried inside a protein) to polar (exposed to solvent), the peak and trough positions shift to the blue (shorter wavelengths). There are also changes in amplitude, which are best described by calculating the ratio (r = a/b) of the two peak-to-trough values m a r k e d in Figure 2 [6,7]. For tyrosine, the value of r decreases as solvent polarity decreases, while the value of r for tryptophan is almost independent of solvent polarity. Thus, in the second derivative spectrum of a protein, the ratio of the peak to trough values and the positions of the peaks and troughs are a function of the relative amounts of the two amino acids and of the average polarity of the environments of the tyrosines as well as that of the tryptophans. For the protein shown in Figure 2, increasing pressure from 0.28 to 200 MPa has decreased the average polarity around the tryosine residues, while not changing the environment around the tryptophan residues. Extracting the physical m e a n i n g of the changes in second derivative spectra relies on knowing the amino acid composition of the protein and u s i n g the constituent spectra to simulate the observed spectra. Simulations were performed by combining the second derivative spectra of tyrosine- and tryptophan-ethylester in solvents of various dielectric constants, in a molar ratio of 9 tyr to 5 trp (that of enolase). Figure 3 shows the observed spectra of native (dimeric) yeast enolase and that of tyr in water and trp in 100 and 50 % ethanol. The simulations produce average values for changes in polarity. When an Xray structure is also available, it may be possible to assign the changes in polarity to changes in the environment of specific tyrosines or tryptophans.
556
Figure 3. Simulated spectra of enolase. ( - - ) native enolase ; a) tyr in w a t e r and trp in 100 % ethanol, b) tyr in w a t e r and trp in 50 % ethanol
Figure 4. X-ray structure of enolase
P r e s s u r e has previously been shown to dissociate the enolase dimer into monomers [8] ; the pressure range in which this occurs is the same as that in which we observe changes in the second derivative spectra. Dissociation is accompanied by a net hydration of the surface buried at the subunit interface. The second derivative results indicate that the average tyrosine polarity decreases, the opposite of w h a t one would expect if hydration is o c c u r r i n g . Using the X-ray s t r u c t u r e (Figure 4 ) [ 9 ] , we can resolve this a p p a r e n t contradiction [10]. In the dimeric form, there is a water-filled cleft between the two monomers. Two tyrosines from each monomer point into this cleft. As dissociation occurs, these tyrosines go from a region in which they are exposed to relatively rigid waters - ergo fixed dipoles - to one in which they are exposed to bulk water. The net change is a decrease in polarity that accompanies a net increase in hydration. The use of second derivative spectroscopy allows us to observe the effects of p r e s s u r e on this enzyme, to quantitate the changes occuring (we can use the values of r to calculate Keq as a function of pressure and hence AG and AV for dissociation) and to interpret those changes in terms of the known s t r u c t u r e of the enzyme. Second derivative spectroscopy has both advantages a n d disadvantages vis-a-vis fourth derivative spectroscopy. With second derivatives, one uses the ratio of the amplitudes, which is independent of the absolute absorbance. Therefore, one does not have to correct the spectra for c o m p r e s s i o n effects and can easily compare samples studied at different times. The changes, however, are dominated by the contributions of tyrosines and provide much less information about the tryptophan residues.
557 3,2 F o u r t h d e r i v a t i v e s p e c t r o s c o p y The selective resolution e n h a n c e m e n t in derivative spectroscopy is p u s h e d even f u r t h e r in the fourth derivative mode. As in the case of second derivative spectroscopy, the a m p l i t u d e and the position of the derivative spectral bands of the a r o m a t i c a m i n o acids are related to the polarity of the m e d i u m . We have u n d e r t a k e n a s y s t e m a t i c investigation of these spectral features of the N-acetyl O-ethyl esters of tyrosine and t r y p t o p h a n in various solvents of different polarity (from cyclohexane to water). Astonishingly, a simple r e l a t i o n s h i p between the spectral p a r a m e t e r s of the fourth derivatives and the dielectric c o n s t a n t w a s found [11]. As shown in Figure 5, for tyrosine it is the position of kmax, and for t r y p t o p h a n it is the derivative a m p l i t u d e which depends linearly on the dielectric c o n s t a n t e r. Since in addition the fourth derivative spectra of these model c o m p o u n d s do not depend significantly on p r e s s u r e (at least up to 500 MPa), these spectral features m a y be used as an intrinsic probe to sense the dielectric c o n s t a n t in the vicinity of tyrosine and t r y p t o p h a n . 288 286
"<
3
< <]
2
...im
El ,'< 2 8 4 282 ,
0
,
,,
I
2O
. . . .
I
4O
. . . .
I
6O
.....
1~
80
0
20
dielectric constant,
40
60
80
er
F i g u r e 5. Relation between a m p l i t u d e and kma x of the 4th derivative spectra of tyrosine and t r y p t o p h a n on the dielectric constant er. A good e x a m p l e of application is given by the protein s t r u c t u r a l changes of bovine ribonuclease A in the course of its d e n a t u r a t i o n by p r e s s u r e . The U V s p e c t r u m of R N a s e is d o m i n a t e d by the absorbance of tyrosine - this RNase does not contain t r y p t o p h a n . As shown in Figure 6, an increase of p r e s s u r e from 1 to 500 M P a r e s u l t s in a blue-shift of the 4th derivative m a x i m u m from 285.7 + 0.05 to 283.5 + 0.05 nm. This shift of 2.2 n m corresponds to an increase of the m e a n dielectric constant from 25 to 59. It is characteristic of the exposure to the aqueous solvent of part of the 6 tyrosines, as it is expected for a partly d e n a t u r a t i o n . The t r a n s i t i o n is fully reversible with clear isosbestic points. The p r e s s u r e effect can therefore be described by a simple two-state model between the native (er = 25) and the partially d e n a t u r e d (er = 59) state. A s i m u l a t i o n on the basis of this model p e r m i t t e d us to d e t e r m i n e the t h e r m o d y n a m i c p a r a m e ters of this t r a n s i t i o n : AG~ = 10.3 kJ/mol and AV = - 52 ml/mol. A c o m p a r i s o n w i t h r e s u l t s obtained by other m e t h o d s indicates t h a t the (~ = 59) state corresponds to an i n t e r m e d i a t e in the defolding process which h a s m o l t e n globule like c h a r a c t e r i s t i c s [12]. It t h u s a p p e a r s t h a t fourth derivative
558 spectroscopy under high pressure is a suitable technique to investigate protein structural changes occurring during the folding or defolding processes. 0.2 ....
~'
4
...1 M P a
0.1
c<3
<~
O0
-0
1
t 275
i
i 280
i
!.
285 wavelength,
~
! 290
l, 295
nm
Figure 6. Effect of pressure on the fourth derivative spectra of RNase A at pH2. 4. ACKNOWI~DGMENTS The authors thank Mrs N. Bec for her technical help and NSERC Canada for financial support. 5. REFERENCES 1 V.V. Mozhaev, K. Heremans, J. Frank, P. Masson and C. Balny, Proteins : Structure, Function, and Genetics, 24 (1996) 81. 2 J-L. Saldana and C. Balny in High Pressure and Biotechnology (C. Balny et al. eds) J. Libbey Eurotext/INSERM, Montrouge, France, vol. 224 (1992) 529. 3 C. Balny, J-L. Saldana, and N. Dahan, Anal. Biochem., 139 (1984) 178. 4 C. Balny, J-L. Saldana, and N. Dahan, Anal. Biochem., 163 (1986) 309. 5 C. Balny, P. Masson and F. Travers, High Pres. Res., 2 (1989) 1. 6 K. Ruckpaul, H. Rein, D.P. Ballou and M.J. Coon, Biochem. Biophys. 7 R. Ragone, G. Colonana, C. Balestrieri, L. Servillo and G. Irace, 8 A.A. Paladini and G. Weber, Biochemistry 20 (1981) 2587. 9 B. Stecand L. Lebioda, J. Mol. Biol 211 (1990) 235. 10 J.A. Kornblatt, M.J. Kornblatt and G. Hui Bon Hoa, Biochemistry, 34 (1995) 1218. 11 R. Lange, J. Frank, J.-L. Saldana and C. Balny, Eur. Biophys. J. (1996) in press. 12 R. Lange, N. Bec, V.V. Mozhaev and J. Frank, Eur. Biophys. J. (1996) in press.
553
UV/vis Biochemical Spectroscopy under High Pressure C. Balny a, J.-L. Saldana a, R. Lange a, M. J. Kornblatt b and J. A. Kornblatt b aINSERM, Unit~ 128, BP 5051, 34033 Montpellier, Cedex 1, France bConcordia University, 1455 de Maisonneuve Blvd W, PQ H3G 1M8 Canada Abstract
High hydrostatic pressure induces changes in protein conformation, solvation and enzyme activities via reversible and non-reversible effects on intra- and inter-molecular interactions (noncovalent bonds) [1]. To have access to these structural modifications, spectroscopic investigations are required which necessitate special spectroscopic adaptations. Two improvements are presented : first for enzyme reactions and second for structural determination. 1. I N T R O D U C T I O N
A complete study of catalytic reactions involving a succession of very rapid different steps, consists of the exploration of the properties of these steps, including thermodynamic parameters obtained by the action of temperature and pressure. One difficulty in measuring kinetics using spectroscopic detection method is the relatively long dead-time of the high-pressure techniques. Different devices have been proposed to eliminate sources of errors in time and temperature. These techniques include in situ initiation of the reaction after the heat of compression has been dissipated. To reduce the deadtime with respect to the reactions studied, we have combined low temperature which decreases reaction velocities according to the Arrhenius expression, with the stopped-flow method which provides the rapid mixing of two compounds. The second recent spectroscopic improvement is second and fourth derivative spectroscopies in the ultraviolet region of proteins. Derivative spectroscopy is a new tool for analyzing the effects of pressure on proteins. It permits one to enhance selectively spectral changes due to the UV absorbance of phenylalanine, tyrosine and tryptophan. The solvent polarity affects the amplitude, the position and the shape of the second and fourth derivative spectral bands. 2. THE HIGH PRESSURE STOPPED-FLOW APPARATUS The relatively long dead-time of high-pressure techniques using spectroscopic detection is the first important limitation to exploiting enzyme kinetics. If the system under study can be characterized via optical detection
554 (absorbancy, fluorescence), the stopped-flow method is easily used. The second limitation deals with the t e m p e r a t u r e control which m u s t be efficient to compensate the heat of compression. To solve these problems, devices were designed to mix samples under high pressure, at controlled temperatures. The first a p p a r a t u s was described by Grieger and Eckert and modified by Sasaki et al.. Both systems were thermostated and used the breakage of a foil d i a p h r a g m for the mixing of two components. After different improvements, the Heremans' group described a stopped-flow a p p a r a t u s designed for spectroscopic detections of fast reactions at pressures up to 120 MPa by m e a n s of i m m e r s i n g a stopped-flow unit in a high-pressure bomb. Since the, other high-pressure stopped-flow devices have been described [2]. To reduce the dead-time with respect to the reactions studied, we have developped an a p p a r a t u s which permits rapid mixing. The high p r e s s u r e stopped-flow system functions at low t e m p e r a t u r e thereby slowing reaction velocities according to the A r r h e n i u s equation. The design of our i n s t r u m e n t incorporates certain features of previous stopped-flow systems described for cryoenzymological studies and for investigations under high-pressure. The general design, already published [3,4], consists of a powerful p n e u m a t i c system driving the syringe mechanism, two vertical drive syringes c o n t a i n i n g the samples to be mixed, a mixing chamber, an observation chamber with quartz windows, and a waste syringe. Both t e m p e r a t u r e and p r e s s u r e homogeneities are m a i n t a i n e d by housing the whole a p p a r a t u s in a highpressure thermostated bomb. The stopped-flow a p p a r a t u s can operate in absorbance or fluorescence mode over temperature and pressure ranges of + 40 to - 35 ~ C and of 1 to 300 MPa, respectively. The system is mounted either on a n Aminco DW2 spectrophotometer or on a spectrofluorometer specially designed in the laboratory (wavelength limits : 230 - 650 nm). The dead-time, nearly independent of pressure, is less than 5 ms using aqueous solutions at room temperature. Using this device, different biological systems have been examined allowing the development of cryobaroenzymatic studies [1, 5]. 3. DERIVATIVE S P E C T R O S C O P Y METHODS
The main problems for a protein chemist or enzymologist who wishes to study the effects of pressure on a protein are a) to observe and quantitate the effects and b) to interpret them. The majority of proteins do not contain easily studied chromophores but they do contain substantial amounts of the a r o m a t i c amino acids, tryptophan and tyrosine. These amino acids are characterized by strong but overlapping absorption bands in the ultraviolet. A protein that contains five to ten of each of the aromatics presents a spectrum that is a composite of the contributions of each. A change in a protein, such as a conformational change, dissociation, or denaturation, may result in a c h a n g e in the environment of one or more of these residues. However, the UV spectrum of a protein is broad and featureless; the changes of interest are m a s k e d by the contributions from all the other aromatics whose e n v i r o n m e n t has not changed.
555 3.1 Second derivative spectroscopy Second derivative spectroscopy is a means to extract information from the spectrum. In contrast to the relatively featureless UV spectra, the second derivatives of UV spectra are characterized by 2 sharp peaks and troughs [6,7]. Figure 1 shows the ultraviolet spectra of yeast enolase at 0.28 and 200 MPa and Figure 2 shows the second derivative of those spectra.
Figure 1. UVspectra of yeast enolase at 0.28 and 200 MPa
Figure 2. Second derivative of the UV spectra
Ragone [7] has determined the effects of solvent polarity on the second derivative spectra of model compounds. For both tyrosine and tryptophan, as the solvent polarity goes from non-polar (as when the residue is buried inside a protein) to polar (exposed to solvent), the peak and trough positions shift to the blue (shorter wavelengths). There are also changes in amplitude, which are best described by calculating the ratio (r = a/b) of the two peak-to-trough values m a r k e d in Figure 2 [6,7]. For tyrosine, the value of r decreases as solvent polarity decreases, while the value of r for tryptophan is almost independent of solvent polarity. Thus, in the second derivative spectrum of a protein, the ratio of the peak to trough values and the positions of the peaks and troughs are a function of the relative amounts of the two amino acids and of the average polarity of the environments of the tyrosines as well as that of the tryptophans. For the protein shown in Figure 2, increasing pressure from 0.28 to 200 MPa has decreased the average polarity around the tryosine residues, while not changing the environment around the tryptophan residues. Extracting the physical m e a n i n g of the changes in second derivative spectra relies on knowing the amino acid composition of the protein and u s i n g the constituent spectra to simulate the observed spectra. Simulations were performed by combining the second derivative spectra of tyrosine- and tryptophan-ethylester in solvents of various dielectric constants, in a molar ratio of 9 tyr to 5 trp (that of enolase). Figure 3 shows the observed spectra of native (dimeric) yeast enolase and that of tyr in water and trp in 100 and 50 % ethanol. The simulations produce average values for changes in polarity. When an Xray structure is also available, it may be possible to assign the changes in polarity to changes in the environment of specific tyrosines or tryptophans.
556
Figure 3. Simulated spectra of enolase. ( - - ) native enolase ; a) tyr in w a t e r and trp in 100 % ethanol, b) tyr in w a t e r and trp in 50 % ethanol
Figure 4. X-ray structure of enolase
P r e s s u r e has previously been shown to dissociate the enolase dimer into monomers [8] ; the pressure range in which this occurs is the same as that in which we observe changes in the second derivative spectra. Dissociation is accompanied by a net hydration of the surface buried at the subunit interface. The second derivative results indicate that the average tyrosine polarity decreases, the opposite of w h a t one would expect if hydration is o c c u r r i n g . Using the X-ray s t r u c t u r e (Figure 4 ) [ 9 ] , we can resolve this a p p a r e n t contradiction [10]. In the dimeric form, there is a water-filled cleft between the two monomers. Two tyrosines from each monomer point into this cleft. As dissociation occurs, these tyrosines go from a region in which they are exposed to relatively rigid waters - ergo fixed dipoles - to one in which they are exposed to bulk water. The net change is a decrease in polarity that accompanies a net increase in hydration. The use of second derivative spectroscopy allows us to observe the effects of p r e s s u r e on this enzyme, to quantitate the changes occuring (we can use the values of r to calculate Keq as a function of pressure and hence AG and AV for dissociation) and to interpret those changes in terms of the known s t r u c t u r e of the enzyme. Second derivative spectroscopy has both advantages a n d disadvantages vis-a-vis fourth derivative spectroscopy. With second derivatives, one uses the ratio of the amplitudes, which is independent of the absolute absorbance. Therefore, one does not have to correct the spectra for c o m p r e s s i o n effects and can easily compare samples studied at different times. The changes, however, are dominated by the contributions of tyrosines and provide much less information about the tryptophan residues.
557 3,2 F o u r t h d e r i v a t i v e s p e c t r o s c o p y The selective resolution e n h a n c e m e n t in derivative spectroscopy is p u s h e d even f u r t h e r in the fourth derivative mode. As in the case of second derivative spectroscopy, the a m p l i t u d e and the position of the derivative spectral bands of the a r o m a t i c a m i n o acids are related to the polarity of the m e d i u m . We have u n d e r t a k e n a s y s t e m a t i c investigation of these spectral features of the N-acetyl O-ethyl esters of tyrosine and t r y p t o p h a n in various solvents of different polarity (from cyclohexane to water). Astonishingly, a simple r e l a t i o n s h i p between the spectral p a r a m e t e r s of the fourth derivatives and the dielectric c o n s t a n t w a s found [11]. As shown in Figure 5, for tyrosine it is the position of kmax, and for t r y p t o p h a n it is the derivative a m p l i t u d e which depends linearly on the dielectric c o n s t a n t e r. Since in addition the fourth derivative spectra of these model c o m p o u n d s do not depend significantly on p r e s s u r e (at least up to 500 MPa), these spectral features m a y be used as an intrinsic probe to sense the dielectric c o n s t a n t in the vicinity of tyrosine and t r y p t o p h a n . 288 286
"<
3
< <]
2
...im
El ,'< 2 8 4 282 ,
0
,
,,
I
2O
. . . .
I
4O
. . . .
I
6O
.....
1~
80
0
20
dielectric constant,
40
60
80
er
F i g u r e 5. Relation between a m p l i t u d e and kma x of the 4th derivative spectra of tyrosine and t r y p t o p h a n on the dielectric constant er. A good e x a m p l e of application is given by the protein s t r u c t u r a l changes of bovine ribonuclease A in the course of its d e n a t u r a t i o n by p r e s s u r e . The U V s p e c t r u m of R N a s e is d o m i n a t e d by the absorbance of tyrosine - this RNase does not contain t r y p t o p h a n . As shown in Figure 6, an increase of p r e s s u r e from 1 to 500 M P a r e s u l t s in a blue-shift of the 4th derivative m a x i m u m from 285.7 + 0.05 to 283.5 + 0.05 nm. This shift of 2.2 n m corresponds to an increase of the m e a n dielectric constant from 25 to 59. It is characteristic of the exposure to the aqueous solvent of part of the 6 tyrosines, as it is expected for a partly d e n a t u r a t i o n . The t r a n s i t i o n is fully reversible with clear isosbestic points. The p r e s s u r e effect can therefore be described by a simple two-state model between the native (er = 25) and the partially d e n a t u r e d (er = 59) state. A s i m u l a t i o n on the basis of this model p e r m i t t e d us to d e t e r m i n e the t h e r m o d y n a m i c p a r a m e ters of this t r a n s i t i o n : AG~ = 10.3 kJ/mol and AV = - 52 ml/mol. A c o m p a r i s o n w i t h r e s u l t s obtained by other m e t h o d s indicates t h a t the (~ = 59) state corresponds to an i n t e r m e d i a t e in the defolding process which h a s m o l t e n globule like c h a r a c t e r i s t i c s [12]. It t h u s a p p e a r s t h a t fourth derivative
558 spectroscopy under high pressure is a suitable technique to investigate protein structural changes occurring during the folding or defolding processes. 0.2 ....
~'
4
...1 M P a
0.1
c<3
<~
O0
-0
1
t 275
i
i 280
i
!.
285 wavelength,
~
! 290
l, 295
nm
Figure 6. Effect of pressure on the fourth derivative spectra of RNase A at pH2. 4. ACKNOWI~DGMENTS The authors thank Mrs N. Bec for her technical help and NSERC Canada for financial support. 5. REFERENCES 1 V.V. Mozhaev, K. Heremans, J. Frank, P. Masson and C. Balny, Proteins : Structure, Function, and Genetics, 24 (1996) 81. 2 J-L. Saldana and C. Balny in High Pressure and Biotechnology (C. Balny et al. eds) J. Libbey Eurotext/INSERM, Montrouge, France, vol. 224 (1992) 529. 3 C. Balny, J-L. Saldana, and N. Dahan, Anal. Biochem., 139 (1984) 178. 4 C. Balny, J-L. Saldana, and N. Dahan, Anal. Biochem., 163 (1986) 309. 5 C. Balny, P. Masson and F. Travers, High Pres. Res., 2 (1989) 1. 6 K. Ruckpaul, H. Rein, D.P. Ballou and M.J. Coon, Biochem. Biophys. 7 R. Ragone, G. Colonana, C. Balestrieri, L. Servillo and G. Irace, 8 A.A. Paladini and G. Weber, Biochemistry 20 (1981) 2587. 9 B. Stecand L. Lebioda, J. Mol. Biol 211 (1990) 235. 10 J.A. Kornblatt, M.J. Kornblatt and G. Hui Bon Hoa, Biochemistry, 34 (1995) 1218. 11 R. Lange, J. Frank, J.-L. Saldana and C. Balny, Eur. Biophys. J. (1996) in press. 12 R. Lange, N. Bec, V.V. Mozhaev and J. Frank, Eur. Biophys. J. (1996) in press.