- Email: [email protected]
[2] High-pressure techniques
14
C O N F O R M A T I O N A N D TRANSITIONS
[2]
protein unfolding,:' and the other three do not cause significant CD change in the region of 220 nm. The latter transitions may be due at least in part to lipid regions that are organized in close proximity to certain integral membrane proteins/' They demonstrate the high sensitivity to local anesthetics expected for lipid order-disorder transitions. As mentioned earlier, one of these transitions takes place in a membrane domain that appears to be involved in anion transport. It has been found that three different covalent inhibitors of anion transport induce large changes in the C transition upon binding to the membrane. This is shown in the upper curve of Fig. 5 for the inhibitor 4, 4'-diisothiocyano2,2'-stilbenedisulfonic acid (DIDS). The C transition has shifted to higher temperature by about 13° with no detectable change in any of the other transitions. Examination of membranes reacted with less than saturating amounts of DIDS shows that the interaction of the inhibitor with the membrane is an "'all-or-none'" process. That is, the C transition is never seen at temperatures intermediate between 67 ° and 80 ° . The transitions of the erythrocyte membrane also display a high sensitivity to pH and salt concentration. Illustrative of this is the observation that the B1 and B2 transitions fuse together to form a single B transition at slightly lower salt concentration than that employed in the solutions of Fig. 5. E r y t h r o c y t e membranes from ten different mammals have now been examined. All of these show transitions analogous to those seen for human membranes. Interestingly enough, the transitions show some fundamental differences in their response to changes in ionic strength. For example, in the case of sheep and cow membranes, the B2 transition fuses with the C transition in going from below physiological to physiological osmolarity at pH 7.4. Thus, scanning calorimetry can identify multiple domains in the erythrocyte membrane and can, in addition, see the fusion of two independent domains into a single domain. The latter phenomenon is a known characteristic of lipid phase behavior. More extensive studies might help to elucidate in more detail the functional significance of these interesting domains.
[2] High-Pressure Techniques By S. A. HAWLEY It has been known since the early part of this century that protein denaturation can be produced by the application of several thousand kilograms of hydrostatic pressure per square centimeter.' Until a decade or so ago this discovery was only of occasional interest. The relatively ' P. W. Bridgman, J. Bhd. Chem. 19, 511 (1914).
[2]
HIGH-PRESSURE TECHNIQUES
15
modest level of activity in high-pressure biochemistry that has followed is not altogether surprising in view of the fact that observable interactions with biopolymers often require pressures that are substantially in excess of any found ambient to terrestrial biological systems, The relevance of the pressure as an environmental factor in this extreme is, of course, all but lost. The direction of recent work has been toward the delineation of highpressure water as a special kind of aqueous system. In this respect one may compare the behavior of solutions under high pressures to those containing urea, hydrogen ions, or any of a number of chemical additives that may be employed to selectively perturb conformational structure. In studies of protein denaturation, for example, the phenomenology and experimental strategy are often quite similar--one hopes to surmise which factors are important determinants of tertiary structure by exploring the limits of stable native behavior. Beyond this admittedly limited similarity. there are important differences. One of the most appealing aspects of pressure investigations is the relative simplicity with which the thermodynamic nature of experimental results may be established. A knowledge of the volume of a substance over a range of pressure and temperature provides a nearby complete thermodynamic description of that substance, lacking only the specific heat. This also applies to transition processes for which the incremental changes are of primary concern. In certain cases, which include most pressure-induced protein transitions studied thus far, it is possible to estimate the specific heat increment directly from high-pressure data. In any given circumstance the relative thermodynamic simplicity must be weighed against some of the practical difficulties that are encountered in the laboratory. The generation of high pressures requires a constraint on the volume, often taking the form of a fairly massive steel structure. Many simple laboratory operations that are taken for granted in atmospheric pressure experiments, such as stirring of the sample or its visual inspection, cannot be done at high pressures without making special provision to do so. No attempt will be made here to review the general practice of high pressure experimentation: the discussion will be restricted to techniques that appear to be particularly useful in biopolymer studies. There are several informative surveys'-'-:' of pressure techniques, including one in this -' S. D. Hamann, "'Physico-Chemical Effects of Pressure." Butterworths, London, 1957. C. C. Bradley. "High Pressure Methods in Solid State Research." Plenum, New York. 1969. E. W. Comings, "'High Pressure Technology." McGraw-Hill, New York, 1956. D. C. Monro. in "High Pressure Physics and Chemistry" (R. S. Bradley, ed.), Vol. t, p. II. Academic Press, New York, 1963.
16
CONFORMATION
AND TRANSITIONS
[2]
series, '~ which the interested reader will find helpful. Although written over 40 years ago, the book by Bridgman 7 remains a valuable guide to high-pressure work. The apparatus necessary for the generation and measurement of high pressures and a variety of closed reactor vessels are readily available from several manufacturers. A vessel suitable for determination of various physical properties of bipolymer solutions usually requires some degree of independent development by the investigator. For almost every significant physical probe, one can usually find an example of its use at high pressure, in many cases with water as the material under study. However, when working with biopolymers it is often necessary to measure not the bulk property of a solution, but rather the variations of a small increment of that property attributable to the solute. In such cases special attention to the sensitivity and stability of the system is required. At present the most useful techniques have involved optical probes, particularly absorbance, s-'2 fluorescence, j'~ and scattering.'4""~ Because of stress birefringence that inevitably occurs to some degree in the highpressure windows, optical rotation measurements 16'17 are difficult, but nonetheless have provided valuable data on nucleic acids and protein transition processes. Volume
Measurements
The complete assessment of a pressure-induced effect requires an experimental determination of the volume changes. For a pure substance, pressure-volume measurements usually do not represent serious experimental problems. Direct measurement of the volume associated with a substance in dilute solution represents a substantially more difficult task. In particular for a 1% solution (by volume), an experimental resolution of '; K. Suzuki, this series Vol. 26, p. 424. 7 p. W. Bridgman, - T h e Physics of High P r e s s u r e . " Dover, N e w York, 1970. K. Suzuki, Y. M i y o s a w a , and C. Suzuki, Arch. Biochem. Biophys. 101,225 (1963). " J. F. Brandts, R. J. Oliveira, and C. Westort, Biochemistry 9, 1038 (1970). '" S. A. Hawley, Biochemistry 10, 2436 (1971). '~ A. Zipp and W. K a u z m a n n , Biochemistry 12, 4217 (1973). re K. A. H. H e r e m a n s , J. Snauwaert, H. Vandersypen, and Y. Van Nuland, Proc. Int. Conf. High Pressures, 4th, Kyoto, 1974, p 623. ':~ T. M. Li, J. W. Hook, H. G. Drickamer, and G. Weber, Biochemistry 15, 3205 (1976). ,4 T. E. Gunter and K. K. Gunter, Biopolymers 11,667 (1972). ':' K. A. H. H e r e m a n s , Proc. Conf. High Pressures, 4th, Kyoto, 1974, p. 627. "; S. J. Gill and R. L. GIogovsky, J. Phys. Chem. 69, 1515, (1965). ,T B. Weida and S. J. Gill, Biochim. Biophys. Acta 112, 179 (1966),
[2]
HIGH-PRESSURE
TECHNIQUES
17
I% of the volume contribution would require a sensitivity of nominally 10-4 . Often it is of interest to resolve the transition increment associated with a conformational change. Protein denaturation represents a case in which direct volume measurements would be of considerable value. Indirect estimates, however, indicate that 1-2% of the protein volume is involved in the transition process. Thus, for a 1% solution direct measurement of the transition volume would require that the solution volume be resolved to one part in 10~. With a pressure vessel of convenient size. only a fraction of a microliter would be involved. Although a very high experimental sensitivity is required, one might expect that a carefully designed piezometer would approach these requirements. A number of devices were developed early in this century that are capable of this level of precision and are reviewed by Bridgman5 Often the technique involves displacement of the sample with mercury during compression. Recently, for example, Yayanos '~ examined the partial specific compressibility of simple amino acids using a piezometer of a design originated by Tait and developed by Amagat. This device is simply a glass bulb with capillary stem that is inverted so that the open end of the capillary is immersed in a pool of mercury. Electrical contacts are placed in the stem so that, as mercury is drawn up the capillary, the pressure is recorded at several known volumes. In the Yayanos investigation, a resolution of one part in 10'~ was achieved with a 12-ml sample volume. A few direct measurements have been made on proteins ''~ ~ with mercury piezometers. Recently, a magnetic densimeter ~' was employed to measure the compressibility of ribonuclease and other biological preparations. Apparently, however, no attempts have been made thus far to develop a differential device for measuring solution properties. This is interesting insofar as the absolute volume measurements, if nothing else, have demonstrated that the necessary sensitivity may be achieved. It is also possible to assess the transition volume, ~V, of a chemical reaction if the pressure dependence of the equilibrium constant, K, can be determined. This is done most simply by using Planck's equation, - R T ( O In K / O P ) T = A V . If data are available over a range of pressures, and temperatures, a more useful expression can be obtained by integrating the relation d A G = A V d P - A S dT, which yields: AG = (A/3/2)(P - P o ) ~ + Ao~(P - P o ) ( T -
To) + ( A C p / 2 T o ) ( T - To)-' + A V , g P - P,,) - A S , > ( T - To) + A G o
n~ A. A. Yayanos, J. Phys. Chem. 76, 1783 (1972). ~' S. Palitzsch, J. A m . Chem. Soc. 3, 346 (1919). ~' L. J. Henderson and F. N. Brink, A m . J. Physiol. 21, 248 (19081. ~ G. R. Andersson, Ark. K e m i 20, 513 (1963). e-' P. F. Fahey, D. W. Kupke, and J. W. Beams, Proc. Natl. Acad. Sci. U.S.A. 63,548 ( 1969].
]8
C O N F O R M A T I O N A N D TRANSITIONS
[2]
where AGo=AG(Po,To), AV0= V(Po,T.), ASo=S(Po,To) with A/3= (0 AV/OP)T, IAa = (0 AV/OT)e = - ( 0 AS/OP)r, and ACp = T(O AS/OT)p. Figure 1 shows the transition map of chymotrypsinogen on the pressure-temperature plane, which is reasonably well described by the above equation." The equivalence of free energy and extent of reaction is established via the relation &G = - R T In K, and to be applicable requires a knowledge of the stoichiometry of the reaction. Sometimes, as is the case for the melting of DNA, a simple stoichiometric statement is not appropriate. Ther-
4000
%
5OO0
D ZOO0
I000
TEMPERATURE %
FIG. I. Pressure-temperature transition map of chymotrypsinogen A (pH 2.08). Contour lines connect points of equal fraction denatured, XD. The free-energy increment between native and denatured states, AG, vanishes along the line connecting transition midpoints, XD = 0.5. The salient thermodynamic features of the process can be deduced by inspection. At atmospheric pressure the transition occurs with AS > 0. Since (dT/dP)m is positive, then A V is also positive. At about 1600 kg/cm ~, the transition temperature is pressure independent (i.e., (dT/dP)m = 0), thereby indicating that ,,.%V = 0 at this point. Because A V is evidently decreasing with increased pressure, the incremental compressibility, A/3, is negative. However, since A V increases with temperature about this point, the incremental expansivity, As, is positive. At about 25 ° the transition pressure becomes temperature independent, (dP/ dT)m = 0, indicating the AS vanishes. Since AS increases with increasing temperature about this point, ACv is positive. Adapted from S. A. Hawley, Biochemistry 10, 2436 (1971).
[2]
HIGH-PRESSURE
TECHNIQUES
19
modynamic information is still accessible, although to a limited degree, by employing the Clausius-Clapeyron relation. The approach is formally the same as that used in assessing the properties of phase transitions. It is necessary, however, to locate a path on the pressure-temperature plane for which AG is constant. An example of this might be the loci of transitions midpoints (e.g., Fig. 1). Since d A G = A V d P - A S d T -- 0 along such a line, the Clausius-Clapeyron relation, (dT/dP),,, = A V / A S obtains. Thus if calorimetric data at atmospheric pressure are available, the direction of the coexistence lines provide a measure of the volume increment A V. The curvature of coexistence lines may also be a source of useful thermodynamic data. By differentiating the relation of d A G and making use of the fact that the result, d e A G , also vanishes along a coexistence line, one obtains the relation: A a dT,,, dP,,, + Aft dP,,," + A V d2P,,, = AC~, dT,,,2/T + A S d"-T,,
where the subscript indicates that the path is at constant G. When the process is pressure independent, then A V = 0 and (d2T/dP2),, = Aft~AS. This situation arises in certain protein transitions '"'~ (Fig. 1) as well as in DNA melting in low buffer salts, e:~ In both cases the curvature of the coexistence lines provides a simple and direct means of assessing the differential compressibility. Another interesting case occurs when the transition process is temperature independent, as has been observed for several protein transitions at high pressures. "'~'':~ From the ClausiusClapeyron equation, it is evident that k S = 0, and to second order (d"-P/dT'),,, = A C e / T A V.
Gel Electrophoresis Of several high-pressure techniques that have been tried by the author, none has proved to be as simple or reliable as gel electrophoresis. It is also interesting to note that in investigations of pressure-induced conformational changes of chymotrypsinogen, '':"~4 the resolution compares favorably to UV difference spectroscopy. The basic equipment required is a pressure vessel with electrical leads to the interior and a sufficiently large working volume to accommodate the electrophoresis cell. The actual configuration will be determined by a number of factors. Usually the buffer reservoirs will be the primary determinant of cell volume and must be large enough to accommodate the necessary charge transfer without producing a prohibitively high cell re~:~ S. A. Hawley and R. M. Macleod. Biopolymers 13, 1417 (1975). -" S. A. Hawley. Biochim. Biophys. Acta 317, 236 (1973).
20
CONFORMATION AND TRANSITIONS
[2]
5
5 6
4
FIG. 2. Electrophoresis cell for high-pressure experiments. Pyrex test tube (1) serves as a container for the cell assembly. Inner reservoir (2) contains the anodal buffer for chymotrypsinogen experiments. The glass tube (3) also contains buffer providing a conductive bridge to the gel tube (4); the two are joined by a short segment of latex tubing (5). Initial location of the protein solution (6). Platinum electrodes (not shown) are inserted from above through the hydraulic fluid (7). Reproduced from S. A. Hawley and R. M. Mitchell, Biochemistry 13, 1417 (1975).
sistance. The breakdown of the electrical feedthroughs is a possibility that must be considered before simply increasing the driving voltage. Figure 2 shows a cell used for high-pressure studies of protein folding. -'4:'5 The geometry reflects an effort to make efficient use of the cylindrical bore of the pressure vessel. Pyrex tubing was used throughout in the construction. A potential difficulty, which was anticipated but never materialized, is the pressure-induced shrinkage of the gel column away from support tube. Evidently the increase in the matrix density occurs by the flow of a small amount of buffer into the column from the reservoirs. A practical difference between experiments conducted at elevated pressures and their counterpart at ambient pressures is the manner in which the solution is loaded onto the end of the column. At high pressures is no longer possible to simply apply the sample solution via pipette. An alternative is to layer the sample onto the gel at atmospheric pressure and then fix the solution in place by ~'stoppering" the end of the gel tube with a short gel 'cylinder. When pressure is applied, the '~stopper" gel will slide into the tube, displacing any residual air. If ends are cut squarely, the ~:' S. A. Hawley and R. M. Mitchell, Biochemistry 14, 3257 (1975).
[2]
HIGH PRESSURE-TECHNIQUES
21
sample solution will be held in a thin laminal volume appropriate for electrophoresis.
Optical M e a s u r e m e n t s The construction of a pressure vessel with optical ports is relatively straightforward, and several useful discussions of the general principles are available. 2 '; When attempting m e a s u r e m e n t s in aqueous biopolymer solutions, additional problems may arise in addition to those usually encotmtered in high-pressure work. For e x a m p l e , it is often desirable to m a k e the pressure vessel small enough so that m e a s u r e m e n t s can be conducted in existing instrumentation without the need for extensive modification. The size of the vessel is determined by a n u m b e r of factors: particularly important is the strength of the material used for its construction, the upper limit of pressure desired, and the internal volume. In several investigations conducted thus far with an upper limit in the range 3000-7000 kg/cm'-', the pressure vessel, typically, has been a tool-steel cylinder 4-7 inches long and 3-5-inches in diameter. In the authors" laboratory, a piece of high-pressure tubing several feet long is used to connect the pressure-generating apparatus to the pressure vessel. Although the tubing is fairly stiff, the length and contouring provide sufficient flexibility to allow the vessel to be inserted or removed from the c o m p a r t m e n t of a s p e c t r o p h o t o m e t e r , simply and without disconnecting it from the highpressure line. A m o r e serious p r o b l e m is that of preventing contamination of the sample by interactions with the vessel, the gaskets, or the hydraulic fluid. In studies of RNase, Brandts e l al.,:' used a stainless-steel vessel with an inert liner to hold the sample, In order to provide a physical barrier between the hydraulic fluid in the p u m p and the sample solution, a piston separator was incorporated into the pressure line. This approach is somewhat c u m b e r s o m e insofar as complete disassembly and reassembly is required for cleaning and reloading. A preferred approach is to isolate the sample in a separate Teflon or stainless-steel c h a m b e r inside the high-pressure vessel, a method adopted by several investigators.'"" '"-"; '-''~In addition to windows, it is impol-tant to provide a pressure-release surface that allows the solution volume to change freely with pressure. An example of this arrangement is shown in Fig. 3, which illustrates the essential characteristics high-pressure -"; H. Kliman, Ph.D. Thesis, Princeton Univ. Princeton. New Jersey. 1969. : S. J. Gill and W. Rummel, Rev. Sci. l n s l r , m . 32,752 (1961). "-'*G. Weber, F. Tanaka, B. Y. Okamoto. and H. G. Dickamer. Proc. Natl. Acad. Sci. U.S.A. 71, 1264 (1974).
22
CONFORMATION AND TRANSITIONS
/
[2]
/
j /
FIG. 3. A schematic representation of a high-pressure cell used for fluorescence measurements. Above: the pressure vessel. Below: the stainless-steel sample chamber with piston-mounted sapphire windows. Reproduced from G. Weber, F. Tanaka, B. Y. Okamoto, and H. G. Dickamer, Proc. Natl. Acad. Sci. U.S.A. 71, 1264 (1974).
fluorescence cell used by Weber, Dfickamer, and co-workers.28 The sample is held in a stainless-steel cylinder with windows mounted as movable pistons. Volume changes are accommodated by displacement of the windows within the bore of the cylinder. Usually crystalline sapphire is used for high-pressure windows. It is a material with good mechanical strength and has a transmission band that extends from about 6 /zm to a lower limit approaching 150 nm with selected material. Windows may be mounted without gaskets in a manner described by Poulter. '-':~':~° The mode of sealing is a simple application of the unsupported area principle of Bridgman. 7 The window is supported from the low-pressure side on a surface with a contact area smaller than the window by the amount of the aperture. This means that the contact pressure between window and the supporting surface will always be greater than the internal pressure by a corresponding amount, thereby assuring a pressure-tight seal, The contact surfaces must be of near optical quality, however. Even a relatively fine scratch can cause the seal to " w e e p " at high pressures. If the window is mechanically fastened onto 2, D. M. Warshauer and W. Paul, Rev. Sci. Instrum. 29, 675 (1958). :~" T. C. Poulter, Phys. Rev. 40, 860 (1932).
[2]
HIGH-PRESSURE TECHNIQUES
23
the support, it may be used a number of times without further attention after it has been properly seated. A slight modification of this idea was adopted by the author in developing a cell for UV absorbance measurements'" (Fig. 4). The basic idea here was to incorporate the high-pressure windows into the sample chamber so that hydraulic fluid does not appear in the optical path. The chamber is formed from thin-walled (0.1 ram) heat-shrinkable Teflon tubing with an inside diameter equal to the outside diameter of the windows. The tubing is heated on a mandril in order to uniformly decrease the diameter by 5 mm over a 1 cm length midway along the cylinder. A cylindrical aluminum ring is fitted into the resulting depression and provides structural rigidity as well as a path of relatively low thermal conductivity from the sample to the pressure vessel. Because the chamber is loaded while outside the pressure vessel, it is necessary to " m a k e " the high-pressure seal on the windows with each experimental run. In order to eliminate problems resulting from improper seating of the windows, a thin (0.7 mm) O ring was placed on the face of each window support.
FIG. 4. High-pressure cell for absorbance measurements. (1) Thermostatic jacket: thermostatting fluid passes from one side of the vessel to the other through a 5-ram port (2). Sample (3) is located between sapphire windows (4) and is contained in a thin-walled. contoured Teflon sheath (thickness exaggerated for purposes of illustration) (5). Brass compression rings (6) seal Teflon sheath to windows. Structural rigidity is imparted to the chamber by an annular aluminum ring (7).
24
CONFORMATION AND TRANSITIONS
[3]
In pressure-jump experiments any flexural stiffness in the sample c h a m b e r will result in a pressure gradient between inside and outside of the chamber. Joints that are under uniform pressure at equilibrium will be stressed during the transient and if not properly sealed will result in contamination of sample by hydraulic fluid and vice versa. Simple friction seals are usually not adequate. In the sample c h a m b e r used by the author, the joint between the Teflon tubing and the windows was found to be susceptible to this problem. In order to ensure an effective barrier, the windows were fitted with circumferential 0 rings so that the Teflon could be tightly sealed to the windows by sliding compression rings o v e r the outside of the tube.
[3] H y d r o g e n - T r i t i u m
Exchange
By S. W. ENGLANDER and J. J. ENGLANDER H y d r o g e n exchange (HX) m e a s u r e m e n t s on proteins and nucleic acids can now provide several kinds of unusual structural information. Peptide groups that are involved in internal H bonds can be distinguished from those that are exposed and H - b o n d e d to solvent water, and the n u m b e r of groups of each kind can be accurately counted. The analogous measurements can be performed for nucleotides in nucleic acids. HX methods can separately detect the individual structural changes that together constitute an allosteric transition in a protein, and, among other parameters, the free energy associated with each change can be measured. H X methods can measure the so-called breathing b e h a v i o r of macromolecules. This article discusses these capabilities especially from the point of view of experimental design and data interpretation. T h e H y d r o g e n - T r i t i u m Exchange Method The exchange techniques discussed here use tritium (T) as a label to follow the exchange of protons (H) between m a c r o m o l e c u l e s and solvent, Previous articles in this series ~'' and elsewhere :~'4 have dealt with basic H - T exchange methodology, and these and some more recently published papers should be consulted for details of experimental techniques, which s. w. Englander and J. J. Englander, this series Vol. 12B, p. 379. S. W. Englander and J. J. Englander, this series Vol. 26, p, 406. :~W. F. Harrington, R. Josephs, and D. M. Segal, Annu. Rev. Biochem. 35, 599 (1966). 4 B. McConnell and P. H. yon Hippel, in "Procedures in Nucleic Acid Research" (G. L. Cantoni and D. R. Davies, eds.), Vol. 2, p. 389. Harper, New York, 1972.
C O N F O R M A T I O N A N D TRANSITIONS
[2]
protein unfolding,:' and the other three do not cause significant CD change in the region of 220 nm. The latter transitions may be due at least in part to lipid regions that are organized in close proximity to certain integral membrane proteins/' They demonstrate the high sensitivity to local anesthetics expected for lipid order-disorder transitions. As mentioned earlier, one of these transitions takes place in a membrane domain that appears to be involved in anion transport. It has been found that three different covalent inhibitors of anion transport induce large changes in the C transition upon binding to the membrane. This is shown in the upper curve of Fig. 5 for the inhibitor 4, 4'-diisothiocyano2,2'-stilbenedisulfonic acid (DIDS). The C transition has shifted to higher temperature by about 13° with no detectable change in any of the other transitions. Examination of membranes reacted with less than saturating amounts of DIDS shows that the interaction of the inhibitor with the membrane is an "'all-or-none'" process. That is, the C transition is never seen at temperatures intermediate between 67 ° and 80 ° . The transitions of the erythrocyte membrane also display a high sensitivity to pH and salt concentration. Illustrative of this is the observation that the B1 and B2 transitions fuse together to form a single B transition at slightly lower salt concentration than that employed in the solutions of Fig. 5. E r y t h r o c y t e membranes from ten different mammals have now been examined. All of these show transitions analogous to those seen for human membranes. Interestingly enough, the transitions show some fundamental differences in their response to changes in ionic strength. For example, in the case of sheep and cow membranes, the B2 transition fuses with the C transition in going from below physiological to physiological osmolarity at pH 7.4. Thus, scanning calorimetry can identify multiple domains in the erythrocyte membrane and can, in addition, see the fusion of two independent domains into a single domain. The latter phenomenon is a known characteristic of lipid phase behavior. More extensive studies might help to elucidate in more detail the functional significance of these interesting domains.
[2] High-Pressure Techniques By S. A. HAWLEY It has been known since the early part of this century that protein denaturation can be produced by the application of several thousand kilograms of hydrostatic pressure per square centimeter.' Until a decade or so ago this discovery was only of occasional interest. The relatively ' P. W. Bridgman, J. Bhd. Chem. 19, 511 (1914).
[2]
HIGH-PRESSURE TECHNIQUES
15
modest level of activity in high-pressure biochemistry that has followed is not altogether surprising in view of the fact that observable interactions with biopolymers often require pressures that are substantially in excess of any found ambient to terrestrial biological systems, The relevance of the pressure as an environmental factor in this extreme is, of course, all but lost. The direction of recent work has been toward the delineation of highpressure water as a special kind of aqueous system. In this respect one may compare the behavior of solutions under high pressures to those containing urea, hydrogen ions, or any of a number of chemical additives that may be employed to selectively perturb conformational structure. In studies of protein denaturation, for example, the phenomenology and experimental strategy are often quite similar--one hopes to surmise which factors are important determinants of tertiary structure by exploring the limits of stable native behavior. Beyond this admittedly limited similarity. there are important differences. One of the most appealing aspects of pressure investigations is the relative simplicity with which the thermodynamic nature of experimental results may be established. A knowledge of the volume of a substance over a range of pressure and temperature provides a nearby complete thermodynamic description of that substance, lacking only the specific heat. This also applies to transition processes for which the incremental changes are of primary concern. In certain cases, which include most pressure-induced protein transitions studied thus far, it is possible to estimate the specific heat increment directly from high-pressure data. In any given circumstance the relative thermodynamic simplicity must be weighed against some of the practical difficulties that are encountered in the laboratory. The generation of high pressures requires a constraint on the volume, often taking the form of a fairly massive steel structure. Many simple laboratory operations that are taken for granted in atmospheric pressure experiments, such as stirring of the sample or its visual inspection, cannot be done at high pressures without making special provision to do so. No attempt will be made here to review the general practice of high pressure experimentation: the discussion will be restricted to techniques that appear to be particularly useful in biopolymer studies. There are several informative surveys'-'-:' of pressure techniques, including one in this -' S. D. Hamann, "'Physico-Chemical Effects of Pressure." Butterworths, London, 1957. C. C. Bradley. "High Pressure Methods in Solid State Research." Plenum, New York. 1969. E. W. Comings, "'High Pressure Technology." McGraw-Hill, New York, 1956. D. C. Monro. in "High Pressure Physics and Chemistry" (R. S. Bradley, ed.), Vol. t, p. II. Academic Press, New York, 1963.
16
CONFORMATION
AND TRANSITIONS
[2]
series, '~ which the interested reader will find helpful. Although written over 40 years ago, the book by Bridgman 7 remains a valuable guide to high-pressure work. The apparatus necessary for the generation and measurement of high pressures and a variety of closed reactor vessels are readily available from several manufacturers. A vessel suitable for determination of various physical properties of bipolymer solutions usually requires some degree of independent development by the investigator. For almost every significant physical probe, one can usually find an example of its use at high pressure, in many cases with water as the material under study. However, when working with biopolymers it is often necessary to measure not the bulk property of a solution, but rather the variations of a small increment of that property attributable to the solute. In such cases special attention to the sensitivity and stability of the system is required. At present the most useful techniques have involved optical probes, particularly absorbance, s-'2 fluorescence, j'~ and scattering.'4""~ Because of stress birefringence that inevitably occurs to some degree in the highpressure windows, optical rotation measurements 16'17 are difficult, but nonetheless have provided valuable data on nucleic acids and protein transition processes. Volume
Measurements
The complete assessment of a pressure-induced effect requires an experimental determination of the volume changes. For a pure substance, pressure-volume measurements usually do not represent serious experimental problems. Direct measurement of the volume associated with a substance in dilute solution represents a substantially more difficult task. In particular for a 1% solution (by volume), an experimental resolution of '; K. Suzuki, this series Vol. 26, p. 424. 7 p. W. Bridgman, - T h e Physics of High P r e s s u r e . " Dover, N e w York, 1970. K. Suzuki, Y. M i y o s a w a , and C. Suzuki, Arch. Biochem. Biophys. 101,225 (1963). " J. F. Brandts, R. J. Oliveira, and C. Westort, Biochemistry 9, 1038 (1970). '" S. A. Hawley, Biochemistry 10, 2436 (1971). '~ A. Zipp and W. K a u z m a n n , Biochemistry 12, 4217 (1973). re K. A. H. H e r e m a n s , J. Snauwaert, H. Vandersypen, and Y. Van Nuland, Proc. Int. Conf. High Pressures, 4th, Kyoto, 1974, p 623. ':~ T. M. Li, J. W. Hook, H. G. Drickamer, and G. Weber, Biochemistry 15, 3205 (1976). ,4 T. E. Gunter and K. K. Gunter, Biopolymers 11,667 (1972). ':' K. A. H. H e r e m a n s , Proc. Conf. High Pressures, 4th, Kyoto, 1974, p. 627. "; S. J. Gill and R. L. GIogovsky, J. Phys. Chem. 69, 1515, (1965). ,T B. Weida and S. J. Gill, Biochim. Biophys. Acta 112, 179 (1966),
[2]
HIGH-PRESSURE
TECHNIQUES
17
I% of the volume contribution would require a sensitivity of nominally 10-4 . Often it is of interest to resolve the transition increment associated with a conformational change. Protein denaturation represents a case in which direct volume measurements would be of considerable value. Indirect estimates, however, indicate that 1-2% of the protein volume is involved in the transition process. Thus, for a 1% solution direct measurement of the transition volume would require that the solution volume be resolved to one part in 10~. With a pressure vessel of convenient size. only a fraction of a microliter would be involved. Although a very high experimental sensitivity is required, one might expect that a carefully designed piezometer would approach these requirements. A number of devices were developed early in this century that are capable of this level of precision and are reviewed by Bridgman5 Often the technique involves displacement of the sample with mercury during compression. Recently, for example, Yayanos '~ examined the partial specific compressibility of simple amino acids using a piezometer of a design originated by Tait and developed by Amagat. This device is simply a glass bulb with capillary stem that is inverted so that the open end of the capillary is immersed in a pool of mercury. Electrical contacts are placed in the stem so that, as mercury is drawn up the capillary, the pressure is recorded at several known volumes. In the Yayanos investigation, a resolution of one part in 10'~ was achieved with a 12-ml sample volume. A few direct measurements have been made on proteins ''~ ~ with mercury piezometers. Recently, a magnetic densimeter ~' was employed to measure the compressibility of ribonuclease and other biological preparations. Apparently, however, no attempts have been made thus far to develop a differential device for measuring solution properties. This is interesting insofar as the absolute volume measurements, if nothing else, have demonstrated that the necessary sensitivity may be achieved. It is also possible to assess the transition volume, ~V, of a chemical reaction if the pressure dependence of the equilibrium constant, K, can be determined. This is done most simply by using Planck's equation, - R T ( O In K / O P ) T = A V . If data are available over a range of pressures, and temperatures, a more useful expression can be obtained by integrating the relation d A G = A V d P - A S dT, which yields: AG = (A/3/2)(P - P o ) ~ + Ao~(P - P o ) ( T -
To) + ( A C p / 2 T o ) ( T - To)-' + A V , g P - P,,) - A S , > ( T - To) + A G o
n~ A. A. Yayanos, J. Phys. Chem. 76, 1783 (1972). ~' S. Palitzsch, J. A m . Chem. Soc. 3, 346 (1919). ~' L. J. Henderson and F. N. Brink, A m . J. Physiol. 21, 248 (19081. ~ G. R. Andersson, Ark. K e m i 20, 513 (1963). e-' P. F. Fahey, D. W. Kupke, and J. W. Beams, Proc. Natl. Acad. Sci. U.S.A. 63,548 ( 1969].
]8
C O N F O R M A T I O N A N D TRANSITIONS
[2]
where AGo=AG(Po,To), AV0= V(Po,T.), ASo=S(Po,To) with A/3= (0 AV/OP)T, IAa = (0 AV/OT)e = - ( 0 AS/OP)r, and ACp = T(O AS/OT)p. Figure 1 shows the transition map of chymotrypsinogen on the pressure-temperature plane, which is reasonably well described by the above equation." The equivalence of free energy and extent of reaction is established via the relation &G = - R T In K, and to be applicable requires a knowledge of the stoichiometry of the reaction. Sometimes, as is the case for the melting of DNA, a simple stoichiometric statement is not appropriate. Ther-
4000
%
5OO0
D ZOO0
I000
TEMPERATURE %
FIG. I. Pressure-temperature transition map of chymotrypsinogen A (pH 2.08). Contour lines connect points of equal fraction denatured, XD. The free-energy increment between native and denatured states, AG, vanishes along the line connecting transition midpoints, XD = 0.5. The salient thermodynamic features of the process can be deduced by inspection. At atmospheric pressure the transition occurs with AS > 0. Since (dT/dP)m is positive, then A V is also positive. At about 1600 kg/cm ~, the transition temperature is pressure independent (i.e., (dT/dP)m = 0), thereby indicating that ,,.%V = 0 at this point. Because A V is evidently decreasing with increased pressure, the incremental compressibility, A/3, is negative. However, since A V increases with temperature about this point, the incremental expansivity, As, is positive. At about 25 ° the transition pressure becomes temperature independent, (dP/ dT)m = 0, indicating the AS vanishes. Since AS increases with increasing temperature about this point, ACv is positive. Adapted from S. A. Hawley, Biochemistry 10, 2436 (1971).
[2]
HIGH-PRESSURE
TECHNIQUES
19
modynamic information is still accessible, although to a limited degree, by employing the Clausius-Clapeyron relation. The approach is formally the same as that used in assessing the properties of phase transitions. It is necessary, however, to locate a path on the pressure-temperature plane for which AG is constant. An example of this might be the loci of transitions midpoints (e.g., Fig. 1). Since d A G = A V d P - A S d T -- 0 along such a line, the Clausius-Clapeyron relation, (dT/dP),,, = A V / A S obtains. Thus if calorimetric data at atmospheric pressure are available, the direction of the coexistence lines provide a measure of the volume increment A V. The curvature of coexistence lines may also be a source of useful thermodynamic data. By differentiating the relation of d A G and making use of the fact that the result, d e A G , also vanishes along a coexistence line, one obtains the relation: A a dT,,, dP,,, + Aft dP,,," + A V d2P,,, = AC~, dT,,,2/T + A S d"-T,,
where the subscript indicates that the path is at constant G. When the process is pressure independent, then A V = 0 and (d2T/dP2),, = Aft~AS. This situation arises in certain protein transitions '"'~ (Fig. 1) as well as in DNA melting in low buffer salts, e:~ In both cases the curvature of the coexistence lines provides a simple and direct means of assessing the differential compressibility. Another interesting case occurs when the transition process is temperature independent, as has been observed for several protein transitions at high pressures. "'~'':~ From the ClausiusClapeyron equation, it is evident that k S = 0, and to second order (d"-P/dT'),,, = A C e / T A V.
Gel Electrophoresis Of several high-pressure techniques that have been tried by the author, none has proved to be as simple or reliable as gel electrophoresis. It is also interesting to note that in investigations of pressure-induced conformational changes of chymotrypsinogen, '':"~4 the resolution compares favorably to UV difference spectroscopy. The basic equipment required is a pressure vessel with electrical leads to the interior and a sufficiently large working volume to accommodate the electrophoresis cell. The actual configuration will be determined by a number of factors. Usually the buffer reservoirs will be the primary determinant of cell volume and must be large enough to accommodate the necessary charge transfer without producing a prohibitively high cell re~:~ S. A. Hawley and R. M. Macleod. Biopolymers 13, 1417 (1975). -" S. A. Hawley. Biochim. Biophys. Acta 317, 236 (1973).
20
CONFORMATION AND TRANSITIONS
[2]
5
5 6
4
FIG. 2. Electrophoresis cell for high-pressure experiments. Pyrex test tube (1) serves as a container for the cell assembly. Inner reservoir (2) contains the anodal buffer for chymotrypsinogen experiments. The glass tube (3) also contains buffer providing a conductive bridge to the gel tube (4); the two are joined by a short segment of latex tubing (5). Initial location of the protein solution (6). Platinum electrodes (not shown) are inserted from above through the hydraulic fluid (7). Reproduced from S. A. Hawley and R. M. Mitchell, Biochemistry 13, 1417 (1975).
sistance. The breakdown of the electrical feedthroughs is a possibility that must be considered before simply increasing the driving voltage. Figure 2 shows a cell used for high-pressure studies of protein folding. -'4:'5 The geometry reflects an effort to make efficient use of the cylindrical bore of the pressure vessel. Pyrex tubing was used throughout in the construction. A potential difficulty, which was anticipated but never materialized, is the pressure-induced shrinkage of the gel column away from support tube. Evidently the increase in the matrix density occurs by the flow of a small amount of buffer into the column from the reservoirs. A practical difference between experiments conducted at elevated pressures and their counterpart at ambient pressures is the manner in which the solution is loaded onto the end of the column. At high pressures is no longer possible to simply apply the sample solution via pipette. An alternative is to layer the sample onto the gel at atmospheric pressure and then fix the solution in place by ~'stoppering" the end of the gel tube with a short gel 'cylinder. When pressure is applied, the '~stopper" gel will slide into the tube, displacing any residual air. If ends are cut squarely, the ~:' S. A. Hawley and R. M. Mitchell, Biochemistry 14, 3257 (1975).
[2]
HIGH PRESSURE-TECHNIQUES
21
sample solution will be held in a thin laminal volume appropriate for electrophoresis.
Optical M e a s u r e m e n t s The construction of a pressure vessel with optical ports is relatively straightforward, and several useful discussions of the general principles are available. 2 '; When attempting m e a s u r e m e n t s in aqueous biopolymer solutions, additional problems may arise in addition to those usually encotmtered in high-pressure work. For e x a m p l e , it is often desirable to m a k e the pressure vessel small enough so that m e a s u r e m e n t s can be conducted in existing instrumentation without the need for extensive modification. The size of the vessel is determined by a n u m b e r of factors: particularly important is the strength of the material used for its construction, the upper limit of pressure desired, and the internal volume. In several investigations conducted thus far with an upper limit in the range 3000-7000 kg/cm'-', the pressure vessel, typically, has been a tool-steel cylinder 4-7 inches long and 3-5-inches in diameter. In the authors" laboratory, a piece of high-pressure tubing several feet long is used to connect the pressure-generating apparatus to the pressure vessel. Although the tubing is fairly stiff, the length and contouring provide sufficient flexibility to allow the vessel to be inserted or removed from the c o m p a r t m e n t of a s p e c t r o p h o t o m e t e r , simply and without disconnecting it from the highpressure line. A m o r e serious p r o b l e m is that of preventing contamination of the sample by interactions with the vessel, the gaskets, or the hydraulic fluid. In studies of RNase, Brandts e l al.,:' used a stainless-steel vessel with an inert liner to hold the sample, In order to provide a physical barrier between the hydraulic fluid in the p u m p and the sample solution, a piston separator was incorporated into the pressure line. This approach is somewhat c u m b e r s o m e insofar as complete disassembly and reassembly is required for cleaning and reloading. A preferred approach is to isolate the sample in a separate Teflon or stainless-steel c h a m b e r inside the high-pressure vessel, a method adopted by several investigators.'"" '"-"; '-''~In addition to windows, it is impol-tant to provide a pressure-release surface that allows the solution volume to change freely with pressure. An example of this arrangement is shown in Fig. 3, which illustrates the essential characteristics high-pressure -"; H. Kliman, Ph.D. Thesis, Princeton Univ. Princeton. New Jersey. 1969. : S. J. Gill and W. Rummel, Rev. Sci. l n s l r , m . 32,752 (1961). "-'*G. Weber, F. Tanaka, B. Y. Okamoto. and H. G. Dickamer. Proc. Natl. Acad. Sci. U.S.A. 71, 1264 (1974).
22
CONFORMATION AND TRANSITIONS
/
[2]
/
j /
FIG. 3. A schematic representation of a high-pressure cell used for fluorescence measurements. Above: the pressure vessel. Below: the stainless-steel sample chamber with piston-mounted sapphire windows. Reproduced from G. Weber, F. Tanaka, B. Y. Okamoto, and H. G. Dickamer, Proc. Natl. Acad. Sci. U.S.A. 71, 1264 (1974).
fluorescence cell used by Weber, Dfickamer, and co-workers.28 The sample is held in a stainless-steel cylinder with windows mounted as movable pistons. Volume changes are accommodated by displacement of the windows within the bore of the cylinder. Usually crystalline sapphire is used for high-pressure windows. It is a material with good mechanical strength and has a transmission band that extends from about 6 /zm to a lower limit approaching 150 nm with selected material. Windows may be mounted without gaskets in a manner described by Poulter. '-':~':~° The mode of sealing is a simple application of the unsupported area principle of Bridgman. 7 The window is supported from the low-pressure side on a surface with a contact area smaller than the window by the amount of the aperture. This means that the contact pressure between window and the supporting surface will always be greater than the internal pressure by a corresponding amount, thereby assuring a pressure-tight seal, The contact surfaces must be of near optical quality, however. Even a relatively fine scratch can cause the seal to " w e e p " at high pressures. If the window is mechanically fastened onto 2, D. M. Warshauer and W. Paul, Rev. Sci. Instrum. 29, 675 (1958). :~" T. C. Poulter, Phys. Rev. 40, 860 (1932).
[2]
HIGH-PRESSURE TECHNIQUES
23
the support, it may be used a number of times without further attention after it has been properly seated. A slight modification of this idea was adopted by the author in developing a cell for UV absorbance measurements'" (Fig. 4). The basic idea here was to incorporate the high-pressure windows into the sample chamber so that hydraulic fluid does not appear in the optical path. The chamber is formed from thin-walled (0.1 ram) heat-shrinkable Teflon tubing with an inside diameter equal to the outside diameter of the windows. The tubing is heated on a mandril in order to uniformly decrease the diameter by 5 mm over a 1 cm length midway along the cylinder. A cylindrical aluminum ring is fitted into the resulting depression and provides structural rigidity as well as a path of relatively low thermal conductivity from the sample to the pressure vessel. Because the chamber is loaded while outside the pressure vessel, it is necessary to " m a k e " the high-pressure seal on the windows with each experimental run. In order to eliminate problems resulting from improper seating of the windows, a thin (0.7 mm) O ring was placed on the face of each window support.
FIG. 4. High-pressure cell for absorbance measurements. (1) Thermostatic jacket: thermostatting fluid passes from one side of the vessel to the other through a 5-ram port (2). Sample (3) is located between sapphire windows (4) and is contained in a thin-walled. contoured Teflon sheath (thickness exaggerated for purposes of illustration) (5). Brass compression rings (6) seal Teflon sheath to windows. Structural rigidity is imparted to the chamber by an annular aluminum ring (7).
24
CONFORMATION AND TRANSITIONS
[3]
In pressure-jump experiments any flexural stiffness in the sample c h a m b e r will result in a pressure gradient between inside and outside of the chamber. Joints that are under uniform pressure at equilibrium will be stressed during the transient and if not properly sealed will result in contamination of sample by hydraulic fluid and vice versa. Simple friction seals are usually not adequate. In the sample c h a m b e r used by the author, the joint between the Teflon tubing and the windows was found to be susceptible to this problem. In order to ensure an effective barrier, the windows were fitted with circumferential 0 rings so that the Teflon could be tightly sealed to the windows by sliding compression rings o v e r the outside of the tube.
[3] H y d r o g e n - T r i t i u m
Exchange
By S. W. ENGLANDER and J. J. ENGLANDER H y d r o g e n exchange (HX) m e a s u r e m e n t s on proteins and nucleic acids can now provide several kinds of unusual structural information. Peptide groups that are involved in internal H bonds can be distinguished from those that are exposed and H - b o n d e d to solvent water, and the n u m b e r of groups of each kind can be accurately counted. The analogous measurements can be performed for nucleotides in nucleic acids. HX methods can separately detect the individual structural changes that together constitute an allosteric transition in a protein, and, among other parameters, the free energy associated with each change can be measured. H X methods can measure the so-called breathing b e h a v i o r of macromolecules. This article discusses these capabilities especially from the point of view of experimental design and data interpretation. T h e H y d r o g e n - T r i t i u m Exchange Method The exchange techniques discussed here use tritium (T) as a label to follow the exchange of protons (H) between m a c r o m o l e c u l e s and solvent, Previous articles in this series ~'' and elsewhere :~'4 have dealt with basic H - T exchange methodology, and these and some more recently published papers should be consulted for details of experimental techniques, which s. w. Englander and J. J. Englander, this series Vol. 12B, p. 379. S. W. Englander and J. J. Englander, this series Vol. 26, p, 406. :~W. F. Harrington, R. Josephs, and D. M. Segal, Annu. Rev. Biochem. 35, 599 (1966). 4 B. McConnell and P. H. yon Hippel, in "Procedures in Nucleic Acid Research" (G. L. Cantoni and D. R. Davies, eds.), Vol. 2, p. 389. Harper, New York, 1972.