Biochemistry 1981, 20, 2587-2593 Robinson, R. A,, & Stokes, R. H. (1959) in Electrolyte Solutions, pp 492-493, Butterworth, London. Roxby, R., Miller, K., Blair, D. P., & Van Holde, K. E. (1974) Biochemistry 13, 1662-1668. Scatchard, G., Hammer, W. J., & Wood, S. E. (1938) J . Am. Chem. SOC.60, 3061-3070. Schrier, E. E., & Schrier, E. B. (1967) J . Phys. Chem. 71, 1851-1 860. Schroder, E., Wollmer, A., Kubicki, J., & Ohlenbusch, H. D. (1976) Biochemistry 15, 5693-5697. Siezen, R., & Van Driel, R. (1973) Biochim. Biophys. Acta 295, 131-139.
2581
St. Pierre, T., & Jencks, W. P. (1969) Arch. Biochem. Biophys. 133, 99-102. Tanford, C . (1969) J . Mol. Biol. 39, 539-544. Van Bruggen, E. F. J., Schuiten, V., Wiebenga, E. H., & Gruber, M. (1963) J . Mol. Biol. 7, 249-253. Van Holde, K. E., Blair, D., Eldren, N., & Arisaka, F. (1 977) in Structure and Function of Hemocyanin (Bannister, J. V., Ed.) pp 22-30, Springer-Verlag, West Berlin. von Hippel, P. H., & Schleich, T. (1969) in Structure and Stability of Biological Macromolecules (Timasheff, S. N., & Fasman, G. D., Eds.) pp 457-574, Marcel Dekker, New York.
Pressure-Induced Reversible Dissociation of Enolase? Alejandro A. Paladini, Jr.,f and Gregorio Weber*
ABSTRACT:
A study of the polarization of the intrinsic fluorescence and the fluorescence of dansyl conjugates of enolase shows that an increase in hydrostatic pressure, in the range of 1 bar-3 kbar, promotes the dissociation of this protein into dimers. The dissociation of oligomeric proteins under pressure is predicted to be a general phenomenon by a model that assumes the existence of small “free volumes” at the intersubunit boundaries. The same model predicts a dependence of the standard volume change in the dissociation reaction upon the pressure, owing to the additional surface compressibility of the monomers, and numerical analysis of
the results clearly shows that dependence for enolase. For a midpoint dissociation pressure of 1.5 kbar the standard volume change in the dissociation reaction is AV; = -65 f 8 mL mol-’, and the dependence of the volume change upon pressure (dV;/dp) is approximately -30 mL mol-’ kbar-’. The reversibility of the pressure effects is shown to be better than 95% by either polarization or fluorescence spectrum recovery. The pressure perturbation of the fluorescence polarization is a method of general applicability to studies of protein aggregation, and it can be also of value in characterizing the effect of ligands on the aggregation of oligomeric proteins.
I n the last few years we have conducted a series of studies both on single-chain proteins (Li et al., 1976a,b; Visser et al., 1977b) and on suitable molecular complexes (Weber et al., 1974; Visser et al., 1977a; Torgerson et al., 1979) to ascertain the possible causes of the changes in equilibrium conformation of proteins under high hydrostatic pressures, in the range of 10-3-1 2 kbar. These investigations have pointed definitely to the primary importance of the covalent bond architecture in determining both the sign and magnitude of the characteristic equilibrium changes under pressure. Following their experiments with the inclusion complexes of cyclodextrin polymers, Torgerson et al. (1979) proposed that binding sites be classified as “soft” or “hard” according to whether they can or cannot, respectively, reduce their volume under pressure. This reduction of size can only be achieved by rotation about covalent bonds since both bond angles and bond lengths remain unchanged over the range of pressures at which observations in liquid water solution are possible, 12 kbar. The constancy of bond angles and bond lengths will, in general, restrict the approach of amino acid residues to each other toward the distances that would minimize their free energy of interaction in the absence of constraints and will lead to the appearance of small “free volumes” or “dead spaces” in the interior of a peptide chain when this adopts a globular folded conformation.
Dilatometric studies (Zamyatnin, 1972) show that an increase in volume on the order of 2% should attend the folding of a peptide chain to the globular form, and analysis of X-ray crystallographicdata of Richards (1977) directly validates the concept of small free volumes distributed throughout the protein structure. The existence of these small free volumes gives also an explanation of a finding of Karplus and coworkers in their study of the molecular dynamics of proteins by computer simulation: the appearance of very fast (picosecond) low-amplitude motions of the amino acid residues, faster in fact than those that would be expected in a homogeneous liquid. Viewed from this standpoint the oligomeric proteins present a particularly interesting case: The contact areas between subunits with their multiple points of contact, determined by the folding of each peptide chain into a compact globule, must be considered as typical hard binding sites. We cannot expect that the subunit faces will make perfect van der Waals contacts throughout. Rather minimal distances and optimal atomic packing will prevail at those contacts, probably a small minority, which are the main origin of the free energy of subunit association while at other points it will be on average distinctly larger than the interatomic distances between nearby molecules of a liquid. The small free volumes so created will disappear upon dissociation owing to the much better packing of molecules of the liquid solvent against each subunit interface than the packing of these against each other. Accordingly, the existence of these dead spaces dictates that dissociation of the oligomer into subunits will proceed with decrease in volume (AV,,; < 0). A few observations in the literature indicate that this may well be the general case (Salmon, 1975; En-
From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801. Received September 19, 1980. This work was supported by National Institutes of Health Grant GM 11223. A.R.P. was partly supported during this work by a fellowship from the Organization of American States. *Present address: Instituto de Investigaciones Bioquimicas, Obligado 2490, 1429 Buenos Aires, Argentina.
0006-296018 110420-2587$01.25/0 0 1981 American Chemical Society
2588
B 10 C H E M I S T R Y
PALADIN1 A N D WEBER
gelborghs et al., 1976). However, most of these observations refer to tubulin which involves not only indefinite aggregation but also more than one mode of aggregation and thus lends itself poorly to a rigorous thermodynamic analysis. We present here a study of the effect of pressure upon the aggregation of a simple dimeric protein, enolase, which we analyze according to the ideas sketched in this introduction and further developed in the text. Detection of Changes in Aggregation under Pressure. The change in the equilibrium constant K with pressure is given by eq 1 where AV: is the standard volume change in the -RT d In K/dp = AV:
(1)
reaction at pressure p. For the simplest case of association, that of two monomers into a dimer, K , the dissociation constant, is related to a, the degree of dissociation, and C,, the total protein concentration as dimer, by the relation K = 4c0[a2/(l - a ) ]
(2)
From the last two equations d l n K -AvP” d a 2 - a -----dp a(l - a )
dp
RT
(3)
According to (3), a fixed change in pressure produces a maximal change in the degree of dissociation when a = 2 21/2= 0.5858, that is, when dissociation is just over half accomplished. At this point of maximal sensitivity of the system Aa
E
0.17AV:Ap/(RT)
(4)
where Ap is the finite increase in pressure required to change the degree of dissociation by the amount Pa. Considering the accuracy that is practically attainable in the analysis of chemical equilibria of proteins and ligands, we can hardly expect to do much better than detect a change in a of 0.05, giving AV:Ap/(RT)
N
0.29
(5)
The last relation indicates that for AV; = -50 mL mol-’ and T = 285 K, Ap will be 0.14 kbar. Pressures on the order of tenths of a kilobar are reached in the ultracentrifuge, and the pressure perturbation of the sedimentation of aggregating systems has been repeatedly observed (Harrington & Kegeles, 1973). Detection of dissociation changes under pressure will become increasingly difficult as a I ,the degree of dissociation at atmospheric pressure, approaches 0 or 1. Thus, for a relatively stable dimer for which a I = 0.02 and AV; = -50 mL mol-’, the pressure required to reach the significantly different value of a (0.07) is 3.3 kbar. These considerations indicate that we can only expect to demonstrate the effect of pressure upon the state of aggregation by operating under conditions of solvent composition, temperature, and particularly protein concentration such that there is already some perceptible dissociation at atmospheric pressure. Operation in this range is favorable on another importent account: Detection of changes in association by observations of some spectroscopic variable under pressure, be it absorption or emission, requires demonstration that the the observed changes do not arise as a result of the direct effect of pressure up0 the chromophore or fluorophore. The equivalence of the effects produced by pressure changes at fixed protein concentration and by concentration changes at fixed pressure provides the means of excluding such artifactual effects. Fluorescence Polarization as the Method of Choice. Of the methods by which protein association may be measured, we require one which is sufficiently sensitive to permit ap-
plication to aggregates of weight 105-106 daltons at protein concentrations on the order of micromolar. The only spectroscopic method that qualifies for these purposes is that based on the dependence of the fluorescence polarization, measured under either stationary or time-dependent conditions, upon the rotational diffusion of the protein (Weber, 1952). This method has been satisfactorily applied to the study of many cases of temperature or concentration dependence of protein association (Weber & Young, 1964; Wahl & Frey, 1966; Wahl & Weber, 1967; Yguerabide et al., 1970; Shore & Chakrabarti, 1976; Reinhart & Lardy, 1980). In general, a foreign fluorophore is covalently attached to the protein, and the conjugate is purified by conventional methods. The fluorophore to be attached must have a lifetime sufficiently long to permit a sizable change in the stationary polarization upon change of the state of aggregation of the protein. As a general rule the protein tryptophans have lifetimes of 2-4 ns, not long enough for a reliable measurement of the size of particles with weight larger than lo4 daltons. However, in most proteins the polarization of the intrinsic fluorescence is determined primarily by the local freedom of rotation of these residues which typically involve amplitudes of 20-30° during the fluorescence lifetime and only secondarily by the particle size (Lakowicz & Weber, 1980; Munro et al., 1979). Upon dissociation of oligomeric proteins we expect the local motions of at least some of the tryptophan residues to increase as the restrictions to motion at the interfaces are lessened upon dissociation, and this phenomenon has indeed been observed (Anderson & Weber, 1966; Brewer et al., 1978). It is to be noticed that if we are not interested in establishing the exact size of the particles, but simply the degree of dissociation through the polarization measurements, we only require that the average polarization observed be the composite of fixed polarizations corresponding to the aggregated and disaggregated forms, irrespective of the origin of the depolarizing motions. As shown below, the polarization of the intrinsic fluorescence of enolase as well as the polarization of dansyl conjugates have been investigated as a means of establishing the degree of dissociation under pressure. Thermodynamics of Enolase Dissociation Determination of Degree of Dissociation from Polarization of Emitted Fluorescence. The equilibrium between monomers and dimers is described by
= K
[Dl m41 (6) where [D] and [MI stand for the equilibrium concentrations of dimer and monomer, respectively, and K is the dissociation constant. The fractions of monomer c f M ) and dimer cfD) present at equilibrium are f M = [M1/2C0 fD = (7) with Co being the total molar concentration of protein expressed as dimer. In general, our system is a heterogeneous one consisting of appreciable monomer and dimer populations. In deriving a relation to obtain fM and f D from the observed polarization, it is convenient to replace polarizations x by a related quantity, the anisotropy of emission A, since the latter, but not the former, is weighted according to the contributions of the components to the total emission (Weber, 1952). A and x are related by
-( !-)
A =2 1 3 x
-1
If A D and AM are the anisotropies of the fluorescence from
VOL. 20, N O . 9 , 1 9 8 1
PRESSURE DISSOCIATION OF ENOLASE
pure dimer and monomer populations, respectively, the anisotropy observed when both are present is ADID+ A M ~ M (9) ID + I M where ZM and ID are intensities of the fluorescence from the monomer and dimer populations, respectively, as registered by the detector. Clearly A=
= QD(1 - a) (10) where QM and QD are proportional to the fluorescence yields of monomer and dimer, respectively. Defining the quantity (11) @ I ZM/(ZM ID) ZM
=
ZD
QMa
+
we have from (9) and ( 1 1 ) (12) 9 = (AD - A)/(AD - AM) and with Q = QD/QM,from (10) and (1 l), the reciprocal relations
9=
a
+ Q(1 - a) 9
ff=
9+
and a=
[+ 1
Q (
py1 - 9)
=)Iv1
(13)
(14)
Plots of a against p that include the value p 1 / 2at which a = ' I 2permit a simple estimate of AV;/(RT) from the slope d a l d p at a = 'I2,since for this value of a d In K/dp = 6 da/dp (15) Signijicance of Volume Changes in "Dead Spaces" Model. We sketched briefly in the introduction the consequences of the existence of dead spaces within the structure for the effects of pressure upon the aggregation of oligomeric proteins. We restate that the interactions of two folded peptide chains uniting to form the dimer cannot lead to packing of the boundary atoms as close as that found for the packing of solvent around the corresponding monomer interfaces because the inflexible peptide framework will inevitably lead to the appearance of small free volumes at places other than the favorable contact points. Dissociation into monomers will decrease the volume on account of two separate effects: First, the more efficient packing of the solvent molecules against the amino acid residues at the newly formed surfaces, as compared with the packing of these against each other in the dimer, will eliminate the dead spaces. Secondly, the compression of the residues on the new monomer surfaces appearing on dissociation will produce a further decrease in volume which increases with the applied pressure. Assuming that the compressibility of the solvent at the new surfaces is not different than the bulk compressibility and calling AVlo the standard volume change on dissociation at atmospheric pressure AV; = AV10 PVg (16)
+
In this last equation V, is the effective volume of the amino acid residues at the interfaces that become exposed to solvent upon dissociation, and /3 = l/Vs(dVs/dp) is their average compressibility. Combining this equation with (2) we obtain 2-a AV," PVg da = dP (17) ff(1 - a) RT To obtain an expression describing the change in degree of
2589
dissociation over the whole pressure range, we integrate the last equation between p and atmostpheric pressure, which can be taken as p = 0. The corresponding degrees of dissociation are aband a l , respectively. The integration yields In [$/(I
- ab)]=
In [a12/(1 - a1)l + (Av1"p+ P/2Vg2)/(RT) (18) AVlo and the product PV, may be obtained by best fitting of the data to the last equation. Materials and Methods Chemicals. Enolase was a generous gift from Dr. John M. Brewer of the Biochemistry Department, University of Georgia, Athens, GA. The samples were received as frozen water solutions stored in dry ice, each of them of -2 mL with a protein concentration of lo4 M. The enzyme was highly pure and deionized; therefore, experiments without magnesium as well as those with magnesium were possible. Sample concentration was determined by UV absorption at 280 nm using an extinction coefficient of 80 600 M-' cm-2. Enolase molecular weight was considered to be 90 000. The enzyme stock was kept as small batches at -20 OC and thawed as required. Some of the samples that showed turbidity upon thawing were centrifugated at +4 OC for 15-30 min at 15 000 rpm. For verification of the enzyme purity, a slab gel was run. The gel was 15% acrylamide running gel and 6% acrylamide stacker (Laemmli, 1970). Eight samples of the stock solution of -2.4 mg/mL, ranging in volume from 3 to 15 pL, were run in each well of the gel. There was no significant contamination visible. In the overload samples there was a single band given by the dimer and a hint of some more bands of lower molecular weight. Ultraviolet absorption and fluorescence spectra were measured before and after a pressure run to check reversibility as well as to detect contamination from the alcohol surrounding the sample in the high-pressure bomb. Unless several experiments were to be performed within 2 or 3 days, the buffer solutions were always freshly prepared. Use of detergents was specially avoided in the cleanup of the glassware. Millipore water (10l8 D cm-2) was used in all experiments. Under normal conditions a protein sample could be thawed, diluted, and placed in the bomb to proceed with an experiment within 0.5 h. This includes the time required by the termostatic bath to regain the equilibrium temperature of the bomb after handling. Tris(hydroxymethy1)aminomethane (Tris) buffer (from Sigma Chemical Co.) was used because of the low pK dependency upon hydrostatic pressure (Neuman et al., 1973). Apparatus. (a) High-pressure Bomb. The high-pressure bomb is illustrated in Figure 1. A full description as well as the determination of the correction factors required to obtain fluorescence polarization values free of error due to the birefringence of the windows (induced by the hydrostatic pressure) can be found in Paladini (1980) and Paladini & Weber (1981). Essentially, the bomb has four window ports and one pressure inlet. The samples are placed in an inner cylindrical bottle-shaped quartz cuvette capped with a collapsible polyethylene tube sealed at one end that doubles as a stopper and as a pressure equilibration valve. The four windows are in a plane and placed at 90' with respect to each other; therefore, the samples can be studied by absorption or fluorescence spectrophotometry. Absorption observations were
-
+
Abbreviations used: Tris, tris(hydroxymethy1)aminomethane; EDTA, ethylenediaminetetraacetic acid; Dns, 5-dimethylamino-1 naphthalenesulfonyl residue.
-
2590
BIOCHEMISTRY
PALADIN1 A N D WEBER
0
05
IO 15 PRESSURE ( k bor 1
2’0
25
FIGURE 3:
1: Side-view diagram of high-pressure bomb (A) with inner cell (F) and its holder (E), the window ports (C), and pressure inlet (B). Sealing rings I, J, and K and extractor H are shown making no contact with the walls and ports to facilitate visualization. (D) Quartz windows; (G) polyethylene flexible stopper. FIGURE
FIGURE 2: Red shifts of fluorescence spectra of enolase maintained at room temperature, induced by changes in ionic strength and M, in 0.05 M Tris, pH 7.0; (---) pressure. (-) Enolase, 5 X enolase, 5 X 10” M, in 0.05 M and 1 M KC1, pH 7.0 (normalized); enolase, 5 X 10” M, in 0.05 M Tris, pH 7.0, at 2 kbar. = 280 nm. (-e-)
made by using a Beckman Model ACTA MVI spectrophotometer. Fluorescence excitation and emission were done by using an analog spectrofluorometer (Wehrly, 1979). Fluorescence polarization measurements were made by using a photon-counting polarization instrument of the type described by Jameson et al. (1978). The instrument photon-counting electronics is described elsewhere (Paladini, 1980). Lifetime determinations were done with a cross-correlation fluorometer (SLM Instruments, Urbana, IL). (b) Pressure Generation. The pump used was from High Pressure Equipment, Erie, PA, and consists of a manually operated piston screw pump. It was specifically designed for use in applications where a liquid is to be compressed within a small volume in order to develop pressure. The gauge, of the Bourdon type, was from Olson Engineering and Sales Corp., Burlington, IA. Results Fluorescence Spectrum. Figure 2 shows the fluorescence emission from a 5 X lo4 M solution of enolase in 0.05 M Tris buffer, pH 7.0, at 25 O C , excited at 280 nm. When the same conditions are used with a buffer of much higher ionic strength (0.05 M Tris, 1 M KCI), a red shift and decrease in intensity
Fluorescence polarization changes induced by pressure in enolase solutions (4.9 X 10” M) of different ionic strength, with and without magnesium ions present. (0)0.05 M Tris plus MgZ+,pH 7.3; ( 0 ) 0.05 M Tris and 1 M KC1 plus MgZ+,pH 7.7; (0)0.05 M Tris and 1 M KCl plus 1 mM EDTA; (A)experimental points obtained upon reduction of the pressure for the first sample.
are shown. The same figure shows the effect of a pressure of 2 kbar upon the solution with the low ionic strength as a red shift and decrease in fluorescence yield exactly matching the effects of high ionic strength at atmospheric pressure. The fluorescence spectrum taken 5 min after release of the pressure showed reversibility better than 95%. It is known (Brewer et al., 1978) that the enzyme is largely dissociated at atmospheric pressure in solutions of high ionic strength and the identity of the spectra indicates that a rise in pressure has similar effects. All the samples in these experiments had excess magnesium (1 mM). Intrinsic Fluorescence Polarization. The polarization of the intrinsic fluorescence of enolase as a function of pressure was studied with the enzyme solubilized in Tris buffers of different ionic strength, at different temperatures, and with or without Mg2+ions. Figure 3 shows experiments that compare variations of these conditions. It can be seen that the lower the ionic strength of the solvent the higher the hydrostatic pressure required to achieve a fixed amount of change in polarization. The points are experimental values of polarization x corrected for the scrambling by the windows. Near atmospheric pressure (plotted as 0 kbar) the system does not show a polarization value that corresponds to the dimer form: Polarization values required extrapolation to infinite protein concentration to obtain AD, the anisotropy characteristic of the dimer. The lower polarization plateau achieved under the higher pressures shows virtually the same value for the three curves, 0.09 at -2 kbar. At the time of these experiments the attainable pressure limit was 2.2 kbar. More recently, employing a pump that permitted pressurization of the bomb to its theoretical design limit of 4 kbar, we confirmed the existence of a plateau of polarization at and beyond 2 kbar. Figure 3 shows also that when Mg2+is removed from the system by addition of 1 mM EDTA, the polarization changes indicate a shift in equilibrium toward the monomer in agreement with observations of Brewer & Weber (1968). The same figure shows the extent of the reversibility of the changes in polarization caused by the increase in pressure. The measurements made upon reducing the pressure of the system were determined after allowing 10 min for sample stabilization at each pressure. The observed reversibility was better than 96% as judged by comparison of experimental points obtained upon increasing or reducing pressure. When identical samples were studied at 2 and 25 O C , the latter showed a shift in equilibrium toward the mo-
VOL. 20, NO. 9 , 1981
PRESSURE DISSOCIATION O F ENOLASE
2591
0.20
20-1
I
,
I
I
0.15
2
r--‘ A J
z
0 I-
a
I
;0.10 a J
0 0
.ooI I
260
1
270
200 XEXC (nm)
290
Excitation polarization spectra of enolase at 1 bar (0)and at 2 kbar (A). Concentration, 4.9 X 10” M, in 0.05 M Tris and 1 M KC1 plus 1 mM EDTA, pH 7.30; temperature, 2 OC; excitation bandwidth, 3 nm. Polarization data were corrected for window scrambling. Excitation filter, Corning 7.54; emission filters, 0.54.
0.05
FIGURE 4:
nomer, but a low polarization plateau was not observed at high pressure indicating that at this temperature the dissociated monomers might not be completely stable. A lower limit polarization was clearly observable in the experiments at 12 OC,and this temperature was adopted in many further experiments. It is known that indole and tryptophan (Valeur & Weber, 1977), as well as proteins (Weber, 1960), exhibit a complex excitation polarization spectrum that results from overlap of the L, and Lb transitions in the So-SI absorption. Particularly at those wavelengths at which the overlap is most conspicuous, pressure could change their relative contribution to the absorption resulting in a change in polarization from purely spectroscopic causes if excitation is carried at these wavelengths. This will falsify, to some extent, the computation of the changes in aggregation from the polarization data. To exclude this possibility, we studied the excitation polarization spectrum of enolase by using an excitation bandwidth of 3 nm, at atmospheric pressure and at 2 kbar. The polarization spectra determined in Figure 4 were parallel over the entire range of excitation wavelengths (260-295 nm) showing that, to the accuracy required for our purpose, the relative contributions to the absorption of the La and Lb transitions are not modified by pressure. Similar results were obtained in the absence and presence of MgZf ions. The experiments described point to the dissociation as the cause of the decrease in polarization but do not exclude all possible spectroscopic causes of depolarization with increase in pressure. Direct proof can be obtained by comparing the effects of pressure and total protein concentration upon the observed polarizations. At a constant degree of dissociation, we have from eq 2 In K = In Co + In [4a2/(1 - a ) ]
(19) which introduced into eq 18 yields an expression of the form d In (Co/2K)/dp, = a + bp, (20) where p, is the pressure at which a fixed degree of dissociation is reached, a = AVlo, and b = PV,. Over a relatively small range of pressures, as will be regularly the experimental case, the last relation predicts a straight-line relationship between In Coand pa, but with a slope dependent on both the pressure-independent and pressure-dependent terms. Figure 5 shows the parallel displacement of the x vs. pressure curves toward higher pressures as Cois increased, and the inset the approximate linear relation predicted by eq 2. This can be taken as proof that the process that gives rise to the changes in polarization is one of first order in the total protein con-
i/:,\j
P
.2
k bs f I
, I.o I .4 PRESSURE ( k bar)
.6
,
,
1.8
2.2
Enolase polarization changes induced by application of hydrostatic pressure at different total protein concentration. Temperature, 12 “C; buffer, 0.05 M Tris at pH 7.3 with excess Mg2+ present. (0)1.5 X M; (0) 5 X 10” M; (A) 2 X 10” M; (X) 1 X 10” M; ( 0 )6 X lo-’ M. FIGURE 5:
Table I: Effect of Protein Concentration upon Standard Change in Volume upon Dissociation of Enolase (D -+ M + M) by Measurements of Intrinsic Fluorescence Polarization
C,, (pM)
10 5 2 1 0.6 a
PllZ (kbar)
(mL mol-’)
6cyStb
AVP1,,O (calcd)c
(mL mol-’)
1.28 1.17 1.05 0.88 0.69
-52 -45 -42 -37 -32
0.024 0.005 0.013 0.024 0.014
-50 -47 -44 -39 -33.5
AVPl,20 a
that best fits the experimental data. of measurements of degree of dissociaValue calculated from the equation A V p O = - 1 8 -
Values of AVp
112
.
as$= standard deviation
tion, cy. 3OP,lz.
112
centration. Limiting polarization values were 0.185 and 0.080 for the dimer and monomer, respectively. The data shown in the five curves of Figure 5 were analyzed by fitting them to eq 18 by employing variation of the parameters AVlo and PV, = dV/dp. These, together with p1/2,the interpolated value of p at a = define A V p I lfor ~ each concentration. The standard deviation of the experimental values of a from the computed curves (6ast)were -0.02 which agrees with the precision expected in the measured polarization. Table I, which collects the results of the analyses, shows that AV,, 20 increases systematically with pressure. In fitting any of the five curves shown in Figure 5, we can vary AVlo and dV/dp broadly with only small change in sast,provided the sum permits approach to the value of AVPI,: within 10% error. Because of this circumstance we attach little importance to the values of AVlo and dV/dp required to fit any single curve and retain only the best value of AVpIIIO. It is from the variation of this value with pllZthat we obtain estimates for AVIo and dV/dp (see Table I). Dns-enolase Experiments. Excitation polarization spectra of Dns-enolase at 1 atm and at 2 kbar were parallel, indicating no change in the relative contributions of adjacent transitions in the range of 350-380 nm (see inset of Figure 6). A wavelength of excitation of 360 nm was selected for subsequent polarization studies. Figure 6 shows the polarization changes induced by pressure upon a 6 X 10“ M solution of the conjugate dissolved in 0.05 M Tris and 0.1 M KCl with excess Mg2+. The curve to fit the data has AV10= -18 mL mol-’
-
2592
BI OCHEM ISTR Y
PALADIN1 AND WEBER 31
0 301
26
24
z 0
380
360
340
t
< c! 5522 0
a
20 0
2
6
1'0 14 18 PRESSURE (k bar )
22
26
FIGURE 6:
Fluorescence polarization changes of Dns-enolase induced by hydrostatic pressure. The enolase concentration was 5.6 X 10" M in 0.05 M Tris and 0.1 M KC1, pH 7.4, with excess Mg2+present. Temperature, 25 OC; Xexcit = 360 nm; excitation bandwidth, 5 nm. The solid line represents averaged changes in polarization. Inset: Excitation polarization spectra of Dns-enolase con'ugate at 1 bar and 2 kbar. The enolase concentration was 5 X 10-2M in 0.05 M Tris with excess of Mgzt. Temperature, 25 OC; excitation bandwidth, 1.25 nm; excitation filter, none; emission filters, Coming 3-73. Polarization values were corrected for scrambling of the windows.
0:2
0'6
IO
I'4
I6
2'2
PRESSURE (k bar) FIGURE 7:
Degree of dimer dissociation of enolase or Dns-enolase
(a)vs. hydrostatic pressure. Enolase, C = 4 X 10" M, in 0.05 M Tris buffer with Mg2+ present. Temperature, 12 OC. (0)Experimental points; ( 0 )Dns-enolase, C = 5.6 X 10" M, same solvent. Both theoretical curves were drawn for AVIo = -18 mL mol-': @V, = -36 mL mol-' kbar-'; cyI = 0.24 (-); a i = 0.27 (---).
and PV, = -36 mL mol-' kbar-', cyI = 0.275, which give AV,,,: = -54 mL mol-' at p112= 1 kbar (Figure 7). The effect of increasing enolase concentration at fixed temperature is shown in Figure 8. Lower limit polarization values were similar, and the pressure for half-dissociation, p I l 2 ,was shifted to higher pressures with an increase in concentration by the expected amount. The fluorescence lifetime of the Dns conjugates was measured by the phase and modulation technique at atmospheric pressure and at 2 kbar. Identical values of 13.2 f 0.1 ns were found by both phase and modulation and at the two pressures. Thus, there was no pressure dependence of the lifetime of the excited state, and the identity of phase and modulation lifetimes indicates that from this point of view the emission was remarkably homogeneous. Further, no correction for differences in fluorescence yields was required. Discussion and Conclusions The similarity of the changes in the fluorescence spectrum and intrinsic fluorescence polarization brought about by high
4
8
12 16 20 PRESSURE ( k b a r )
24
2.8
32
FIGURE 8: Dns-enolase fluorescence polarization changes measured at different total protein concentrations. hex,,,= 360 nm with a bandwidth of 5 nm. Buffer, 0.05 M Tris and 0.1 M KCI with excess Mg2+present. Polarization values were corrected for scrambling of M at 12 'C; (0)4 X M at 12 the windows. (A) 1.6 X OC.
pressure, on one hand, and by low Mg2+or high ionic strength, on the other, indicates that pressure has a dissociating effect upon enolase. A direct proof is obtained by observations on the shift of the range of pressures over which polarization changes occur with changes in the total protein concentration. This shift is characteristic of a process of first order in protein concentration, and, by adjustment of this so that there is already a detectable dissociation at atmospheric pressure, a reasonable sensitivity in the changes of polarization with pressure can be achieved and the pressure limit for complete dissociation kept below 2 kbar. Two objectives are thus accomplished: First, the changes in polarization take place over the range of pressure where reliable corrections for the depolarizing effect of the windows of the high-pressure bomb are available (Paladini, 1980). Secondly, a number of observations on single-chain proteins (Brandts et al., 1970; Zipp & Kauzmann, 1973; Hawley, 1971; Li et al., 1976a,b; Visser et al., 1977a) indicate that in virtually all the cases the detectable effects of pressure upon the conformation are confined to the region of pressures above 4 kbar. Thus, the effects on the aggregation of oligomers below 2 kbar may be considered, to a satisfactory first approximation, as independent of effects of pressure upon the conformation of the chains. The similarity of the results of observations on the polarization of intrinsic protein fluorescence and of Dns conjugates of enolase is also to be recalled to exclude purely spectroscopic effects as causing appreciable artifacts. Similar values of the pressure-independent (AVlo) and pressure-dependent (PVg) terms could be used to fit within experimental errors the observations on enolase and Dns-enolase shown in Figure 7. To gauge the accuracy and predictive value of these observations, we cite a numerical analysis employing the eight central values of (Y shown in the plot of Figure 8 for Co= 4 X M. Best fitting with standard deviation 6a,,= 0.028 was obtained for AV10 = 32 f 16 mL mol-'; PV, = -30 f 20 mL mol-' kbar-' and AV,., = -66 f 8 mL mol-'. If /3V,is assumed null, best fitting gives 6a,,= 0.06, and a similar bad fit is obtained with AVlo = 0. Thus, the inclusion of pressure-dependent and pressure-independent volume changes is required by the data. It also follows from these considerations that quotation of a standard volume change upon dissociation, AV:, requires a statement of the pressure assigned to this value. It seems natural to assign the volume change extracted from a plot of the degree of dissociation vs. pressure to the pressure p1/2since the precision of the measurements is maximal for a = ' / p The compressibility term of the standard volume change indicates that the monomers are more compressible than the dimer. If
VOL. 20, NO. 9 , 1981
PRESSURE DISSOCIATION OF ENOLASE
proteins underwent a homogeneous decrease in volume with a rise in pressure, one could not expect the compressibilities of dimer and monomers to differ. On the other hand, the excess compressibility of the monomers is easily understood if we are dealing essentially with surface compressibilities affecting only the solvent-exposed residues. A gross, but nonetheless interesting, estimate of p can be made from the enolase data. A cubic dimer of molecular weight M and mean amino acid residue weight m has M / m residues. Upon splitting across one face into two equal monomers, the volume of residues in the newly exposed areas equals (M/m)2f3m0, where B is the partial specific volume. If we assume the compression to act appreciably upon one-sixth of the total surface of the residues V, = M2/3m‘i3B/6
with m = 110 and 0 = 0.75, which apply to globular proteins in general, V , = O.6M2I3. For enolase M = 90000, V, = 1200, and /3 = 0.025 kbar-’. This value of 0 should be compared with the bulk compressibilities of hexane (0.09 kbar-’), benzene (0.07 kbar-’), and water (0.045 kbar-’). The figure for enolase, even if found to be incorrect by a large fraction, would still be a good demonstration of the low compressibility of the protein surface dictated by the rigid covalent bonding holding the amino acid residues. Finally, we wish to stress the general applicability of the fluorescence polarization techniques to the study of protein dissociation under pressure, the speed, accuracy, and ease of the measurements, and the almost ideal reversibility of pressure effects upon the delicate protein oligomeric systems. Observations on other oligomeric proteins should permit a test of the validity and generality of our proposed “free volume” model and ultimately provide some figures for comparison with the X-ray crystallographicdata of oligomeric proteins. Equally promising appears to us the use of pressure perturbation of the polarization to study the changes in aggregation of oligomers owing to binding of enzymic allosteric effectors. Acknowledgments We thank Professors H. G. Drickamer and J. Jonas for their help in the design of the instrument for measurement of fluorescence polarization at high pressure and for valuable discussions. References Anderson, S., & Weber, G. (1966) Arch. Biochem. Biophys. 116, 207-223. Brandts, J. F., Oliveira, R. J., & Westort, C. (1970) Biochemistry 9, 1038. Brewer, J. G., Faini, G. J., Wu, C . A., Gross, L. P., Carreira, L. A., & Wojcik, R. (1978) Dev. Biochem. 3, 57-78.
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