J . Phys. Chem. 1988, 92, 2850-2853
2850
Protein Dynamlcal Structure by Tryptophan Phosphorescence and Enzymatic Activity in Reverse Micelles: 1. Liver Alcohol Dehydrogenase Giovanni Battista Strambini* and Margherita Gonnelli CNR, Istituto di Biofisica, via S. Lorenzo 26, 56100 Pisa, Italy (Received: August 13, 1987; In Final Form: December 9, 1987)
Subtle perturbations in the conformational state of liver alcohol dehydrogenase incorporated in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate in isooctane were monitored by the phosphorescence lifetime of Trp-314. The enzyme in the micellar milieu possesses an overall structure not dissimilar to the native state except for a considerably greater flexibility of the coenzymebinding domain. While this change in dynamical structure of the macromolecule is shown to account entirely for the decrease in turnover rate with the larger micelles, with the smaller ones inhibition of the catalytic function points to an additional probably direct role of micellar water.
Introduction In the past decade an increasing number of enzymes have been introduced in reverse micelles, aggregates formed by ionic surfactants in organic The ability to solubilize various amounts of water in their interior confers to these structures a topology and physicochemical properties typical of biological membranes. The retention of catalytic activity in reverse micelles has stimulated great interest in them for they provide simple models for studying conformation and activity of biopolymers in membrane-like environments' and open new possibilities for studying enzymes at low temperature3 as well as promise new potentialities for technological a p p l i c a t i ~ n . ~ , ~ A salient feature displayed by enzymes in reverse micelles is the dramatic dependence of their catalytic rate constant on the size of the water pool. In reverse micelles of sodium bis(2ethylhexyl) sulfosuccinate (AOT) in isooctane, the most common and best characterized solvent system, enzyme activity displays a typical bell-shape dependence on wo (wo = [H20]/[AOT]).* The maximum activity, which can sometimes be. much larger than in aqueous solution, often coincides with a diameter of the inner cavity roughly of the same dimension as the macromolecule.2 In general, the structure of the biopolymer, as detected by spectroscopic techniques, is not significantly modified in the micellar environment, and modulation of enzyme activity by the water content is thought to derive from rather subtle changes in conformation/flexibility of the protein molecule. Whether these presumed perturbations are due to variation in the properties of micellar water at different wo or to the direct interaction between biopolymer and charged double-layer, surfactant and apolar solvent is not known. In this study we focus attention on the dynamical structure of liver alcohol dehydrogenase, LADH, in the micellar state as a possible indicator of subtle changes in its conformational state. The flexibility of the coenzyme-binding domain of LADH is inferred from the remarkable sensitivity that the triplet-state lifetime of tryptophan has for the fluidity of its microenvironment.6 LADH possesses a tryptophan residue in the coenzyme-binding domain displaying unusually long-lived phosphorescence in aqueous solutions at room temperature.' Most important, minor perturbations in the dynamical structure of this region were shown to result in a remarkable alteration of the catalytic activity.s The findings of this investigation do reveal uniquivocal changes in the flexibility of the protein structure when confined in the micellar environment. Furthermore, while at large values of w o the catalytic inefficiency of the enzyme can be fully accounted for by the alteration in the conformational state of the macromolecule with the smaller micelles, inhibition of the catalytic function points to an additional contribution arising most likely from the unavailability of micellar water.
* To whom correspondence should be addressed. 0022-3654/88/2092-2850$01.50/0
Materials and Method Materials. Liver alcohol dehydrogenase from a horse (LADH) was obtained as a crystalline suspension from Boehringer (Mannheim, FRG). Sodium bis(2-ethylhexyl) sulfosuccinate (AOT) obtained from Sigma Chemical Co. (St. Louis, MO) was purified according to Wong et al.9 and dried in vacuo over P205. The purity of AOT was checked by the procedure of Luisi et al.' Spectroscopic grade isooctane and glycerol from Merck (Darmstadt, FRG) were used without further purification. The enzyme was dialyzed for at least 24 h under nitrogen against 0.1 M glycine-NaOH buffer (pH 8.8). Any remaining insoluble precipitate was removed by centrifugation. Fresh preparations were made weekly, and no loss of activity was found during that time. The activity of LADH in water, measured by Dalziel's method,1° ranged between 130% and 145%. Active site concentrations were also determined by spectrophotometric titration of LADH coenzyme-binding sites with NAD+ in the presence of excess pyrazole." On the basis of molar extinction coefficient of EZg0= 3.53 X lo4 M4 cm-', the coenzyme-binding capacity was typically 95% or better. The enzyme activity in reverse micelles was assayed according to the method of Meir and Luisi.12 Sample Preparationfor Luminescence Measurements. Micellar solutions of desired wo were obtained by adding, with a microsyringe, concentrated protein in glycine-NaOH (0.1 M, pH 8.8) to a 100 mM AOT solution in isooctane. The protein concentration, evaluated by using the extinction coefficient in water, was kept roughly constant to a value of 5.8 X loa M. To obtain reproducible phosphorescence data in fluid solutions, it is of paramount importance to thoroughly remove all dissolved oxygen. Satisfactory deoxygenation was achieved by placing about 0.5 mL of the protein solution in the short arm of an L-shaped quartz cell provided with a small stirrer where gas exchange and equilibration are to take place. The solution is finally transferred to the other arm (4-mm i.d.) for emission studies. The short arm (1) Luisi, P. L.; Meier, P.; Imre, V. E.;Pande, A. In Reverse Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum: New York, 1984. (2) Martinek, K.; Levashov, A. V.; Klyachko, N.; Khmelnitski, Y. L.; Berezin, I. Eur. J. Biochem. 1986, 155, 453-468. (3) Douzou, P. Adv. Enzymol. 1980, 51, 1-74. (4) Liithi, P.; Luisi, P. L. J . Am. Chem. SOC. 1984, 106, 7285-7286. (5) Meier, P.; Imre, V. E.; Fleschar, M.; Luisi, P. L. In Proceedings ofthe Lund International Symposium on Surfactants in Solution; Hal, K. L., Lindman, B., Eds.; Plenum: New York, 1984. (6) Strambini, G. B.; Gonnelli, M. Chem. Phys. Lett. 1985, 115, 196-201. (7) Saviotti, M. L.; Galley, W. C. Proc. Narl. Acad. Sci. U.S.A. 1974, 71, 4 154-41 58. (8) Strambini, G. B.; Gonnelli, M. Biochemistry 1986, 25, 2471-2476. (9) Wong, M.; Thomas, J. K.; Gratzel, M. J . Am. Chem. SOC.1976, 98, 2391-2397. (10) Dalziel, K. Acta Chem. Scand. 1975, 11, 397-398. (1 1 ) Theorell, H.; Yonetani, T. Biochem. 2.1963, 338, 537-553. (12) Meier, P.; Luisi, P. L. J . Solid-Phase Biochem. 1980, 5, 269-282.
0 1988 American Chemical Society
The Journal of Physical Chemistry, Vol. 92, No. 10, 1988
Liver Alcohol Dehydrogenase
is connected to a vacuum line for gas exchange by means of a vacuum-tight steel cap (Swagelock patent D-316), which, upon detachment from the line, avoids air leakage. Satisfactory removal of O2from the solution was obtained in about 10 min by repeated application of moderate vacuum followed by inlet of very pure nitrogen (0.1 ppm in 02,SIO, Florence, Italy) at a pressure of 3 atm and gentle stirring. A check on the thoroughness of deoxygenation is provided by the dependence of phospohorescence lifetime on the amount of excitation absorbed by the ~ a m p 1 e . I ~ As a further control that the lifetime obtained with this procedure is the oxygen-free value, known amounts of O2were added to the solution by equilibrating it with given oxygen pressures. Extrapolation of the lifetime value to zero oxygen pressure confirmed that the deoxygenation procedure is adequate. Enzyme activity measurements before and after degassing showed no alteration in the sample. Spectroscope Measurements. Fluorescence and phosphorescence spectra were obtained with a conventionally designed instrument.I3 The excitation was selected by a 250-mm grating monochromator (Jarrel-Ash) employing a band-pass of 2 nm for fluorescence and 10 nm for phosphorescence. The emission was dispersed by a 250-mm grating monochromator (Jobin-Yvon H25) and detected with an EM1 9635 Q2B photomultiplier. Variations in the lamp output were corrected for by normalizing fluorescence and phosphorescence intensities with the intensity of the exciting light. Phosphorescence decays were monitored at 440 nm by a double-shutter arrangement permitting the emission to be detected 2 ms after the excitation cutoff. The decaying signal was stored and on occurrence averaged in a Varian C-1024 time-averaging computer and successively transferred to an Apple I1 computer for exponential decay analysis by a least-squares method. Fluorescence anisotropy measurements were carried out by inserting linear polarizers, Polaroid type HNP'B, in both the excitation and the emission beam. The excitation wavelength was 303 nm while the emission was centered at 330 nm. The anisotropy, r , was calculated in the usual way from the formula r=
Ill
Ill
- GI,
+ 2GI,
where Il and l I , are the emission intensities polarized parallel and perpendicular, respectively, to the vertically polarized exciting beam. The correction factor G is the ratio of the vertically to horizontally polarized emission intensities obtained with horizontal excitation. Circular dichroism measurements were carried out in a Jasco Model 5-500 A recording spectropolarimeter utilizing a cell of 1-cm path length. All experiments were conducted at 20 "C.
Results Enzyme Aciiuity. The turnover number of LADH in reverse micelles of AOT in isooctane as a function of water content was first reported by Meier and Luisi12 in 1980. Since the impurity content of AOT can heavily affect the catalytic rate of some enzymes' and the purity of AOT available today is considerably better, we have carefully repeated these activity determinations. The catalytic rate constant, kcat, determined for values of wo ranging from 10 to 50 is displayed in Figure 1. The results essentially confirm the values reported in the previous study. At pH 8.8 of the stock aqueous solution in glycine-NaOH buffer, k,, shows the characteristic bell-shape dependence upon the water content of the micelles (Figure 2). Maximum activity is found around about wo = 40 where k,,, is about 70% of the value in buffer. Tryptophan Steady-State Emission Properties. The emission properties of tryptophan in proteins may be a useful indicator of possible conformational changes in the macromolecule. Upon excitation at 303 nm the luminescence from LADH is dominated
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Figure 1. Dependence of the catalytic rate constant k,, (A) and the phosphorescence lifetime ( 0 )of Trp-314 of LADH on the water content, wo,of reverse micelles. Lifetime-normalized phosphorescence intensities, Zp/7 (m), relative to the value of this parameter in buffer are also reported.
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TIME (SEC) Figure 2. Decay of the phosphorescence intensity of LADH in glycineN a O H (0.1 M, p H 8.8) and in micelles a t some representative ww A,, = 300 nm, ,X, = 440 nm. At w, = 11.1 the decay is also reported for a sample in which the surfactant concentration is [AOT] = 300 mM.
by the contribution of Trp-314.I4 The wavelength of the maxima and the width of vibronic bands in fluorescence and phosphorescence spectra of the protein solubilized in reverse micelles faithfully reproduce the emission observed in aqueous solution. These spectral features suggest that the macromolecule is confined in the water pool of the micelle and that no alteration in structure has occurred that might affect the polarity of the chromophores' environment. Further, fluorescence anisotropy measurements at 330 nm yield a value of r (r = 0.26 f 0.003) which is constant at every w,,and only slightly smaller than in solution ( r = 0.27 f 0.004). If one allows for the depolarizing effect due to the greater light scattering of micellar solutions, the difference in r is probably insignificant. Thus, phenomena such as dimer-tomonomer dissociation and/or independent local rotational motions of the tryptophanyl side chains, both of which would reduce the value of r , can be ruled out. The conclusion that LADH in the micellar solution has not undergone gross conformational changes also finds support from circular dichroism measurements. The almost constancy of the CD spectrum at various wostands to indicate that only very minor alterations are detectable in the secondary/tertiary structure arrangement of the macromolecule. Kinetics of Tryptophan Phosphorescence Emission. The room-temperature phosphorescence of LADH is due entirely to (14) Abdallah, M. A,; Biellman, J. F.; Wiget, P.; Joppich-Kuhn, R.; Luisi,
(13) Strambini, G. B. Biophys. J . 1983, 43, 127-130.
P.L. Eur. J . Biochem. 1978, 89, 397-405.
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The Journal of Physical Chemistry, Voi, 92, No. 10, 1988
Strambini and Gonnelli
TABLE I: Dependence of the Phosphorescence Lifetime of LADH on the Viscosity of a Glycerol/Glycine-NaOH Buffer Solvent glycerol, wt% viscosity," CP phosphorescence lifetime, s 0 1.005 0.68 f 0.02 70 22.5 0.65 f 0.03 80 109 0.67 f 0.03 OViscosities were taken from: Miner, C. S.; Dalton, N. N.In Glycerol; Reinhold: New York, 1953.
Trp-314, and because the two subunits of the enzyme are equivalent, the kinetics of this emission in glycine-NaOH buffer at 20 OC is represented by a single lifetime, T , of 0.68 s. The decay of the phosphorescence intensity with time for LADH in micelles at a few representative w o values is shown in Figure 2. The intensity obeys an exponential law at all wo,indicating that the phosphorescing protein constitutes a homogeneous sample in each micellar system investigated. That is, all tryptophan chromophores are equivalent in the dynamical properties of their environment, when averaged over a time window of the order of T . Furthermore, such a phosphorescence lifetime is strictly a property of the macromolecule as it was shown not to depend on second-order processes that might derive from the collision with empty or enzyme-containing micelles. In fact, when both the micellar concentration and the fraction of protein-filled micelles are changed by over a factor of 10, T remains constant. The dependence of the phosphorescence lifetime of Trp-3 14 on the water content of the micelles is reported in Figure 1. We note that the value of T in the micellar state is always considerably smaller than observed in solution, the largest departure occurring at the smallest wo. Below w o = 10 T is too short ( r C 4 ms) for detection. As the water content of the micelle becomes greater, T increases toward the aqueous solution limit, reaching a maximum at wo = 31 beyond which it drops again. The smaller value of r on going from the aqueous solution to the micellar state signals a loosening of protein structure surrounding Trp-314. When this change is quantified using the dependence of T on the viscosity of the chromophore's environment,6 we obtain a decrease in viscosity of the coenzyme-binding domain which at wo = 31 and at wo = 11 corresponds to a factor of 2.4 and 10, respectively. It is significant that the largest change in T occurs at the onset of the catalytic function and that at w o < 10 where r is too short for detection the enzyme is no longer functional. It is important to recognize that a more flexible internal structure of LADH cannot result simply from a greater viscosity of the water pool. In fact, lifetime measurements in water/glycerol solvent mixtures in which the viscosity is varied by a factor of 100 (Table I) demonstrate that to a large extent T is unaffected by the viscosity of the medium. (If anything, the dampening effect on structural fluctuations of a more viscous solvent causes a lengthening of T . ) The greater flexibility, therefore, of the coenzyme-binding domain reveals unambiguously that the enzyme solubilized in micelles is subject to perturbations in its conformation and, significantly, these alterations persist even at w ofor which the properties of micellar water approach closely those of bulk ~ a t e r . ~ , ' ~ , ' ~ Homogeneity of the Protein Sample in Reverse Micelles. Control on the homogeneity of the protein sample is paramount in this multiphasic solvent system where in principle heterogeneity may arise from repartition of the biopolymer among different microphases and the fact that micelles of AOT with different structural arrangements3-" can be produced by minor variations in experimental conditions. Furthermore, some enzymes were reported to undergo a degree of denaturation during the solubilization process.'* (15) Zinsli, P. J . Phys. Chem. 1979,83,3223-3228. (16)Politi, M. J.; Chaimovich, H. J . Phys. Chem. 1986,90, 282-287. (17) Gamboa, C.; Sepulveda, L J. Colloid Interface Sci. 1986, 113, 566-576. (18) Wolf, R.; Luisi, P. L. In Solution Behauior ofSurfacrants. Theoretical and Applied Aspect; Mittal, K. L., Fendler, E. J., Eds ; Plenum: New York. 1982
[NADtiIx loe M
Figure 3. Quenching of the fluorescence intensity (Aex = 303, A,, = 330 nm) of LADH (1.4 X 10" M) upon binding of NADH ( 0 ) glycineN a O H (0.1 M, pH 8.8). (*) wo = 11.1; (A)w o = 20;).( wo = 31.1; (A) w o = 40; (0) wo = 50.
The phosphorescence lifetime of LADH can be a sensitive monitor of the state of the macromolecule. As an example in Figure 1 we show the heterogeneity in the phosphorescence decay induced by concentrating the micellar solution to [AOT] = 300 mM. At this concentration of AOT and w o = 11 the phosphorescence decays in a clearly nonexponential fashion with short-lived components characteristic of LADH in dilute micelles and a long-lived component, representing roughly 20% of macromolecules, with a lifetime of 2.2 s even longer than the value of 0.65 s found in aqueous solutions. Clearly under these conditions the protein sample is partitioned between single molecules in "normal" micelles and single molecules or aggregates in a new microphase. The exponential decay of the phosphorescence of Trp-314 is an indication of homogeneity in the sample only if each macromolecule contributes to the total phosphorescence intensity. A parameter which is directly related to the fraction of emitting macromolecules in the sample is the lifetime-normalized phosphorescence intensity, Z , / T . * ~ ' ~ In particular, the value of I,/T observed in micelles relative to that obtained in buffer is a measure of the integrity of the protein sample in the micellar state. A value smaller than one implies that some macromolecules, due to pronounced perturbations in structure, possess a T too short for detection. In Figure 1 we note that at wo = 11.1 only about 65% of the LADH population contributes to the phosphorescence emission. This fraction increases monotonically with wo until at w o I45 I,/T becomes one. Reversibility tests in which micelles prepared at wo = 11.1 were successively brought to w o = 50 by the addition of water show that the structural change causing the phosphorescence quenching is not a permanent one in that the phosphorescence intensity is completely restored. Additional evidence that the nonphosphorescing protein molecules have not undergone irreversible damage is provided by the coenzymebinding capacity. From Figure 3 we can appreciate that although coenzyme binding is slightly altered in the micellar state, showing a somewhat sigmoidal behavior, the variation in Z,/T with w o is not paralleled by the changes in the number of binding sites. In conclusion, according to Zp/r data LADH is present in micelles in more than one conformer in dynamic equilibrium and at least one of them is nonphosphorescing. We may also add that the equilibrium is established slowly relative to T because for interconversion rates of magnitude similar to T the decay of phosphorescence would be biexponential.20 Of course, much faster (19) Domanus, J.; Strambini, G. B.; Galley, W. C. Photochem. Photobiol. 1980,31, 15-21. (20) Laws, W. R.; Brand, L. J . Phys. Chem. 1979,83,795-802.
The Journal of Physical Chemistry, Vol. 92, No. IO, 1988 2853
Liver Alcohol Dehydrogenase -
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