Langmuir 1993,9, 912-915
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Electric Percolation of Enzyme-Containing Microemulsions Vassiliki Papadimitriou,t Aristotelis Xenakis,*it and Panagiotis Lianost National Hellenic Research Foundation, Institute of Biological Research & Biotechnology, 48 Vas. Constantinou Avenue, 11635 Athens, Greece, and Physics Section, School of Engineering, University of Patras, 26500 Patras, Greece Received July 13,1992. In Final Form: December 28, 1992 Water in oil microemulsions containing chymotrypsin or trypsin have been studied by conductivity and luminescence decay measurements. A percolation process has been demonstrated to occur in all cases. The percolation threshold, however, was obtained at higher water content values on increasing the enzyme concentration. The luminescence decay profiles of Ru(bpy)32+in the presence of quencher also depended on the enzyme presence and its concentration. The resulta are discussed in terms of a "percolation" model.
Introduction Water in oil microemulsions, sometimes called reverse micelles, are fine dispersions of water in a nonpolar organic solvent stabilized by surfactant molecules. When A O T is used as the surfactant, the reverse micelles are spherical aqueous droplets surrounded by a monolayer of AOT molecules.' T h e size of the reverse micelles is directly related to the water content, which can be expressed by t h e ratio w o = [H201/[AOTl, and varies within the range of 2-20 nmS2 The dispersed water pools can solubilize various biomolecules, such as enzymes, which may retain, or even enhance, their catalytic ability.%' T h e presence of enzymes in t h e microemulsions results in structural changes that occur in both t h e biomolecule and the reverse micelles.a11 It has been recently reported that t h e presence of cytochrome c induces stronger attractive interactions between t h e reverse micelles, resulting in a percolation proces~.~~J~ I n the present work we have studied t h e behavior of water in oil microemulsions containing chymotrypsin or trypsin, two enzymes well studied in this medium.14J5 The effect of the presence of enzyme on the structure of AOT reverse micelles was investigated both by electric conductivity measurements and by time-resolved luminescence spectroscopy. For t h e latter method, we have used
* To whom correspondence should be addressed. t
Institute of Biological Research & Biotechnology.
* University of Patras.
(1) Reverse Micelles, Luisi, P. L., Straub, B., Eds.; Plenum Press: London, 1984. (2) Zulauf, M.; Eicke, H. F. J. Phys. Chem. 1979,83,480. (3) Structure and reactivity in reverse micelles; Pileni, M. P., Ed.; Elsevier: Amsterdam, 1989. (4) Luisi, P. L.; Magid, L. CRC Crit. Rev. Biochem. 1986,20, 409. (5) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Khmelnitaki, Y. L.; Berezin, Y. V. Eur. J. Biochem. 1986, 155, 453. (6) Xenakis, A.; Valis, T. P.; Kolisis, F. N. Prog. Colloid Polym. Sci. 1989, 79, 88. (7) Kolisis, F. N.; Valis, T. P.; Xenakis, A. Ann. N.Y. Acad. Sci. 1990, 613, 674.
(8)Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 209. (9) Sheu, E.; Gdklen, K. E.; Hatton, T. A.; Chen, S.-H. Biotechnol. Prog. 1986, 2, 175. (10) Cazianis, C . T.; Xenakis, A.; Evangelopoulos, A. E. Biochem. Biophys. Res. Commun. 1987,148, 1151. (11) Xenakis, A.; Cazianis, C. T. B o g . Colloid Polym. Sci. 1989, 76, 159. (12) Huruguen, J. P.; Authier, M.; Greffe, J. L.; Pileni, M. P. Langmuir 1991, 7, 243. (13) Huruguen, J. P.; Pileni, M. P. Eur. Biophys. J. 1991, 19, 103. (14) Walde, P.; Peng, Q.; Fadnavis, N. W.; Battistel, E.; Luisi, P. L. Eur. J. Biochem. 1988, 173, 401. (15) Barbaric, S.; Luisi, P. L. J. Am. Chem. SOC.1981, 103, 4239.
0743-7463/93/2409-0912$04.00/0
Ru(bpy)S2+ as t h e lumophore and Fe(CN)& as t h e quencher, both well known as probes of water in oil microemulsions made of anionic ~ u r f a c t a n t s . ' ~ JT~h e analysis of the luminescence decay profiles was done with a "percolation" model,18 which is particularly adapted to water in oil microemulsions.
Experimental Section Materials. a-Chymotrypsin (EC 3.4.21.1) from bovine pancreas type I1 was purchased from Sigma. Trypsin (EC 3.4.21.4) from bovine pancreas type I11was from Merck. Bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) was from Sigma. It WBB used without further purification, since the degree of purity was shown to be acceptable for this type of experiment.19 Cetyltrimethylammonium bromide (CTAB) was from Serva, Heidelberg. Isooctane was purchased from Merck, Darmstadt, while butanol, pentanol, and hexanol were from Ferak, Berlin. Tris(2,2'bipyridine)ruthenium dichloride hexahydrate (Ru(bpy)a*+)was from GFS Chemicals and potassium hexacyanoferrate(II1) (KsFe(CN)e)from Merck. All chemicals were of the highest available degree of purity. Double-distilled water was used throughout this study. Preparation of Microemulsions. In the case of water/AOT/ isooctane microemulsions a stock solution of 0.1 M AOT in isooctane was prepared and stocked. The content in water was periodically checked by Karl-Fischer titrations. The amount of water (in general less than 1% ) was taken into consideration in the calculation of the global water content. Reverse micelles were formed with the addition of the appropriate amount of a 50 mM Tris/HCl (pH 9) buffer solution containing chymotrypsin or trypsin. Solubilization was achieved by gentle shaking within a few seconds. The total amount of buffer was adjusted to give the desired value of the ratio wo = [H20l/[AOTl. In the case of the cationic systems based on CTAB, microemulsions were obtained with the addition of butanol as a cosurfactant. In all cases the initial concentration of CTAB was 0.2 M and the molar ratio [cosurfactant]/[CTAB] was 5.5. Conductivity Measurements. The conductivity of the microemulsions was measured with a Metrohm 644 conductometer using a thermostated microcell. The cell constant, c, was equal to 0.97 cm-I. Luminescence Decay Measurements. Nanosecond decay profiles were recorded with the photon counting technique using a specially constructed hydrogen flash, ORTEG and Schlumberger-Enertec electronics, and a Nucleus Multichannel scaler card with an IBM-PC. A Melles-Griot interference filter was (16) Attik, S. S.; Thomas, J. K. J. Am. Chem. SOC.1981, 103, 7403. (17) Lianos, P.; Zana, R.;Lang, J.; Cazabat, A. M. In Surfactants in Solution; Mittal, K. L., Bothorel, P., Eds.; Plenum: New York, 1986; Vol. 6, p 1365. (18) Lianos, P.; Modes, S.; Staikos, G.; Brown, W. Langmuir 1992,8, 1054. (19) Fletcher, P. D. I.; Perrins, N. M.; Robinson, B. H.; Toprakcioglu,
C. In Reverse Micelles; Luisi, P. L., Straub, B., Eds.; Plenum Press: London, 1984.
0 1993 American Chemical Society
Electric Percolation of Microemulsions ,- 500
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Langmuir, Vol. 9, NO.4,1993 913
A
i
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V
:::D -
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100
0 45
%v 5% Figure 1. Variation of the conductivity of water in oil microemulsions as a function of water content for different temperatures: (A) AOT/isooctane system, wo = [H2Ol/[AOTl; (B) CTAB/butanol/isooctane,cp1 is the volume fraction of water; ( 0 ) 15 "c; ( 0 )25 O C ; ( 0 )30 "c; (A)35 O C ; (A)40 O C ; (*) 45 O C .
50
Results and Discussion Conductivity. The conductivities of various microemulsion systems were measured in the presence and abeence of enzyme. Figure 1 shows the variation of the conductivity as a function of the water content of the microemulsions for different temperatures. In the case of cationic systems (Figure 1B)the water content is expressed in terms of the volume fraction, since the ratio wo = [HzOI/ [surfactant] does not reflect the real composition of the amphiphilic membrane. This is due to the presence of butanol which not only is located at the interface but also partitions between the different microdomains. As expected for these oil continuous systems the conductivity is practically zero for relatively low temperatures. The water microphase which contains the surfactant counterions and the ions of the buffer is compartmentalized in the reverse micelles surrounded by the nonconducting organic phase. However, when the temperature is increased above 30 "C,a sharp increase of the conductivityappears at high wovalues. This abrupt change of the conductivity is attributed to the increased probability of the reverse micelles to form large clusters, allowing the exchange of the conducting species. This phenomenon is attributed to the appearance of a percolation procedure of the distinct droplets, when the volume fraction of the dispersed phase attains a certain value, known as the percolation t h r e ~ h o l d . ~ lWith - ~ ~ increasing temperature the percolation threshold appears a t lower w o values for the anionic systems (Figure 1A)and at lower water content (cpw) for the cationic ones (Figure 1B). We have also measured the conductivity of the abovementioned microemulsions in the presence of various enzyme concentrations [El at constant temperature. Figure 2A shows the variation of the conductivity as a (20) Grinvdd, A.; Steinberg, 1. 2.Anal. Biochem. 1974, 59, 583. (21) Lagues, M.; Ober, R.; Taupin, C. J. Phys. Lett. 1978, 39, L-487. (22) Eicke, H. F.; Hilfiker, R.; Holz, M. Helo. Chim. Acta 1984,67,361. (23) Van Dijk, M. A. Phys. Reo. Lett. 1985, 55, 1003. (24) Bhattacharaya,S.; Stokes, J. P.; Kim, M. W.; Huang, J. S. Phys. Reo. Lett. 1984,55, 1884. (25) Safran, S. A.; Webman, I.; Crest, G. S. Phys. Rev. A 1986,32,506.
60
65
45 0.0
0.5
1 .o
[o-chymotrypsin].104 M
WO
WO
used for excitation (450 nm) and a cutoff filter (600 nm) for emission. All samples were deoxygenated by the freeze-pumpthaw method. The decay profiles were recorded in 500 channels at 5 ns/channel and were analyzed by least-squaresfib using the distribution of the residuals and the autocorrelationfunction of the residualsas fitting criterion." The lumophorewas Ru(bpy)s2+. Ita concentration was maintained at 1od M . All measurements were performed in thermostated cella at 35 O C . The decay time of free Ru(bpy)Sz+ranged from around 530 ns. The quencher was Fe(CN)& at a constant concentration equal to 5 X lo-'M.
55
Figure 2. (A) Variation of the conductivity of AOT microemulsion systems containinga-chymotrypsinvs WO,for different enzyme concentrations: ( 0 )0.16X lo-' M;( 0 )0.32 X 104 M;(A) 0.38 X W M ;(A)0.63 X 104 M;(V)1.10X lo-'M. (B)Variation of the percolation threshold as a functionof enzyme concentration. The temperature was 35 O C .
- 1 I
5CO
I' -: - - I
A
0.0 WO
I
561
0.5
1 .o
[Trypsin].104 M
Figure 3. (A) Variation of the conductivity of AOT microemulsion systems containingtrypsin vs WO,for different enzyme concentrations: (v)0.16 X lo-' M;( 0 )0.39 X lo-' M;(A)0.79 X lo-' M ( 0 ) 1.10 X lo-' M. (B)Variation of the percolation threshold as a function of enzyme concentration. The temperature was 35 "C. function of WO,for different a-chymotrypsin concentrations. It is seen that a percolation is also observed for all enzyme concentrations. However, the percolation threshold is shifted toward higher w o values when the chymotrypsin concentration increases (Figure 2B). It is interesting to note the quite linear relationship between woand [El. On the other hand, the conductivity values decrease by increasing the enzyme concentration at constant WO. We have studied the effect of another enzyme, such as trypsin, on the conductivity of microemulsions. Figure 3 shows, for example, the variation of the conductivity of trypsin-containing AOT microemulsions as a function of WO,and the effect of the enzyme concentration on the appearance of the percolation. It is seen that the pattern is very similar to that in the case where chymotrypsin was used. Furthermore, when cationic microemulsions were studied, the addition of trypsin seemed to induce the same effect (Figure 4). It then seems that the presence of enzyme, either chymotrypsin or trypsin, in the microenvironment of the water core of the reverse micelles (anionic or cationic) affects the mobility of the various conducting species. This could be explained by a possible localization of the buffer ions on the vicinity of specific enzyme sites, limiting the exchange between the droplets, and leading to a conductivity decrease. Such an assumption is justified by the luminescence data presented below. Luminescence Quenching Data. In order to clarify the effect of the enzyme molecules on the reverse micelles, in light of the above observations, we have undertaken a
Papadimitriou et al.
914 Langmuir, Vol. 9, No.4, 1993 400
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luminescence quenching study. For this, AOT-based microemulaions with various chymotrypsinconcentrations were studied, before and after the appearanceof the electric percolation. The analysis of the luminescence decay profiles was done with a percolation model given by the following equation:18*z6.z7
+ c2t2')
I
800
600
I
lo00
1 .o
[Trypsin].1O4 M
Figure 4. (A) Variation of the conductivity of CTAB/butanol microemuleion systemscontaining trypsin vs the volume fraction , different enzyme concentratione: ( 0 )0.10 X lo-' of water (Gfor M (0)0.54 x l(r M, (A)1.10 x lo-' M. (B) Variation of the percolation threshold as a functionof enzyme concentration. The temperature was 35 OC.
I(t) = I,, exp(-kot) exp(-C,tf
400
Time
nr
Figure5. Luminescence decay profilesof AOT/ieooctane/water microemulsions, wo = 25,[AOT]= 0.096 M, in the absence of enzyme (lower curve) and in the presence of 1.2 X lo-' M of a-chymotrypsin(upper curve). Ru(bpy)s*+( 1 VM)was used aa the lumophore and 5 X lo-'M KsFe(CN)e as the quencher. The temperature was 35 OC.
Table 1. Values of 4 4, KL,and 9 . . for the System with wo= 25 Obtained by Analyzing Luminescence Decay Profiles of 10-6 M Ru(bpy)a*+in the Presence of 6 X lo-' M Fe(CN)& According to Eauation 1 at 36 OC
0 0.15 0.48 0.87 1.20
0.38 0.38 0.27 0.23 0.18
54 54 72 73
0 0 0 0 0
1.2 1.2 1.0 1.0 0.7
(1) This equation applies to infinitely short pulse excitation. 56 ko is the decay rate in the absence of quenching, C1 and CZare constants, and f is a noninteger exponent, 0 C f < progressively. The second column gives the values of the 1. The application of this model is made under the exponent f ,which is a measure of the restrictions imposed assumption that the dispersed phase, i.e., water droplets on the reaction.18 The reaction is more restricted, Le., with surrounding surfactant molecules, constitutes a more local, when f is smaller. Note that, in the presence percolation cluster either below or above the percolation of enzyme, the environment becomes progressively more threshold. Such an amumption is justified by computer restrictive. In other words, the diffusion of the reactants simulationsz8 and verified by e ~ p e r i m e n t . ' If ~ ~we~ ~ ~ ~is more difficult in the presence of enzyme. This phefurther assume that the percolation cluster is self-similar, nomenon, however, is detectable at an enzyme conceni.e., fractal, then f = d$2 = dfjd,, where d, is the spectral tration >1.45 X le5 M. The third column gives the values dimension, df the fractal dimension, and d, the fractal of the first-order reaction rate constant K1, Le., the dimension of the random walk. The reaction between the quenching rate immediately after the shining of the minority species, i.e., the excited Ru(bpy)a2+, and the excitation pulse. Apparently, K1 is larger when the majority species, Le., the quencher Fe(CN)8, occurs by simultaneous presence of an excited lumophore and a diffusion within the percolation cluster. In fact, for the quencher is more probable. Thus, for a given quencher derivation of eq 1 diffusion is represented by a random concentration higher K1 values would mean smaller walk. The analysis of the data with eq 1 allows the micellar concentration. We then interpret the values of calculation of C1, CZ,and f which are then used to obtain K1 in Table I to indicate that within experimental error the first-order reaction rate through the following the micellar concentration does not change or, at the limit, equation:18 it slightly decreases in the presence of enzyme. The fourth column gives the reaction probability at long times, Le., K(t) = felt'-' - 2tC2t2f-ls-1 (2) by diffusion through long distances. It is always zero, which is expected, for (electrically) nonpercolating miThe reaction rate K(t) is time dependent, as expected for celles. Finally the last column gives an average estimate all reactions in restricted geometries. In order to present of the reaction rate. Clearly, the latter decreases in the its values, we have chosen to tabulate K1 (for channel no. presence of enzyme. Apparently, quenching is lees efficient l),KL(for the last channel), and Kay(Le., the average over in the presence of enzyme. The decreased quenching 500 channels of time, 5 ns/channel). efficiency in the presence of 1.2 X lV M enzyme is also Table I shows the data obtained with the use of eqs 1 seen in Figure 5, which shows the luminescence decay and 2 for wo = 25, which corresponds to a micellar system profiles in the absence and in the presence of enzyme. If below the electric percolation threshold. The surfactant we assume anonrestricted distribution of reactants among concentration (0.096 M)and the temperature (35 "C) were micelles, e.g., Poisson statistics, the picture of Figure 5 is kept constant while the added enzyme concentration varied interpreted to indicate that the number of micelles (26)Liana, P. J. Chem. Phys. 1988,89, 6237. increases in the presence of enzyme. Thus, the reactants (27) Duportail, G.; Liana, P. Chem. Phys. Lett. 1990, 165, 35. seem then to be separated by being compartmentalized to (28) Argyrakie, P.; Duportail, G.; Limos, P. J. Chem. Phys. 1991,95, a larger number of micelles, giving less quenching. How3808. (29) Liana, P.; Modes, S. J . Chem. Phys. 1987,91, 6088. ever, an increase in the number of micelles would clearly (30) Liancs, P. h o g . Colloid Polym. Sci. 1988, 76, 140. K1,which is not the case. The number of micelles decrease (31) Modes, S.; Liana, P.; Xenakie, A. J. Phys. Chem. 1990,94,3364. sometimes also affects the values of electric conductivity. (32) Liana, P.;Malliaris, A. Isr. J . Chem. 1991, 31, 177. Even though these two factors might not be strongly (33) Liana, P.; Duportail, G . h o g . Colloid Polym. Sci. 1991,84,151.
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Electric Percolation of Microemulsions
related,the decreaseof electricconductivityin the presence of enzyme certainly is not justified by an increase in the number of micelles. We then conclude that at wo= 25 the presence of enzyme does not substantially affect the micellar concentration. Invariance in the number of micelles implies also invariance in the micellar size. Such a conclusion is in accordance with the recently presented comprehensive model of chymotrypsin solubilization in AOT reverse micelles published by Rahamanand Hatton.% At pH 9.0 a-chymotrypsin is above its isoelectric point; i.e., it is negatively charged. Therefore, it does not favor micellar size shrinking because of repelling like charges. However, the micellar interior is affected by the presence of enzyme. The geometry of the quenching reaction becomes more restrictive, and the quenching decreases. It is as if the enzyme creates within the micelle separate and hardly communicating pockets (compartments). In fact, what probably happens is that the mobility of the reactants is reduced in the presence of enzyme. Such an assumption is then in agreement with the above-presentedconductivity values. We have also tried to analyze the luminescence decay profiles of R~(bpy)3~+ in the presence of Fe(CN)e” in AOT reverse micelles above the electric percolation threshold, i.e., with w o= 55. However, in that case the water content is so high that the micellar interior behaves as if it were (34) Rahaman, R. S.; Hatton, T. A. J. Phys. Chem. 1991,95, 1799.
a continuousphase. Then the luminescencedecay profides are almost single exponentials, and they cannot offer any unambiguous structural information. Nevertheless, the quenching efficiency decreases in the presence of enzyme. Therefore, we believe that the conclusions obtained for wo = 25 can be extrapolated to other water contents. In conclusion, we have detected that the presence of enzyme in water in oil microemulsions affects the electric percolation threshold and causes a progressive decrease of the electric conductivity. Our results indicate that the electric charge carriers may be immobilized within pockets (subcompartments) formed with the help of the cosolubilized macromolecule. Similar results have also been detected with other macromolecules,such as poly(ethy1ene glycol)18solubilized in cyclohexane/SDS/pentanolwater in oil microemulsions. We must finally point out that have reported that the presence of Pileni et certain proteins in reverse micelles induces a bridging between water pools, resulting in an increase of the electric percolation threshold. Our results do not discard this possibility. The two mechanisms should be given simultaneous consideration. al.12J3936936
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(35) Brochette, P.; Petit, C.; Pileni, M. P. J. Phys. Chem. 1988, 92, 3505. (36) Pileni, M. P.;Huruguen,J. P.;Petit, C. In Thestructure,dynamics and equilibriumproperties of colloidalsystems; NATO AS1 Series C324; Bloor, D. M., Wyn-Jones, E., Eds.;Plenum: New York, 1990; p 355.
