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AOT microemulsion structure depending on both apolar solvent and protein concentration
Studies in Surface Science and Catalysis 132 Y. Iwasawa, N. Oyama and H. Kunieda (Editors) c 2001 Elsevier Science B.V. All rights reserved.
165
AOT microemulsion structure depending on both apolar solvent and protein concentration Rika Kawai-Hirai,t Mitsuhiro Hirai,^* Hisae Futatsugi,^ Hiroki Iwase,§ and Tomohiro Hayakawa.S tMeiwa Gakuen Junior College, Maebashi, Gunma 371-0034, Japan. ^Department of Physics, Gunma University, Maebashi 371-8510, Japan. By using synchrotron radiation small-angle X-ray scattering (SR-SAXS) method we studied w/o AOT (sodium bis(2-ethylhexyl)sulfosuccinate) microemulsion systems (water/AOT/n-hexane, /i-heptane, and n-octane) entrapping proteins. We foimd that the structure of the w/o AOT microemulsion clearly depends on the protein concentration and the length of hydrocarbon chain of apolar solvent. 1, INTRODUCTION Enhancement of catalytic activity, so-called super-activity, of enz3nnes entrapped in w/o microemulsions has attracted significant interest concerning not only with future practical applications such as microreactors but also with biophysical catal3rtic mechanisms of enzymes at a limiting condition such as a water pool of w/o microemulsion [1, 2]. By using SR-SAXS and enzymatic activity measurements we clarified that the catalytic activity of a-chymotrypsin entrapped in the water/AOT /isooctane microemulsion is enhanced at low WQ (= [H201/[A0T]) range of 8-16 and that the three different phases (oligomeric phase, transient phase and monomeric phase) appear successively with increasing WQ value [3]. Our neutron spin echo (NSE) study of water/AOT/heptane system showed that the effective diffusion coefficient relating to the bending fluctuation of the microemulsion is significantly enhanced at the transient phase [4]. As shown in other SR-SAXS study [5], there exists the penetration limit of apolar solvent depending on the linear hydrocarbon chain length, which results in the shift of the WQ value of the above phase boundaries. These previous studies suggest that the presence of the transient phase and the enhancement of the bending fluctuation of the microemulsion would induce the increase of an effective surface area of enzymes for the contact with substrates, which would result in the acceleration of the metabolic turnover. Then, we have carried out further SR-SAXS experiments to examine more precisely the structural features of the various AOT microemulsions entrapping enzymes. Corresponding author.
166
2. EXPERIMENTAL AOT was purchased from Nacalai Tesque Inc. Apolar solvents used were 96.5+ % n-hexane, 99.9+ % n-heptane and 97+ % n-octane, which were purchasedfromWako Pure Chemical Industries Ltd. The enzyme used was a-chymotrypsin from bovine pancreas, type 11 produced by Sigma Chemical Co. Water was purified by a Millipore system. The AOT microemulsions were prepared by using an ii^jection method. The wo values of the samples were varied from 0 to 50. The AOT molar concentrations were 0.1 M for all samples. a-Chjrmotrypsin was solubilized in 10 mM Hepes buffer adjusted at pH 8.0. The molar concentrations of o-chymotrypsin in the samples were varied from 2.4x10*5 M to 2.8x10"^ M depending on WQ. S R - S A X S experiments were carried out by using a small-angle X-ray scattering equipment installed at the synchrotron radiation source (PF) at the High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. The measurement condition was the same as the previous experiments [5]. We carried out the following standard analyses for the obtained scattering data as follows. To estimate structural changes of the AOT microemulsion depending on both a-chymotrypsin concentration and apolar solvent, we calculated the distance distribution functions p(r) by the following Fourier transform of the scattering intensity Kq). p(r) = ^-2-J''^/(^)sin(r<7)^
(1)
0
where q is the magnitude of the scattering vector defined as q- (4;r/A)sin(^/2), 6 and Xy are the scattering angle and the X-ray wavelength. The radius of gyration -Rg of the solute particle was obtained from the p(r) function as follows.
R^^kjll
(2)
3. RESULTS AND DISCUSSION Fig. 1 shows the wo dependence of the scattering curves Kq) of the AOT microemulsions with three different apolar solvents (n-hexane, n-heptane and /ioctane) imder the constant protein concentration of 2.4xl0"5 M. The decrease of the scattering intensity below q = 0.02 A'^ is attributed to the beam stopper. With increasing the wo value, the scattering curve shifts to a small-^ region with the rise of the scattering intensity below q = -0.04 A*^ for all cases of the three different AOT microemulsion systems, suggesting the enlargements of the microemulsion structure. The p(r) functions in Fig. 2 using Eq. (1) show this process more clearly. Namely, the position of the maximimi value p(r)niax of the p(jr) function shifts to a long distance side with the increase of the maximum diameter Dmax estimatedfromp(r) = 0 for r
167
0.06
(a) I 0.04
|10* -6
IAS
1, gio*
^N, > ?"
(a)1 1
0
>\-|
0.06
'^S^^v^
1—i_i
11J
rk-
CL 0 . 0 2
n-hex
~" ^
10^
n-hexane 1
r—r-^
rT)
it
1
1
'
•
(b)
j
= 0.04 n-heptane
(0
rr
'S 0.02
0 0.06
0
Fig. 1. wo dependence of the scattering curves of w/o AOT microemulsions (water/AOT//i-hexane, (a); n-heptane. (b); n-octane, (c)) occluding achymotiypsin (2.4x10-5 M).
50
100 150 r(A)
200
^^' 2. Distance distribution functions p(r) of the scattering curves of AOT microemulsions shown ^" ^ - 1./i-hexane, (a);/i-heptane, (b); n-octane, ^«>- V«"°^^ ^^"^« ^^^ ^ ^" ^^- ^•
>^max. Whereupon, the change of the p(r) occurs in the different manner for each system, especially at low water contents. The tailing of the p{r) function to a long distance direction can be seen at wo = 4 for the n-hexane system, at wo = 4 - 8 for the n-heptane system, and wo = 4 - 12 for the n-octane system, respectively. As explained previously [3], the tailing of the p(r) function suggests the presence of a certain amount of oUgomeric AOT microemulsion particles at tiie low water contents. Thus, Pig. 2 shows that the lengthening of the hydrocarbon chain of the apolar solvent extends the transient region from the oligomeric phase to the monomeric phase. Figs. 3(a) and
168
10 20 30 40 [HgOMAOT] (M/M)
50
Pig. 3. wo dependence of the J7g (a) and p{r)max (Jb) of w/o AOT microemulsions occluding achymotiypsin (2.4xl0-^ M).
0.1
0.2 [El (mM)
Fig. 4. Protein concentration dependence ofp(r) function (a) andp(r)inax (b). In (a), water/AOT/nhexane at wo = 20.
3flb) show the WQ dependence of the Rg andp(r)max, respectively. The relation between the WQ andp(r)niax values shows a good linearity for all systems. Whereas, with the lengthening of the hydrocarbon chain the wo vs. Rg relation becomes to deviate from a simple linearity and to separate into three different regions with different slopes. The slope above wo = 16 becomes smaller with the lengthening of the hydrocarbon chain. These results depending on the hydrocarbon chain length are essentially the same as those observed in the w/o AOT microemvdsion systems without proteins [5]. In Fig. 4 the protein concentration dependence of the pir) function and p(r)max shows that the occlusion of the proteins tends to decrease the microemulsion radius, which is more clearly seen with increasing water content or with shortening the hydrocarbon chain length. This would result from the attractive electrostatic interaction between the polar head of AOT and the basic residues of the protein surface.
REFERENCES 1. R. Hilhorst, In Structure and Reactivity in Reversed Micelles; Pileni, M. P. (ed.), Elsevier, Amsterdam, (1989) 323. 2. R. H. Pain (ed.). Mechanisms of Protein Folding, IRL Press, New York, 1994. 3. M. Hirai, et al., J. Chem. Soc. Faraday Trans., 91 (1995) 1081; J. Phys. Chem., 99 (1995) 6652. 4. M. Hirai, et al., J. Phys. Chem. Solids, 60 (1999) 1297. 5. M. Hirai, et al., J. Phys. Chem. B, 103 (1999) 9658.
165
AOT microemulsion structure depending on both apolar solvent and protein concentration Rika Kawai-Hirai,t Mitsuhiro Hirai,^* Hisae Futatsugi,^ Hiroki Iwase,§ and Tomohiro Hayakawa.S tMeiwa Gakuen Junior College, Maebashi, Gunma 371-0034, Japan. ^Department of Physics, Gunma University, Maebashi 371-8510, Japan. By using synchrotron radiation small-angle X-ray scattering (SR-SAXS) method we studied w/o AOT (sodium bis(2-ethylhexyl)sulfosuccinate) microemulsion systems (water/AOT/n-hexane, /i-heptane, and n-octane) entrapping proteins. We foimd that the structure of the w/o AOT microemulsion clearly depends on the protein concentration and the length of hydrocarbon chain of apolar solvent. 1, INTRODUCTION Enhancement of catalytic activity, so-called super-activity, of enz3nnes entrapped in w/o microemulsions has attracted significant interest concerning not only with future practical applications such as microreactors but also with biophysical catal3rtic mechanisms of enzymes at a limiting condition such as a water pool of w/o microemulsion [1, 2]. By using SR-SAXS and enzymatic activity measurements we clarified that the catalytic activity of a-chymotrypsin entrapped in the water/AOT /isooctane microemulsion is enhanced at low WQ (= [H201/[A0T]) range of 8-16 and that the three different phases (oligomeric phase, transient phase and monomeric phase) appear successively with increasing WQ value [3]. Our neutron spin echo (NSE) study of water/AOT/heptane system showed that the effective diffusion coefficient relating to the bending fluctuation of the microemulsion is significantly enhanced at the transient phase [4]. As shown in other SR-SAXS study [5], there exists the penetration limit of apolar solvent depending on the linear hydrocarbon chain length, which results in the shift of the WQ value of the above phase boundaries. These previous studies suggest that the presence of the transient phase and the enhancement of the bending fluctuation of the microemulsion would induce the increase of an effective surface area of enzymes for the contact with substrates, which would result in the acceleration of the metabolic turnover. Then, we have carried out further SR-SAXS experiments to examine more precisely the structural features of the various AOT microemulsions entrapping enzymes. Corresponding author.
166
2. EXPERIMENTAL AOT was purchased from Nacalai Tesque Inc. Apolar solvents used were 96.5+ % n-hexane, 99.9+ % n-heptane and 97+ % n-octane, which were purchasedfromWako Pure Chemical Industries Ltd. The enzyme used was a-chymotrypsin from bovine pancreas, type 11 produced by Sigma Chemical Co. Water was purified by a Millipore system. The AOT microemulsions were prepared by using an ii^jection method. The wo values of the samples were varied from 0 to 50. The AOT molar concentrations were 0.1 M for all samples. a-Chjrmotrypsin was solubilized in 10 mM Hepes buffer adjusted at pH 8.0. The molar concentrations of o-chymotrypsin in the samples were varied from 2.4x10*5 M to 2.8x10"^ M depending on WQ. S R - S A X S experiments were carried out by using a small-angle X-ray scattering equipment installed at the synchrotron radiation source (PF) at the High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. The measurement condition was the same as the previous experiments [5]. We carried out the following standard analyses for the obtained scattering data as follows. To estimate structural changes of the AOT microemulsion depending on both a-chymotrypsin concentration and apolar solvent, we calculated the distance distribution functions p(r) by the following Fourier transform of the scattering intensity Kq). p(r) = ^-2-J''^/(^)sin(r<7)^
(1)
0
where q is the magnitude of the scattering vector defined as q- (4;r/A)sin(^/2), 6 and Xy are the scattering angle and the X-ray wavelength. The radius of gyration -Rg of the solute particle was obtained from the p(r) function as follows.
R^^kjll
(2)
3. RESULTS AND DISCUSSION Fig. 1 shows the wo dependence of the scattering curves Kq) of the AOT microemulsions with three different apolar solvents (n-hexane, n-heptane and /ioctane) imder the constant protein concentration of 2.4xl0"5 M. The decrease of the scattering intensity below q = 0.02 A'^ is attributed to the beam stopper. With increasing the wo value, the scattering curve shifts to a small-^ region with the rise of the scattering intensity below q = -0.04 A*^ for all cases of the three different AOT microemulsion systems, suggesting the enlargements of the microemulsion structure. The p(r) functions in Fig. 2 using Eq. (1) show this process more clearly. Namely, the position of the maximimi value p(r)niax of the p(jr) function shifts to a long distance side with the increase of the maximum diameter Dmax estimatedfromp(r) = 0 for r
167
0.06
(a) I 0.04
|10* -6
IAS
1, gio*
^N, > ?"
(a)1 1
0
>\-|
0.06
'^S^^v^
1—i_i
11J
rk-
CL 0 . 0 2
n-hex
~" ^
10^
n-hexane 1
r—r-^
rT)
it
1
1
'
•
(b)
j
= 0.04 n-heptane
(0
rr
'S 0.02
0 0.06
0
Fig. 1. wo dependence of the scattering curves of w/o AOT microemulsions (water/AOT//i-hexane, (a); n-heptane. (b); n-octane, (c)) occluding achymotiypsin (2.4x10-5 M).
50
100 150 r(A)
200
^^' 2. Distance distribution functions p(r) of the scattering curves of AOT microemulsions shown ^" ^ - 1./i-hexane, (a);/i-heptane, (b); n-octane, ^«>- V«"°^^ ^^"^« ^^^ ^ ^" ^^- ^•
>^max. Whereupon, the change of the p(r) occurs in the different manner for each system, especially at low water contents. The tailing of the p{r) function to a long distance direction can be seen at wo = 4 for the n-hexane system, at wo = 4 - 8 for the n-heptane system, and wo = 4 - 12 for the n-octane system, respectively. As explained previously [3], the tailing of the p(r) function suggests the presence of a certain amount of oUgomeric AOT microemulsion particles at tiie low water contents. Thus, Pig. 2 shows that the lengthening of the hydrocarbon chain of the apolar solvent extends the transient region from the oligomeric phase to the monomeric phase. Figs. 3(a) and
168
10 20 30 40 [HgOMAOT] (M/M)
50
Pig. 3. wo dependence of the J7g (a) and p{r)max (Jb) of w/o AOT microemulsions occluding achymotiypsin (2.4xl0-^ M).
0.1
0.2 [El (mM)
Fig. 4. Protein concentration dependence ofp(r) function (a) andp(r)inax (b). In (a), water/AOT/nhexane at wo = 20.
3flb) show the WQ dependence of the Rg andp(r)max, respectively. The relation between the WQ andp(r)niax values shows a good linearity for all systems. Whereas, with the lengthening of the hydrocarbon chain the wo vs. Rg relation becomes to deviate from a simple linearity and to separate into three different regions with different slopes. The slope above wo = 16 becomes smaller with the lengthening of the hydrocarbon chain. These results depending on the hydrocarbon chain length are essentially the same as those observed in the w/o AOT microemvdsion systems without proteins [5]. In Fig. 4 the protein concentration dependence of the pir) function and p(r)max shows that the occlusion of the proteins tends to decrease the microemulsion radius, which is more clearly seen with increasing water content or with shortening the hydrocarbon chain length. This would result from the attractive electrostatic interaction between the polar head of AOT and the basic residues of the protein surface.
REFERENCES 1. R. Hilhorst, In Structure and Reactivity in Reversed Micelles; Pileni, M. P. (ed.), Elsevier, Amsterdam, (1989) 323. 2. R. H. Pain (ed.). Mechanisms of Protein Folding, IRL Press, New York, 1994. 3. M. Hirai, et al., J. Chem. Soc. Faraday Trans., 91 (1995) 1081; J. Phys. Chem., 99 (1995) 6652. 4. M. Hirai, et al., J. Phys. Chem. Solids, 60 (1999) 1297. 5. M. Hirai, et al., J. Phys. Chem. B, 103 (1999) 9658.