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Protein aggregation in the system “Aerosol-OT-water-octane” and its regulation by pressure application
Trends in High Pressure Bioscience and Biotechnology R. Hayashi (editor) @ 2002 Elsevier Science B.V. All rights reserved.
171
Protein aggregation in the .system "AerosoI-OT-water-octane" regulation by pressure application
and its
N.L.Klyachko 1, S.V.Shipovskov 1, F.Meersman 2, K.Heremans 2 ~Department of Chemical Enzymology, Faculty of Chemistry, University, 119899 Moscow, Russia*
Moscow State
2Department of Chemistry, Katholieke Universiteit Leuven, Belgium
Changes in secondary structure of o~-chymotrypsin were studied in the AOTwater-octane system by Fourier Transform InfraRed spectroscopy in conjunction with pressure. Phase transition in the system, protein structure changes and octane crystallization were observed at different pressure ranges. It was demonstrated that pressure-induced changes in protein structure (formation of partially unfolded states) were responsible for its aggregation when pressure released. This aggregation was accompanied by appearance of special bands (1622 and 1685 cm-1) in the amide I region of the infrared spectra which usually observed upon temperature denaturation. The possibility to regulate the conditions of the formation of protein aggregates is discussed.
1. INTRODUCTION
Protein aggregation plays an important role both in positive and negative ways. For food technology, gel formation of proteins can improve the texture and stability of food [1]. In protein engineering, protein aggregation is one of the factors that can accompany the process of protein folding producing inclusion bodies [2,3]. In pharmacy, protein aggregation is one of the problems in long-term storage or delivery of protein drugs [4,5]. And the most intriguing phenomena involving protein aggregation are a variety of, so called, molecular or conformational diseases, such as amyloidoses, prion encephalopathies, Alzheimer's dementia, etc. (for reviews see [3,6,7]. To follow the structural changes in the protein molecule it is convenient to use Fourier Transform InfraRed (FTIR) spectroscopy elucidating the secondary structure by the analysis of the shape of the amide I band[8]. It was found for several proteins that protein inactivation associated with protein aggregation caused by temperature or temperature with pressure led to appearance of specific bands at 1615-1618 and 1685 cm -1 assigned to an intermolecular antiparallel 13-sheet structure stabilized by hydrogen bonding (see [9] and refs.). It would be interesting to find out the conditions * Fhe work was supported in part by COST Chemistr~ Program. Projects DI0'0003,'98 and D10/0004"98
172
where the appearance of such aggregates can be regulated and studied in more detail. Ternary systems "Surfactant-water-organic solvent" was found to be convenient to study the behavior of proteins and enzymes at ambient and elevated pressures and temperatures (for reviews see, for example, [10,11,12]). Such systems can be easily prepared, they formed spontaneously being an optically transparent (pseudohomogeneous) allowing thus, to use different techniques. For reverse micelles, for example, it was found that the parameters of the system and the size of an inner polar cavity can be varied in wide range by changing water content, and such nanocontainers of a protein size can solubilize biomolecules and other substances of different nature. Depending on the conditions, the situation can be realized when only one protein molecule can be introduced into one micelle. If the size of a micelle is big enough to entrap two or more protein molecules, this can also be realized. The aim of this work is to study the possibility of obtaining the special protein aggregates mentioned above in the AOT-water-octane system under pressure application.
2. MATERIALS AND METHODS
N-octane, o~-chymotrypsin were purchased from Sigma (USA), Bis(2ethylhexyl)sodium-sulfosuccinate (AOT) was from Fluka (Switzerland), D20 was from Cambridge Isotope Laboratories, Inc. (USA). The system AOT-water-octane was obtained by dissolving AOT in octane (0.1-1 M) followed by addition of an enzyme solution and/or D20 to obtain a certain molar ratio of water and surfactant, w0 which was varied from 5 to 40. o~-Chymotrypsin was dissolved in 10 mM deutereted Tris-HCI (pD=7.6) using a protein concentration of 100-200 mg/ml. The solution was centrifuged for a few minutes and stored overnight to allow the H - D exchange of the amide group protons. High pressure was generated in a diamond anvil cell (DAC) (DIACELL products, UK) where the pressure was built up by means of a screw mechanism. For the pressure experiments, the solution was mounted in a stainless steel gasket (0.05 mm thickness) of DAC, and the optical pathlength was 0.05 mm. Barium sulfate was used as an internal pressure standard in all cases [13]. The infrared spectra were recorded by a Bruker IFS66 FTIR spectrometer equipped with a liquid nitrogen cooled broad band MCT solid state detector. 250 interferograms were co-added after registration at a resolution of 2 cm -1. The temperature kept constant at 25~
3. RESULTS AND DISCUSSION
Before to analyze protein-containing AOT-water-octane system we studied phase behavior of the components of the system at different pressures. First of all, the crystallization of pure octane occurred when pressure reached 8 kbar. This phenomenon was observed by FTIR (Fig.l) and visualized also by using light microscope (Fig.2). Fig. 1 shows the changes in the band position of the infrared spectra responsible for the CH2-, CH3-group vibrations as a dependence of applied
173
pressures. As seen, the significant changes occur when pressure is increased up to about 8 kbar. As seen from Fig.2, these changes can be attributed to the appearance of a new phase in the octane solution under high pressure. In our next step, we studied how pressure can affect infrared spectra of the AOTwater-octane system. The band position of the ester bond vibrations in the AOT molecule versus pressure is given in Fig.3. As seen, the sharp shift of this band occurred at pressures about 2 kbar. This experiment was carried out at 1 M AOT and w0=10. The same behavior of the system was found at lower surfactant concentrations down to 0.7 M. There were no changes in the system at 0.6 M AOT and down. The phenomenon observed can be attributed to the phase transition occurred in the system. Indeed, the phase transition in AOT (1 M)-water-octane system at very moderate pressures have been recently observed by using smallangle neutron scattering (SANS) [14].
T-,
1386
o z
1380
oul
1374
w
u.
"
1368
x
9~ 1 3 6 2
0
5
10
PRESSURE, kbar
Fig. 1. Pressure dependence of symmetrical CH3-vibrations band in pure octane.
a
b
Fig.2. Structural changes in pure octane seen in light microscope a - 1 bar, b 10 kbar.
174
E
1735 z
"'
1734
-
"
1733
*
•
1732
0
w ..I
.
.
0
o~176 .
.
.
.
.
# !
I
5
10
. . . .
PRESSURE, kbar
Fig.3. Pressure dependence of AOT ester bond vibrations band in the system AOTwater-octane, w0=10, 1 M AOT.
The o~-chymotrypsin structural changes under pressure in the AOT-water-octane system was studied by recording infrared spectra in the range 1600-1700 cm -1 characterizing the protein amide bond vibrations (amide I region). Fig.4 shows the changes in the peak area of amide I band of c~-chymotrypsin in 0.6 M AOT in octane at w0=40 upon pressure is increasing. At ambient pressure, the strongest maximum in oc-chymotrypsin spectrum appears at 1638 cm 1 that represents ~-structures in accordance with [15]. As seen, an essential shift of the maximum is observed in the pressure range 6-8 kbar. The spectral changes were found to be irreversible; histeresis was observed in all cases when pressure was going down as shown in Fig.4 (shaded squires). The shift to higher frequency 1650 cm ~ can be assigned to the structural changes in the protein molecule, probably the appearance of more disordered fragments [8], indicating that protein unfolds (at least partially) under pressure. Such unfolding is possible at high hydration degrees where the size of the inner cavity of a micelle is much larger than that of a protein entrapped. This was realized in our case; we have chosen hydration degree (w0) 25 and 40. At such hydration degrees we could expect the formation of the protein aggregates because the size of the micelle is big enough to include two and more o~-chymotrypsin molecules [10]. The infrared spectra of ~-chymotrypsin in the AOT-water-octane system at w0 25 and different pressures presented in Fig.5. As seen, new bands at 1622 and 1685 cm -1 appeared in the spectrum when pressure released. Appearance of these bands is characteristic of intermolecular antiparallel 13-structures stabilized by hydrogen bonding which were attributed to the aggregation of unfolded or partially unfolded protein [9,16]. These aggregation specific bands were observed in the case of temperature unfolded proteins [9,16], not observed in the pressure case (pressure caused this phenomenon only with temperature pretreated samples [9]). These results would indicate the process of protein aggregation occurred with pressure treated protein in the surfactant-water-organic solvent system. However, it was shown that experimental conditions can be found where no aggregation observed after pressure released. Thus, the system AOT-water-octane can be a promising one to study the process of protein aggregation at high pressures, to regulate the
175
parameters and conditions in order to favor or prevent such aggregation and understand the mechanism of the process in more detail.
,,-.
1648 -~ [
r Z
N
el#
#
9
9
Bdl
,
tu
1644
T
0
,
W
u. _J
1640
~-
X
J
~<
1 6 : 3 6 ---' , .... 0
10
5 PRESSURE,
kbar
Fig.4. Pressure dependence of maximal frequency in c~-chymotrypsin infrared spectrum. Black circles shows pressure increasing, shaded squires shows pressure decreasing.
1622 P, kbar t ' t tI t
f, tt 'k " "9
1685
: ,...,
t
,," ,.
\
"
,
v
\-., ~
/
.,,,-.-,.. \ ,i
,-"
% .,..,'/" /,'" .,,/"
..;.,,
t
""- /-"
,,. ~
,., kk
...,
0.001
-,
",..'\ ",,, --,..
. ,\,,.,..
_
2
-,, ",-,.... ,,..
6 4 0.001 1600 cm -1
,-,._
1700
1650
Fig.5. Infrared spectra of cz-chymotrypsin in the AOT-water-octane system at different pressures. Spectra presented in chronological order from bottom to top.
176
ACKNOWLEDGMENTS
Authors express their gratitude to Prof. A.V. Levashov for fruitful critics and helpful discussion. REFERENCES
1. 2. 3. 4.
M.G.Semenova, Curr.Opin. Colloid Interface Sci., 3 (1998) 627-632. A.Mitaki, J.King, Biotechnology (N.Y.), 7 (1989) 690-697. A.L.Fink, Folding and Design, 3 (1998) R9-R23. T.Arakawa, S.J.Prestrelski, W.C.Kenney, J.FoCarpenter, Adv. Drug Deliv. Rev., 10 (1993) 1-28. 5. H.R.Costantino, S.P.Schwendeman, R.Langer, A.M.Klibanov, Biokhimiya, 63 (1998) 22-30. 6. J.W.Kelly, Curr. Opin. Struct. Biol., 8 (1998) 101-106. 7. R.W.Carrell, B.Gooptu, Curr. Opin. Struct. Biol., 8 (1998) 799-809. 8. D.M.Byler, H.Susi, Biopolymers, 25 (1986) 469-487. 9. L.Smeller, P.Rubens, K.Heremans, Biochemistry, 36 (1999) 3816-3820. 10. K.Martinek, N.L.Klyachko, A.V.Kabanov, Yu.L.Khmelnitsky, A.V.Levashov, Biochim. Biophys. Acta, 981 (1989) 161-172. 11.P.L.Luisi, M.Giomini, M.P.Pileni, B.H.Robinson, Biochim. Biophys. Acta, 947 (1988) 209-246. 12.C.Balny, N.L.Klyachko. In: High Pressure Molecular Science (R.Winter, J.Jonas, eds.), Kluwer Acad. Publishers, Dordrecht, Boston, London. 1999. Pp. 423-436. 13. P.T.T.Wong, D.J.Moffat, Appl. Spectrosc., 43 (1989) 1279. 14.J.Woenckhaus, R.Kuhling, N.L.Klyachko, R.Winter, Manuscript in preparation. 15. P.T.T.Wong, K.Heremans, Biochim. Biophys. Acta, 956 (1988) 1-9. 16.A.A.Ismail, H.H.Mantsch, P.T.T.Wong, Biochim. Biophys. Acta, 1121 (1992) 183188.
171
Protein aggregation in the .system "AerosoI-OT-water-octane" regulation by pressure application
and its
N.L.Klyachko 1, S.V.Shipovskov 1, F.Meersman 2, K.Heremans 2 ~Department of Chemical Enzymology, Faculty of Chemistry, University, 119899 Moscow, Russia*
Moscow State
2Department of Chemistry, Katholieke Universiteit Leuven, Belgium
Changes in secondary structure of o~-chymotrypsin were studied in the AOTwater-octane system by Fourier Transform InfraRed spectroscopy in conjunction with pressure. Phase transition in the system, protein structure changes and octane crystallization were observed at different pressure ranges. It was demonstrated that pressure-induced changes in protein structure (formation of partially unfolded states) were responsible for its aggregation when pressure released. This aggregation was accompanied by appearance of special bands (1622 and 1685 cm-1) in the amide I region of the infrared spectra which usually observed upon temperature denaturation. The possibility to regulate the conditions of the formation of protein aggregates is discussed.
1. INTRODUCTION
Protein aggregation plays an important role both in positive and negative ways. For food technology, gel formation of proteins can improve the texture and stability of food [1]. In protein engineering, protein aggregation is one of the factors that can accompany the process of protein folding producing inclusion bodies [2,3]. In pharmacy, protein aggregation is one of the problems in long-term storage or delivery of protein drugs [4,5]. And the most intriguing phenomena involving protein aggregation are a variety of, so called, molecular or conformational diseases, such as amyloidoses, prion encephalopathies, Alzheimer's dementia, etc. (for reviews see [3,6,7]. To follow the structural changes in the protein molecule it is convenient to use Fourier Transform InfraRed (FTIR) spectroscopy elucidating the secondary structure by the analysis of the shape of the amide I band[8]. It was found for several proteins that protein inactivation associated with protein aggregation caused by temperature or temperature with pressure led to appearance of specific bands at 1615-1618 and 1685 cm -1 assigned to an intermolecular antiparallel 13-sheet structure stabilized by hydrogen bonding (see [9] and refs.). It would be interesting to find out the conditions * Fhe work was supported in part by COST Chemistr~ Program. Projects DI0'0003,'98 and D10/0004"98
172
where the appearance of such aggregates can be regulated and studied in more detail. Ternary systems "Surfactant-water-organic solvent" was found to be convenient to study the behavior of proteins and enzymes at ambient and elevated pressures and temperatures (for reviews see, for example, [10,11,12]). Such systems can be easily prepared, they formed spontaneously being an optically transparent (pseudohomogeneous) allowing thus, to use different techniques. For reverse micelles, for example, it was found that the parameters of the system and the size of an inner polar cavity can be varied in wide range by changing water content, and such nanocontainers of a protein size can solubilize biomolecules and other substances of different nature. Depending on the conditions, the situation can be realized when only one protein molecule can be introduced into one micelle. If the size of a micelle is big enough to entrap two or more protein molecules, this can also be realized. The aim of this work is to study the possibility of obtaining the special protein aggregates mentioned above in the AOT-water-octane system under pressure application.
2. MATERIALS AND METHODS
N-octane, o~-chymotrypsin were purchased from Sigma (USA), Bis(2ethylhexyl)sodium-sulfosuccinate (AOT) was from Fluka (Switzerland), D20 was from Cambridge Isotope Laboratories, Inc. (USA). The system AOT-water-octane was obtained by dissolving AOT in octane (0.1-1 M) followed by addition of an enzyme solution and/or D20 to obtain a certain molar ratio of water and surfactant, w0 which was varied from 5 to 40. o~-Chymotrypsin was dissolved in 10 mM deutereted Tris-HCI (pD=7.6) using a protein concentration of 100-200 mg/ml. The solution was centrifuged for a few minutes and stored overnight to allow the H - D exchange of the amide group protons. High pressure was generated in a diamond anvil cell (DAC) (DIACELL products, UK) where the pressure was built up by means of a screw mechanism. For the pressure experiments, the solution was mounted in a stainless steel gasket (0.05 mm thickness) of DAC, and the optical pathlength was 0.05 mm. Barium sulfate was used as an internal pressure standard in all cases [13]. The infrared spectra were recorded by a Bruker IFS66 FTIR spectrometer equipped with a liquid nitrogen cooled broad band MCT solid state detector. 250 interferograms were co-added after registration at a resolution of 2 cm -1. The temperature kept constant at 25~
3. RESULTS AND DISCUSSION
Before to analyze protein-containing AOT-water-octane system we studied phase behavior of the components of the system at different pressures. First of all, the crystallization of pure octane occurred when pressure reached 8 kbar. This phenomenon was observed by FTIR (Fig.l) and visualized also by using light microscope (Fig.2). Fig. 1 shows the changes in the band position of the infrared spectra responsible for the CH2-, CH3-group vibrations as a dependence of applied
173
pressures. As seen, the significant changes occur when pressure is increased up to about 8 kbar. As seen from Fig.2, these changes can be attributed to the appearance of a new phase in the octane solution under high pressure. In our next step, we studied how pressure can affect infrared spectra of the AOTwater-octane system. The band position of the ester bond vibrations in the AOT molecule versus pressure is given in Fig.3. As seen, the sharp shift of this band occurred at pressures about 2 kbar. This experiment was carried out at 1 M AOT and w0=10. The same behavior of the system was found at lower surfactant concentrations down to 0.7 M. There were no changes in the system at 0.6 M AOT and down. The phenomenon observed can be attributed to the phase transition occurred in the system. Indeed, the phase transition in AOT (1 M)-water-octane system at very moderate pressures have been recently observed by using smallangle neutron scattering (SANS) [14].
T-,
1386
o z
1380
oul
1374
w
u.
"
1368
x
9~ 1 3 6 2
0
5
10
PRESSURE, kbar
Fig. 1. Pressure dependence of symmetrical CH3-vibrations band in pure octane.
a
b
Fig.2. Structural changes in pure octane seen in light microscope a - 1 bar, b 10 kbar.
174
E
1735 z
"'
1734
-
"
1733
*
•
1732
0
w ..I
.
.
0
o~176 .
.
.
.
.
# !
I
5
10
. . . .
PRESSURE, kbar
Fig.3. Pressure dependence of AOT ester bond vibrations band in the system AOTwater-octane, w0=10, 1 M AOT.
The o~-chymotrypsin structural changes under pressure in the AOT-water-octane system was studied by recording infrared spectra in the range 1600-1700 cm -1 characterizing the protein amide bond vibrations (amide I region). Fig.4 shows the changes in the peak area of amide I band of c~-chymotrypsin in 0.6 M AOT in octane at w0=40 upon pressure is increasing. At ambient pressure, the strongest maximum in oc-chymotrypsin spectrum appears at 1638 cm 1 that represents ~-structures in accordance with [15]. As seen, an essential shift of the maximum is observed in the pressure range 6-8 kbar. The spectral changes were found to be irreversible; histeresis was observed in all cases when pressure was going down as shown in Fig.4 (shaded squires). The shift to higher frequency 1650 cm ~ can be assigned to the structural changes in the protein molecule, probably the appearance of more disordered fragments [8], indicating that protein unfolds (at least partially) under pressure. Such unfolding is possible at high hydration degrees where the size of the inner cavity of a micelle is much larger than that of a protein entrapped. This was realized in our case; we have chosen hydration degree (w0) 25 and 40. At such hydration degrees we could expect the formation of the protein aggregates because the size of the micelle is big enough to include two and more o~-chymotrypsin molecules [10]. The infrared spectra of ~-chymotrypsin in the AOT-water-octane system at w0 25 and different pressures presented in Fig.5. As seen, new bands at 1622 and 1685 cm -1 appeared in the spectrum when pressure released. Appearance of these bands is characteristic of intermolecular antiparallel 13-structures stabilized by hydrogen bonding which were attributed to the aggregation of unfolded or partially unfolded protein [9,16]. These aggregation specific bands were observed in the case of temperature unfolded proteins [9,16], not observed in the pressure case (pressure caused this phenomenon only with temperature pretreated samples [9]). These results would indicate the process of protein aggregation occurred with pressure treated protein in the surfactant-water-organic solvent system. However, it was shown that experimental conditions can be found where no aggregation observed after pressure released. Thus, the system AOT-water-octane can be a promising one to study the process of protein aggregation at high pressures, to regulate the
175
parameters and conditions in order to favor or prevent such aggregation and understand the mechanism of the process in more detail.
,,-.
1648 -~ [
r Z
N
el#
#
9
9
Bdl
,
tu
1644
T
0
,
W
u. _J
1640
~-
X
J
~<
1 6 : 3 6 ---' , .... 0
10
5 PRESSURE,
kbar
Fig.4. Pressure dependence of maximal frequency in c~-chymotrypsin infrared spectrum. Black circles shows pressure increasing, shaded squires shows pressure decreasing.
1622 P, kbar t ' t tI t
f, tt 'k " "9
1685
: ,...,
t
,," ,.
\
"
,
v
\-., ~
/
.,,,-.-,.. \ ,i
,-"
% .,..,'/" /,'" .,,/"
..;.,,
t
""- /-"
,,. ~
,., kk
...,
0.001
-,
",..'\ ",,, --,..
. ,\,,.,..
_
2
-,, ",-,.... ,,..
6 4 0.001 1600 cm -1
,-,._
1700
1650
Fig.5. Infrared spectra of cz-chymotrypsin in the AOT-water-octane system at different pressures. Spectra presented in chronological order from bottom to top.
176
ACKNOWLEDGMENTS
Authors express their gratitude to Prof. A.V. Levashov for fruitful critics and helpful discussion. REFERENCES
1. 2. 3. 4.
M.G.Semenova, Curr.Opin. Colloid Interface Sci., 3 (1998) 627-632. A.Mitaki, J.King, Biotechnology (N.Y.), 7 (1989) 690-697. A.L.Fink, Folding and Design, 3 (1998) R9-R23. T.Arakawa, S.J.Prestrelski, W.C.Kenney, J.FoCarpenter, Adv. Drug Deliv. Rev., 10 (1993) 1-28. 5. H.R.Costantino, S.P.Schwendeman, R.Langer, A.M.Klibanov, Biokhimiya, 63 (1998) 22-30. 6. J.W.Kelly, Curr. Opin. Struct. Biol., 8 (1998) 101-106. 7. R.W.Carrell, B.Gooptu, Curr. Opin. Struct. Biol., 8 (1998) 799-809. 8. D.M.Byler, H.Susi, Biopolymers, 25 (1986) 469-487. 9. L.Smeller, P.Rubens, K.Heremans, Biochemistry, 36 (1999) 3816-3820. 10. K.Martinek, N.L.Klyachko, A.V.Kabanov, Yu.L.Khmelnitsky, A.V.Levashov, Biochim. Biophys. Acta, 981 (1989) 161-172. 11.P.L.Luisi, M.Giomini, M.P.Pileni, B.H.Robinson, Biochim. Biophys. Acta, 947 (1988) 209-246. 12.C.Balny, N.L.Klyachko. In: High Pressure Molecular Science (R.Winter, J.Jonas, eds.), Kluwer Acad. Publishers, Dordrecht, Boston, London. 1999. Pp. 423-436. 13. P.T.T.Wong, D.J.Moffat, Appl. Spectrosc., 43 (1989) 1279. 14.J.Woenckhaus, R.Kuhling, N.L.Klyachko, R.Winter, Manuscript in preparation. 15. P.T.T.Wong, K.Heremans, Biochim. Biophys. Acta, 956 (1988) 1-9. 16.A.A.Ismail, H.H.Mantsch, P.T.T.Wong, Biochim. Biophys. Acta, 1121 (1992) 183188.