Vol. 186, No. 3, 1992 August 14, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1546-1552
SOLUBILIZATION OF RIBOSOMES IN REVERSE MICELLES Giordano Palazzo ° and Pier Luigi Luisi* °Dipartimento di Chimica, Universita" di Bari
* Institut fiir Polymere der ETH-Ziirich, Switzerland Received June i0, 1992
Summary: Pig liver ribosomes have been solubilized in reverse micelles constituted by bis (2-ethyl hexyl) sodium sulfosuccinate (AOT) in isooctane and 3.6% water, v:v. The micellar ribosomal solutions are transparent, show no significant scattering and permit direct spectroscopic observation of the ribosomes to be made. Ultraviolet absorption and circular dichroic spectra have been recorded and indicate that the ribosomes maintain in the micellar environment their structural integrity. Some possible applications of these micellar systems are discussed. ®1 9 9 2 A c a d e m i c P r e s s , I n c .
Enzymes solubilized in reverse micelles have attracted the attention of a number of investigators over the last ten years. Enzymes are thought to be hosted in the water pool of the reverse micelles, and protected from the apolar solvent by a layer of water and surfactant molecules. The very large number of papers dealing with enzymes and proteins in reverse micelles reflects on the one hand the biotechnological expectations, and on the other the fascination for this type of biochemistry with a regulable enzyme microreactor (for reviews see Ref 1-3). One would think that nucleic acids in reverse micelles would have enjoyed similar popularity. It is not so, and actually there are only very few papers dealing with this subject (4-6). The preliminary results were however interesting: for example, it could be shown that high molecular 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
weight DNA could be easily solubilized in AOT reverse micelles, giving rise to optical features which were reminiscent of the "superpackaging" of DNA (4). Also plasmids, up to a molecular weight of 3.106 Dalton, could be solubilized in the AOT/water/isooctane ternary system. Recently, we have taken up again this subject, and this communication is part of this research. The question here is whether, and to what extent, ribosomes can be solubilized in reverse micelles. In particular, one would like to know whether such large assemblies can be hosted in reverse micelles, and whether they remain stable. The outcome is not obvious, as actually one might expect that the micellar microenvironment would induce dissociation of the ribosome subunits and denaturation of the single components. Conversely, if ribosomes were to be solubilized and stabilized in reverse micelles, the question of their reactivity would emerge: for example, whether and to what extent protein biosynthesis could be accomplished in such new types of cell-free systems. The system utilized in this work is the classic AOT/isooctane/ water (AOT stands for bis( 2 ethyl hexyl) sodium sulfosuccinate), at a concentration of 100 mM AOT and a Wo 20-30 (Wo [= H20] /[AOT] ), at room temperature. This system has been used in a number of studies- see Ref. 1-3).
Materials and Methods Chemicals: Sodium bis (2-ethylhexyl) sulfosuccinate (AOT) and isooctane(2,2,4-trimethyl pentane) for UV spectroscopy were from Fluka (Switzerland). Water was twice disttillied. Ribosomes: The cytoplasmic ribosomes were prepared according to Greco et al. (9). Solubilization. Micellar solutions of ribosomes at Wo =20 were prepared by injection of 36 )11 of concentrated ribosomal stock solution into 1 ml of 100 mM AOT solution on isooctane. By this technique, solutions with different w o have all the same overall concentration of ribosomes. Spectroscopic measurements. Circular dichroic (CD) spectra were 1547
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
m e a s u r e d with a JASCO J 600 in a nitrogen atmosphere. All spectra are expressed in molar ellipticity per residue of RNA (deg. cm2/dmol). Absorption measurements were made with Perkin Elmer L a m b d a 9 instrumentation. The biopolymer concentration in water as well as in the micellar solution was calculated using an extinction coefficient of 5 . 3 . 1 0 s M -1 cm-t (8) at 260 nm. Results
Fig.1 shows the uv absorption spectra of a ribosomal preparation in water and in reverse micellar solution at Wo=20 for the same overall ribosomal concentration. Notice that both the position of the m a x i m u m at 260 n m and the form of the spectrum are practically the same in the two solutions. Also the scattering (see the absorption in the 320 n m region) is rather m o d e s t in both cases. The solution of Fig.1 contains 30 m g ribosomes per ml (overall concentration, i.e. referred to the total volume, or 0.83 m g / m l referred to the local concentration, i.e. to the only v o l u m e of the water microphase. The micellar solutions of the ribosomes are optically transparent and thermodynamically stable. There is no significant ribosome precipitation
4
2
[0]
x I o -~
( deg .cm2/mol ) 2~//
1 water 2 RM AOT/iC8/water,Wo=20 I 3 back t r a n s f e r KCI 0.5
-2
225
I 250
I 275
I 300
Fig.1. Ultraviolet absorption spectra of ribosomal preparation in water (curve 1) and in AOT/isooctane reverse micellar (curve 2) solutions, at Wo 20. Both solutions contain 0.03 mg ribosmes per ml (overall concentration). Curve 3 represents the optical density after re-extraction of the biomaterial from the same ribosomal preparation. 1548
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with time, but there is a slow decrease of the optical density of the solution, up to 2.5% in four hours ca. This can be mostly attributed to DNA absorption to the glass walls of the cells: this could be demonstrated by rinsing with fresh water solution the glass vials which had contained the micellar solution. This phenomenon does not significantly affect the spectroscopic measurements which are carried out within one-two hours, as all data presented here. However, a correction for the concetration is needed over longer time intervals. The ribosomes in micellar solution can be transferred back in water, simply by washing the micellar solution with an excess 0.5 M aqueous KC1 solution. The UV spectrum of the ribosomal preparation reextracted in water is shown by the curve 3 of Fig 1: the general features of the spectrum are the same as before-only the ratio of the absorbancies OD260/OD280 is somewhat higher than before, reflecting the loss of some protein component during the re-extraction process. Let us consider now the circular dichroic spectra, which in principle are more informative than UV spectra to the structural changes experienced by the biopolymer on going from the aqueous solution to the micellar solution and back again in water. In fact, CD techniques have been applied already in order to obatain structural informations on ribosomes (7). Spectra are shown in Fig.2. Note again that all spectra are very similar to each other, suggesting that the solubilization in micelles, as well as the process of re-extraction, do not alter the gross conformation of the RNA molecules. We have also studied the CD spectra of ribosomal preparation at Wo=30: no significant changes are observed between the two Wo values. 1549
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.4
OD 0.3
0.2
0.1
0
950
300
wl (nm) Fig.2 Circular dichroic spectra of the ribosomal preparations in water (curveA), in AOT/isooctane micellar solution at w o 20 (curve B) and after re-extraction (curveC). Same concentration as in Fig.1.
The interest on the s t u d y of the influence of Wo on the CD-properties stams from o u r earlier observation concerning the psi-spectra of D N A in reverse micelles. As already mentioned, w h e n high molecular weight D N A or large plasmids are solubilized in reverse micelles, large anomalies in the CD spectra are observed (4-5), which are diagnostic for the occurrence of supercondensed D N A structures. In principle, this could also occurr for RNA. This is h o w e v e r not the case with ribosomes, at least u n d e r our experimental conditions. In the case of DNA, the superpackaging is with all likehood d u e to an intermolecular aggregation of double stranded macromolecules. Ribosomes have a globular structure (also thanks to the accompanying proteins) and for this reason aggregation p h e n o m e n a are less likely. 1550
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Discussion It appears then possible to solubilize ribosomes in reverse micellar solutions. In view of the large dimensions of the pig's liver ribosomes, this is indeed surprising, considering that at w0= 20, we have only 3.6% water (v:v) in the system. This percentage can be further reduced if needed. Notice that at w o = 20 the AOT micelles should have a radius of 35 A ° ca (1), i.e. m u c h smaller than the radius of the pig liver ribosomes, which is a r o u n d 300 A ° ( as judged on the basis of electron microscopy of our o w n preparations. The solubilization of ribosomes in reverse AOT micelles therefore cannot be v i e w e d as a simple insertion of the b i o p o l y m e r in the micelle: rather, large redistribution of material should accompany the solubilization, probably with the formation of v e r y large ribosomecontaining micelles. This is so also inthe case of proteins, as judged from double dye ultracentrifugation (8). Most likely, then, the solubilization of the ribosomes in reverse micelles is due to the same kind of structure which is reponsible for the stabilization of hydrophylic proteins in reverse micelles, the so-called "water-shell" model. Accordingly, the biopolymer is s o r r o u n d e d b y a layer of water, and b y a layer of surfactant molecules, in the order, which are in contact with the organic solvent with their apolar tails. Based on the spectroscopic features of the ribosomes which are reextracted in water from a micellar solution, one can assume that the structural integrity of the ribosomes is not d a m a g e d u p o n solubilization in the micellar system. This is interesting in view of possible applications of the ribosomes. In particular, the ribosome-containing micelle can be seen as a novel microreactor for protein biosynthesis: the surfactant layer 1551
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
around the ribosomes has protecting and stabilizing functions, and at the same time, due to its dynamic properties, should permits the free exchange of substrates to and from the ribosomes. This notion indicates the direction for future work. References
1) P.L. Luisi, M. Giomini, M.P. Pileni, B.H. Robinson,(1988) Biochem. Biophys. Acta, 947, 209 2) K. Martinek, A.V. Levashov, N.L. Klyachko, V.I. Pant-in, I.V. Berezin, (1981) Biochem. Biophys. Acta, 657, 227 3) P.L. Luisi, L.J. Magid,(1986) CRC Crit. Rev. Biochem., 20, 409 4) V.E. Imre, P.L. Luisi(1982) Biochem. Biophys. Res. Comm., 107, 538 5) E. Battistel, E.V. Imre, P.L. Luisi, (1989) in Controlled Release of Drugs: Polymers and Aggregate Systems, edited by M. Rosoff, New York, NY VCH Publishers, 257-275 6) A.B. Hanley, E. Grimfeld, R.L. Baxter,(1990) Biocatalysis, 3,253 7) I.Jr. Tinoco, M.F. Maestre and C. Bustamante, (1980) Trends Biochem. Sci., 8,227 8) G.G. Zampieri, H.J/ickle, P.L.Luisi, (1986) J. Phys. Chem., 90~01849 9) M. Greco, P. Cantatore, G. Pepe, C. Saccone,(1973) Eur. J. Biochem. 37, 171 10) U. Wild, K. Ramm, H.L. Sanher, D. Riesner,(1980) Eur. J. Biochem., 103, 227
1552
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1546-1552
SOLUBILIZATION OF RIBOSOMES IN REVERSE MICELLES Giordano Palazzo ° and Pier Luigi Luisi* °Dipartimento di Chimica, Universita" di Bari
* Institut fiir Polymere der ETH-Ziirich, Switzerland Received June i0, 1992
Summary: Pig liver ribosomes have been solubilized in reverse micelles constituted by bis (2-ethyl hexyl) sodium sulfosuccinate (AOT) in isooctane and 3.6% water, v:v. The micellar ribosomal solutions are transparent, show no significant scattering and permit direct spectroscopic observation of the ribosomes to be made. Ultraviolet absorption and circular dichroic spectra have been recorded and indicate that the ribosomes maintain in the micellar environment their structural integrity. Some possible applications of these micellar systems are discussed. ®1 9 9 2 A c a d e m i c P r e s s , I n c .
Enzymes solubilized in reverse micelles have attracted the attention of a number of investigators over the last ten years. Enzymes are thought to be hosted in the water pool of the reverse micelles, and protected from the apolar solvent by a layer of water and surfactant molecules. The very large number of papers dealing with enzymes and proteins in reverse micelles reflects on the one hand the biotechnological expectations, and on the other the fascination for this type of biochemistry with a regulable enzyme microreactor (for reviews see Ref 1-3). One would think that nucleic acids in reverse micelles would have enjoyed similar popularity. It is not so, and actually there are only very few papers dealing with this subject (4-6). The preliminary results were however interesting: for example, it could be shown that high molecular 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1546
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
weight DNA could be easily solubilized in AOT reverse micelles, giving rise to optical features which were reminiscent of the "superpackaging" of DNA (4). Also plasmids, up to a molecular weight of 3.106 Dalton, could be solubilized in the AOT/water/isooctane ternary system. Recently, we have taken up again this subject, and this communication is part of this research. The question here is whether, and to what extent, ribosomes can be solubilized in reverse micelles. In particular, one would like to know whether such large assemblies can be hosted in reverse micelles, and whether they remain stable. The outcome is not obvious, as actually one might expect that the micellar microenvironment would induce dissociation of the ribosome subunits and denaturation of the single components. Conversely, if ribosomes were to be solubilized and stabilized in reverse micelles, the question of their reactivity would emerge: for example, whether and to what extent protein biosynthesis could be accomplished in such new types of cell-free systems. The system utilized in this work is the classic AOT/isooctane/ water (AOT stands for bis( 2 ethyl hexyl) sodium sulfosuccinate), at a concentration of 100 mM AOT and a Wo 20-30 (Wo [= H20] /[AOT] ), at room temperature. This system has been used in a number of studies- see Ref. 1-3).
Materials and Methods Chemicals: Sodium bis (2-ethylhexyl) sulfosuccinate (AOT) and isooctane(2,2,4-trimethyl pentane) for UV spectroscopy were from Fluka (Switzerland). Water was twice disttillied. Ribosomes: The cytoplasmic ribosomes were prepared according to Greco et al. (9). Solubilization. Micellar solutions of ribosomes at Wo =20 were prepared by injection of 36 )11 of concentrated ribosomal stock solution into 1 ml of 100 mM AOT solution on isooctane. By this technique, solutions with different w o have all the same overall concentration of ribosomes. Spectroscopic measurements. Circular dichroic (CD) spectra were 1547
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
m e a s u r e d with a JASCO J 600 in a nitrogen atmosphere. All spectra are expressed in molar ellipticity per residue of RNA (deg. cm2/dmol). Absorption measurements were made with Perkin Elmer L a m b d a 9 instrumentation. The biopolymer concentration in water as well as in the micellar solution was calculated using an extinction coefficient of 5 . 3 . 1 0 s M -1 cm-t (8) at 260 nm. Results
Fig.1 shows the uv absorption spectra of a ribosomal preparation in water and in reverse micellar solution at Wo=20 for the same overall ribosomal concentration. Notice that both the position of the m a x i m u m at 260 n m and the form of the spectrum are practically the same in the two solutions. Also the scattering (see the absorption in the 320 n m region) is rather m o d e s t in both cases. The solution of Fig.1 contains 30 m g ribosomes per ml (overall concentration, i.e. referred to the total volume, or 0.83 m g / m l referred to the local concentration, i.e. to the only v o l u m e of the water microphase. The micellar solutions of the ribosomes are optically transparent and thermodynamically stable. There is no significant ribosome precipitation
4
2
[0]
x I o -~
( deg .cm2/mol ) 2~//
1 water 2 RM AOT/iC8/water,Wo=20 I 3 back t r a n s f e r KCI 0.5
-2
225
I 250
I 275
I 300
Fig.1. Ultraviolet absorption spectra of ribosomal preparation in water (curve 1) and in AOT/isooctane reverse micellar (curve 2) solutions, at Wo 20. Both solutions contain 0.03 mg ribosmes per ml (overall concentration). Curve 3 represents the optical density after re-extraction of the biomaterial from the same ribosomal preparation. 1548
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with time, but there is a slow decrease of the optical density of the solution, up to 2.5% in four hours ca. This can be mostly attributed to DNA absorption to the glass walls of the cells: this could be demonstrated by rinsing with fresh water solution the glass vials which had contained the micellar solution. This phenomenon does not significantly affect the spectroscopic measurements which are carried out within one-two hours, as all data presented here. However, a correction for the concetration is needed over longer time intervals. The ribosomes in micellar solution can be transferred back in water, simply by washing the micellar solution with an excess 0.5 M aqueous KC1 solution. The UV spectrum of the ribosomal preparation reextracted in water is shown by the curve 3 of Fig 1: the general features of the spectrum are the same as before-only the ratio of the absorbancies OD260/OD280 is somewhat higher than before, reflecting the loss of some protein component during the re-extraction process. Let us consider now the circular dichroic spectra, which in principle are more informative than UV spectra to the structural changes experienced by the biopolymer on going from the aqueous solution to the micellar solution and back again in water. In fact, CD techniques have been applied already in order to obatain structural informations on ribosomes (7). Spectra are shown in Fig.2. Note again that all spectra are very similar to each other, suggesting that the solubilization in micelles, as well as the process of re-extraction, do not alter the gross conformation of the RNA molecules. We have also studied the CD spectra of ribosomal preparation at Wo=30: no significant changes are observed between the two Wo values. 1549
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.4
OD 0.3
0.2
0.1
0
950
300
wl (nm) Fig.2 Circular dichroic spectra of the ribosomal preparations in water (curveA), in AOT/isooctane micellar solution at w o 20 (curve B) and after re-extraction (curveC). Same concentration as in Fig.1.
The interest on the s t u d y of the influence of Wo on the CD-properties stams from o u r earlier observation concerning the psi-spectra of D N A in reverse micelles. As already mentioned, w h e n high molecular weight D N A or large plasmids are solubilized in reverse micelles, large anomalies in the CD spectra are observed (4-5), which are diagnostic for the occurrence of supercondensed D N A structures. In principle, this could also occurr for RNA. This is h o w e v e r not the case with ribosomes, at least u n d e r our experimental conditions. In the case of DNA, the superpackaging is with all likehood d u e to an intermolecular aggregation of double stranded macromolecules. Ribosomes have a globular structure (also thanks to the accompanying proteins) and for this reason aggregation p h e n o m e n a are less likely. 1550
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Discussion It appears then possible to solubilize ribosomes in reverse micellar solutions. In view of the large dimensions of the pig's liver ribosomes, this is indeed surprising, considering that at w0= 20, we have only 3.6% water (v:v) in the system. This percentage can be further reduced if needed. Notice that at w o = 20 the AOT micelles should have a radius of 35 A ° ca (1), i.e. m u c h smaller than the radius of the pig liver ribosomes, which is a r o u n d 300 A ° ( as judged on the basis of electron microscopy of our o w n preparations. The solubilization of ribosomes in reverse AOT micelles therefore cannot be v i e w e d as a simple insertion of the b i o p o l y m e r in the micelle: rather, large redistribution of material should accompany the solubilization, probably with the formation of v e r y large ribosomecontaining micelles. This is so also inthe case of proteins, as judged from double dye ultracentrifugation (8). Most likely, then, the solubilization of the ribosomes in reverse micelles is due to the same kind of structure which is reponsible for the stabilization of hydrophylic proteins in reverse micelles, the so-called "water-shell" model. Accordingly, the biopolymer is s o r r o u n d e d b y a layer of water, and b y a layer of surfactant molecules, in the order, which are in contact with the organic solvent with their apolar tails. Based on the spectroscopic features of the ribosomes which are reextracted in water from a micellar solution, one can assume that the structural integrity of the ribosomes is not d a m a g e d u p o n solubilization in the micellar system. This is interesting in view of possible applications of the ribosomes. In particular, the ribosome-containing micelle can be seen as a novel microreactor for protein biosynthesis: the surfactant layer 1551
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
around the ribosomes has protecting and stabilizing functions, and at the same time, due to its dynamic properties, should permits the free exchange of substrates to and from the ribosomes. This notion indicates the direction for future work. References
1) P.L. Luisi, M. Giomini, M.P. Pileni, B.H. Robinson,(1988) Biochem. Biophys. Acta, 947, 209 2) K. Martinek, A.V. Levashov, N.L. Klyachko, V.I. Pant-in, I.V. Berezin, (1981) Biochem. Biophys. Acta, 657, 227 3) P.L. Luisi, L.J. Magid,(1986) CRC Crit. Rev. Biochem., 20, 409 4) V.E. Imre, P.L. Luisi(1982) Biochem. Biophys. Res. Comm., 107, 538 5) E. Battistel, E.V. Imre, P.L. Luisi, (1989) in Controlled Release of Drugs: Polymers and Aggregate Systems, edited by M. Rosoff, New York, NY VCH Publishers, 257-275 6) A.B. Hanley, E. Grimfeld, R.L. Baxter,(1990) Biocatalysis, 3,253 7) I.Jr. Tinoco, M.F. Maestre and C. Bustamante, (1980) Trends Biochem. Sci., 8,227 8) G.G. Zampieri, H.J/ickle, P.L.Luisi, (1986) J. Phys. Chem., 90~01849 9) M. Greco, P. Cantatore, G. Pepe, C. Saccone,(1973) Eur. J. Biochem. 37, 171 10) U. Wild, K. Ramm, H.L. Sanher, D. Riesner,(1980) Eur. J. Biochem., 103, 227
1552
