ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
171,
Ultrastructure ABRAHAM
AMSTERDAM, The Weizmann
673-677
(197%
of Beaded ZVI
ER-EL,
Institute
of Science,
Received
July
Agarosel AND
SHMUEL
Rehovot,
SHALTIEL2
Israel
14,1975
Beaded agarose was dehydrated and embedded in Epon by a procedure that preserves the size, shape and, most likely, the native ultrastructure of the beads. Thin sections of the embedded beads reveal under the electron microscope a network sponge-like structure, uniform throughout the bead. The matrix skeleton is fairly rigid, though it occupies only a small percentage of the bead volume. This skeleton is composed of thin filaments (-20 A in diameter) bundled in a side-by-side assembly. The pores or channels between the filament bundles vary in shape and diameter (up to 0.3 wm). This structure accounts for some of the known physicochemical properties of beaded agarose.
Agarose (1) is a polysaccharide with high gelling ability obtained from a family of red seaweeds (Rhodophyceae). It is an alternating copolymer of 3-linked P-n-galactopyranose and 4-linked 3,6-anhydro-cyL-galactopyranose residues (2). Several pieces of evidence indicate that the gel structure of agarose is maintained by crosslinks or ‘Ijunction zones” that involve noncovalent cooperative binding of chains in ordered conformations (3, 4). More recent physicochemical studies (optical rotation, X-ray diffraction and computerized molecular model building) indicate that agarose has a double helix structure with 0.95nm axial periodicity (5). Each chain in the double helix forms a left-handed threefold helix of pitch 1.90 nm and is translated axially relative to its partner by exactly half this distance (5). In its commercial beaded form, agarose has found a variety of uses in the separation, purification and characterization of biomolecules in such techniques as gel filtration (6, 71, affinity chromatography (8, 9) and hydrophobic chromatography (lo141, as well as in the preparation of immu’ This work was supported by the Biotechnology Program of the Bundesministerium fiir Forschung und Technologie of the Federal Republic of Germany. * To whom correspondence should be addressed. 673 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
noadsorbents and immobilized enzymes (15-17). The usefulness of beaded agarose as a matrix for gel filtration was suggested to be due to the existence of appropriately sized channels or conical pores within the beads (18-20). It was assumed that when a mixture of molecules or particles is applied on a column of beaded agarose, the smaller ones penetrate into the pores or channels of the beads and thus are retarded while the large ones are excluded and move faster down the column. Arnott et al. (51, who proposed the double helix structure for agarose, assumed that these agarose helices are extensively aggregated, forming a gel framework composed of many chains (lo-lo41 in a side-by-side assembly. In this structure there are relatively large voids into which rather large molecules can penetrate. In a recent report (21) a scanning electron micrograph of beaded agarose was published. This low magnification t--x 400) micrograph shows that commercial Sepharose 4B beads are 15-100 pm in diameter, that they exhibit a furrowed surface and that some of them have slight depressions. At this magnification further ultrastructural details cannot be discerned. We wish to present here high magnification electron micrographs of beaded agarose, obtained by a procedure which
674
AMSTERDAM,
seems not to disrupt of the beads. MATERIALS
the native AND
ER-EL
AND
SHALTIEL
structure
METHODS
Agarose beads (Sepharose 4B, Pharmacia) were washed twice with 10 volumes of 0.1 M sodium cacodylate (pH 7.31, then treated in the same buffer with glutaraldehyde (4%, 2 h, 25°C) and subsequently with 0~0~ (2%, 4 h, 4°C). All operations were carried out in siliconized test tubes to minimize adhesion of the beads to glass surfaces. Dehydration of the beads was carried out by repeated suspension and decantation in the following media: (al 70% ethanol, (b) 95% ethanol, (c) absolute ethanol (twice), (dl propylene oxide (twice). By this gradual transfer into a nonaqueous medium, the beads were dehydrated but never allowed to dry out. The course of dehydration was followed under the light microscope (Ortholux, Leitz) with phase contrast optics, and it was found that the beads did not collapse but retained their size and shape. The dehydrated beads were embedded in Epon following the procedure described by Lufi (22). In order to form a packed pellet, the bead suspension in Epon was centrifuged in a Beem capsule (30 min at 14OOg). Two types of sections were prepared from the Epon-embedded beads, by using an MT-2 ultramicrotome (Sorvall) with glass knives. The first type (1 pm thick) were stained with 1% toluidene blue in sodium borate (1%) and used for light microscopy. The second type were thin sections (silver to gold, -1000 A). These were mounted on a 300-mesh copper grid, then doubly stained with uranyl acetate and lead citrate (231, the latter being known to stain polysaccharides, e.g., glycogen (24). The stained sections were examined and photographed under the electron microscope (Jeolco 100-B) operated at 80 kV. RESULTS
AND
FIG. 1. View of Epon-embedded Sepharose 4B beads under the light microscope. Slight deformations (arrows) are rarely seen. Calibration, 100 km.
DISCUSSION
In an attempt to establish whether the Epon-embedded agarose beads retain their structural integrity, we examined the sections under the microscope. As seen in Fig. 1, the large majority of the beads are perfectly round and only rarely can a slight deformation be detected at the surface (arrows). No intensification of the stain occurs towards the periphery of the bead, and their maximal diameter was found to be 220 pm, in accordance with the specifications of the manufacturers and with the maximal bead size measured by us before dehydration. Under the electron microscope the Eponembedded beads reveal a sponge-like structure (Figs. 2 and 3). The outer edge of a
FIG. edge of Arrows into the
2. Electron micrograph showing the outer a Sepharose 4B bead embedded in Epon. indicate openings of the pores or channels outer surface. Calibration, 1 pm.
representative bead is shown in Fig. 2. It can easily be seen that the network structure of the bead remains unchanged up to the edge and it should also be noted that no difference in structure was observed in different beads. Occasionally, openings of
FIG. 3. High magnification view of Sepharose 4B embedded in Epon, shbwing the network structure of the beads. Circles show the side-by-side assembly of thin filaments in the matrix framework. Arrows point at rounded or elongated electron-dense spots found throughout the section. Calibration, 1 pm. 675
676
AMSTERDAM,
ER-EL
the voids into the outer surface can be seen (arrows). At a higher magnification (Fig. 3) further details can be discerned. The voids, which are cross-sections of pores or channels, are randomly distributed all over the bead and vary in their shape and diameter (up to 0.3 pm). The skeleton network is composed of thin filaments in a side-by-side assembly (circles). The thickness of this matrix skeleton was found to vary between -20 and 300 A. Assuming that Sepharose 4B has the double helix structure proposed by Arnott et al. (5) (helix diameter, -15 A), then the observed bundles of filaments may contain from one to a few hundreds of double helices each. Discrete electron-dense spots are observed throughout the cross-section (Fig. 3). These are either rounded or elongated (arrows) and could represent perpendicular or slanted bundles of filaments in the spongelike three-dimensional structure of the bead. Such a structure is in agreement with the fact that in the gel form, Sepharose beads contain about 96% water. The three-dimensional structure seems to be quite rigid, since it was preserved during dehydration and embedding even without fixation with glutaraldehyde and OsO 4. It could be argued that the structure observed may not be identical with the native structure since glutaraldehyde does not effectively crosslink molecules that do not contain amino groups. However, when the agarose was activated with CNBr (25) then reacted with an excess of an (Y,odiaminoalkane and crosslinked with glutaraldehyde, it exhibited a structure which was indistinguishable from the one described above (26). The structure revealed by the electron microscope accounts for some of the known physicochemical properties of beaded agarose (such as its low matrix volume and high water content) and corroborates some of the structural features proposed (5) on the basis of optical rotation and X-ray diffraction. The procedure described above for embedding agarose beads in Epon and staining them, while preserving their native structure, makes it possible to carry out a direct ultrastructural comparison of
AND
SHALTIEL
various agaroses and thus study the relationship between their structure and their function as molecular sieves. This procedure makes it also possible to carry out electron microscope studies of modified agaroses (Amsterdam, A., Er-el, Z., and Shaltiel, S., in preparation) in an attempt to establish whether the new properties of the modified beads are associated with the chemical modification per se or with an ultrastructural change resulting from this modification. Finally, this procedure makes it possible to study cells adsorbed onto derivatized agaroses and obtain information regarding the ultrastructure of cell surfaces. In particular, these studies may be useful in the elucidation of the structure around cell receptors found at the contact loci between the adsorbed cell and the modified beaded agarose which had been coated with a biospecific ligand. ACKNOWLEDGMENT We thank assistance.
Mr.
S. Gordon
for
skillful
technical
REFERENCES 1. ARAKI, C. (1956) Bull. Chem. Sot. Jup. 29, 543544. 2. ARAKI, C., AND ARAI, K. (1967) Bull. Chem. Sot. Jup. 40, 1452-1456. 3. REES, D. A. (1969) Aduan. Carbohyd. Chem. Biochem. 24, 267-332. 4. DEA, I. C. M., MCKINNON, A. A., AND REES, D. A. (1972) J. Mol. Biol. 68, 153-172. 5. ARNOTT, S., FULMER, A., SCOTT, W. E., DEA, I. C. M., MOORHOUSE, R., AND REES, D. A. (1974) J. Mol. Biol. 90, 269-284. 6. GELLOTTE, B., AND PORATH, J. (1966) in Chromatography (Heftmann, E., ed.), Reinhold, New York. 7. DETERMANN, H. (1968) Gel Chromatography, Springer-Verlag, Berlin, Heidelberg, New York. 8. CUATRECASAS, P., WILCHEK, M., AND ANFINSEN, C. B. (1968) Proc. Nat. Acud. Sci. USA 61, 636-643. 9. JAKOBY, W. B., AND WILCHEK, M. (1974) Method-s Enzymol. 34B, 10. ER-EL, Z., ZAIDENZAIG, Y., AND SHALTIEL, S. (1972) Biochem. Biophys. Res. Commun. 49, 383-390. 11. YON, R. J. (1972) Biochem. J. 126, 765-767. 12. SHALTIEL, S., AND ER-EL, Z. (1973) Proc. Nat. Acad. Sci. USA 70, 778-781. 13. HOFSTEE, B. H. J. (1973) Anal. Biochem. 52, 430-448.
ULTRASTRUCTURE 14. SHALTIEL, S. (1974) Methods Enzymol. 34B, 126140. 15. SILMAN, I. H., AND KATCHALSKI, E. (1966)Annu. Reu. Biochem. 35, 873-908. 16. GOLDMAN, R., GOLDSTEIN, L., AND KATCHALSKI, E. (1971) in Biochemical Aspects of Reactions on Solid Supports (Stark, G. R., ed.), pp. l-78, Academic Press, New York. 17. PORATH, J. (1973) Biochimie 55, 943-951. 18. FLODIN, P. (1961) J. Chromatogr. 5, 103-115. 19. PORATH, J. (1963) Pure Appl. Chem. 6,233-244. 20. SQUIRE, P. G. (1964) Arch. Biochem. Biophys.
OF
BEADED
AGAROSE
677
107,471-478. 21. MALMQVIST, M., AND HOFSTEN, B. v. (1975) J. Gen. Microbial. 87, 167-169. 22. LUFT, J. H. (1961) J. Biophys. Biochem. Cytol. 9, 409-414. 23. VENABLE, J. H., AND COGGESTALL, R. (1965) J. Cell Biol. 25, 407-413. 24. REYNOLDS, E. S. (1963) J. CelZ Biol. 17,208-212. 25. AXEN, R., PORATH, J., AND ERNBACK, S. (1967) Nature (London) 214, 1302-1304. 26. AMSTERDAM, A., ER-EL, Z., AND SHALTIEL, S. (1974) Isr. J. Med. Sci. 10, 1580.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
171,
Ultrastructure ABRAHAM
AMSTERDAM, The Weizmann
673-677
(197%
of Beaded ZVI
ER-EL,
Institute
of Science,
Received
July
Agarosel AND
SHMUEL
Rehovot,
SHALTIEL2
Israel
14,1975
Beaded agarose was dehydrated and embedded in Epon by a procedure that preserves the size, shape and, most likely, the native ultrastructure of the beads. Thin sections of the embedded beads reveal under the electron microscope a network sponge-like structure, uniform throughout the bead. The matrix skeleton is fairly rigid, though it occupies only a small percentage of the bead volume. This skeleton is composed of thin filaments (-20 A in diameter) bundled in a side-by-side assembly. The pores or channels between the filament bundles vary in shape and diameter (up to 0.3 wm). This structure accounts for some of the known physicochemical properties of beaded agarose.
Agarose (1) is a polysaccharide with high gelling ability obtained from a family of red seaweeds (Rhodophyceae). It is an alternating copolymer of 3-linked P-n-galactopyranose and 4-linked 3,6-anhydro-cyL-galactopyranose residues (2). Several pieces of evidence indicate that the gel structure of agarose is maintained by crosslinks or ‘Ijunction zones” that involve noncovalent cooperative binding of chains in ordered conformations (3, 4). More recent physicochemical studies (optical rotation, X-ray diffraction and computerized molecular model building) indicate that agarose has a double helix structure with 0.95nm axial periodicity (5). Each chain in the double helix forms a left-handed threefold helix of pitch 1.90 nm and is translated axially relative to its partner by exactly half this distance (5). In its commercial beaded form, agarose has found a variety of uses in the separation, purification and characterization of biomolecules in such techniques as gel filtration (6, 71, affinity chromatography (8, 9) and hydrophobic chromatography (lo141, as well as in the preparation of immu’ This work was supported by the Biotechnology Program of the Bundesministerium fiir Forschung und Technologie of the Federal Republic of Germany. * To whom correspondence should be addressed. 673 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
noadsorbents and immobilized enzymes (15-17). The usefulness of beaded agarose as a matrix for gel filtration was suggested to be due to the existence of appropriately sized channels or conical pores within the beads (18-20). It was assumed that when a mixture of molecules or particles is applied on a column of beaded agarose, the smaller ones penetrate into the pores or channels of the beads and thus are retarded while the large ones are excluded and move faster down the column. Arnott et al. (51, who proposed the double helix structure for agarose, assumed that these agarose helices are extensively aggregated, forming a gel framework composed of many chains (lo-lo41 in a side-by-side assembly. In this structure there are relatively large voids into which rather large molecules can penetrate. In a recent report (21) a scanning electron micrograph of beaded agarose was published. This low magnification t--x 400) micrograph shows that commercial Sepharose 4B beads are 15-100 pm in diameter, that they exhibit a furrowed surface and that some of them have slight depressions. At this magnification further ultrastructural details cannot be discerned. We wish to present here high magnification electron micrographs of beaded agarose, obtained by a procedure which
674
AMSTERDAM,
seems not to disrupt of the beads. MATERIALS
the native AND
ER-EL
AND
SHALTIEL
structure
METHODS
Agarose beads (Sepharose 4B, Pharmacia) were washed twice with 10 volumes of 0.1 M sodium cacodylate (pH 7.31, then treated in the same buffer with glutaraldehyde (4%, 2 h, 25°C) and subsequently with 0~0~ (2%, 4 h, 4°C). All operations were carried out in siliconized test tubes to minimize adhesion of the beads to glass surfaces. Dehydration of the beads was carried out by repeated suspension and decantation in the following media: (al 70% ethanol, (b) 95% ethanol, (c) absolute ethanol (twice), (dl propylene oxide (twice). By this gradual transfer into a nonaqueous medium, the beads were dehydrated but never allowed to dry out. The course of dehydration was followed under the light microscope (Ortholux, Leitz) with phase contrast optics, and it was found that the beads did not collapse but retained their size and shape. The dehydrated beads were embedded in Epon following the procedure described by Lufi (22). In order to form a packed pellet, the bead suspension in Epon was centrifuged in a Beem capsule (30 min at 14OOg). Two types of sections were prepared from the Epon-embedded beads, by using an MT-2 ultramicrotome (Sorvall) with glass knives. The first type (1 pm thick) were stained with 1% toluidene blue in sodium borate (1%) and used for light microscopy. The second type were thin sections (silver to gold, -1000 A). These were mounted on a 300-mesh copper grid, then doubly stained with uranyl acetate and lead citrate (231, the latter being known to stain polysaccharides, e.g., glycogen (24). The stained sections were examined and photographed under the electron microscope (Jeolco 100-B) operated at 80 kV. RESULTS
AND
FIG. 1. View of Epon-embedded Sepharose 4B beads under the light microscope. Slight deformations (arrows) are rarely seen. Calibration, 100 km.
DISCUSSION
In an attempt to establish whether the Epon-embedded agarose beads retain their structural integrity, we examined the sections under the microscope. As seen in Fig. 1, the large majority of the beads are perfectly round and only rarely can a slight deformation be detected at the surface (arrows). No intensification of the stain occurs towards the periphery of the bead, and their maximal diameter was found to be 220 pm, in accordance with the specifications of the manufacturers and with the maximal bead size measured by us before dehydration. Under the electron microscope the Eponembedded beads reveal a sponge-like structure (Figs. 2 and 3). The outer edge of a
FIG. edge of Arrows into the
2. Electron micrograph showing the outer a Sepharose 4B bead embedded in Epon. indicate openings of the pores or channels outer surface. Calibration, 1 pm.
representative bead is shown in Fig. 2. It can easily be seen that the network structure of the bead remains unchanged up to the edge and it should also be noted that no difference in structure was observed in different beads. Occasionally, openings of
FIG. 3. High magnification view of Sepharose 4B embedded in Epon, shbwing the network structure of the beads. Circles show the side-by-side assembly of thin filaments in the matrix framework. Arrows point at rounded or elongated electron-dense spots found throughout the section. Calibration, 1 pm. 675
676
AMSTERDAM,
ER-EL
the voids into the outer surface can be seen (arrows). At a higher magnification (Fig. 3) further details can be discerned. The voids, which are cross-sections of pores or channels, are randomly distributed all over the bead and vary in their shape and diameter (up to 0.3 pm). The skeleton network is composed of thin filaments in a side-by-side assembly (circles). The thickness of this matrix skeleton was found to vary between -20 and 300 A. Assuming that Sepharose 4B has the double helix structure proposed by Arnott et al. (5) (helix diameter, -15 A), then the observed bundles of filaments may contain from one to a few hundreds of double helices each. Discrete electron-dense spots are observed throughout the cross-section (Fig. 3). These are either rounded or elongated (arrows) and could represent perpendicular or slanted bundles of filaments in the spongelike three-dimensional structure of the bead. Such a structure is in agreement with the fact that in the gel form, Sepharose beads contain about 96% water. The three-dimensional structure seems to be quite rigid, since it was preserved during dehydration and embedding even without fixation with glutaraldehyde and OsO 4. It could be argued that the structure observed may not be identical with the native structure since glutaraldehyde does not effectively crosslink molecules that do not contain amino groups. However, when the agarose was activated with CNBr (25) then reacted with an excess of an (Y,odiaminoalkane and crosslinked with glutaraldehyde, it exhibited a structure which was indistinguishable from the one described above (26). The structure revealed by the electron microscope accounts for some of the known physicochemical properties of beaded agarose (such as its low matrix volume and high water content) and corroborates some of the structural features proposed (5) on the basis of optical rotation and X-ray diffraction. The procedure described above for embedding agarose beads in Epon and staining them, while preserving their native structure, makes it possible to carry out a direct ultrastructural comparison of
AND
SHALTIEL
various agaroses and thus study the relationship between their structure and their function as molecular sieves. This procedure makes it also possible to carry out electron microscope studies of modified agaroses (Amsterdam, A., Er-el, Z., and Shaltiel, S., in preparation) in an attempt to establish whether the new properties of the modified beads are associated with the chemical modification per se or with an ultrastructural change resulting from this modification. Finally, this procedure makes it possible to study cells adsorbed onto derivatized agaroses and obtain information regarding the ultrastructure of cell surfaces. In particular, these studies may be useful in the elucidation of the structure around cell receptors found at the contact loci between the adsorbed cell and the modified beaded agarose which had been coated with a biospecific ligand. ACKNOWLEDGMENT We thank assistance.
Mr.
S. Gordon
for
skillful
technical
REFERENCES 1. ARAKI, C. (1956) Bull. Chem. Sot. Jup. 29, 543544. 2. ARAKI, C., AND ARAI, K. (1967) Bull. Chem. Sot. Jup. 40, 1452-1456. 3. REES, D. A. (1969) Aduan. Carbohyd. Chem. Biochem. 24, 267-332. 4. DEA, I. C. M., MCKINNON, A. A., AND REES, D. A. (1972) J. Mol. Biol. 68, 153-172. 5. ARNOTT, S., FULMER, A., SCOTT, W. E., DEA, I. C. M., MOORHOUSE, R., AND REES, D. A. (1974) J. Mol. Biol. 90, 269-284. 6. GELLOTTE, B., AND PORATH, J. (1966) in Chromatography (Heftmann, E., ed.), Reinhold, New York. 7. DETERMANN, H. (1968) Gel Chromatography, Springer-Verlag, Berlin, Heidelberg, New York. 8. CUATRECASAS, P., WILCHEK, M., AND ANFINSEN, C. B. (1968) Proc. Nat. Acud. Sci. USA 61, 636-643. 9. JAKOBY, W. B., AND WILCHEK, M. (1974) Method-s Enzymol. 34B, 10. ER-EL, Z., ZAIDENZAIG, Y., AND SHALTIEL, S. (1972) Biochem. Biophys. Res. Commun. 49, 383-390. 11. YON, R. J. (1972) Biochem. J. 126, 765-767. 12. SHALTIEL, S., AND ER-EL, Z. (1973) Proc. Nat. Acad. Sci. USA 70, 778-781. 13. HOFSTEE, B. H. J. (1973) Anal. Biochem. 52, 430-448.
ULTRASTRUCTURE 14. SHALTIEL, S. (1974) Methods Enzymol. 34B, 126140. 15. SILMAN, I. H., AND KATCHALSKI, E. (1966)Annu. Reu. Biochem. 35, 873-908. 16. GOLDMAN, R., GOLDSTEIN, L., AND KATCHALSKI, E. (1971) in Biochemical Aspects of Reactions on Solid Supports (Stark, G. R., ed.), pp. l-78, Academic Press, New York. 17. PORATH, J. (1973) Biochimie 55, 943-951. 18. FLODIN, P. (1961) J. Chromatogr. 5, 103-115. 19. PORATH, J. (1963) Pure Appl. Chem. 6,233-244. 20. SQUIRE, P. G. (1964) Arch. Biochem. Biophys.
OF
BEADED
AGAROSE
677
107,471-478. 21. MALMQVIST, M., AND HOFSTEN, B. v. (1975) J. Gen. Microbial. 87, 167-169. 22. LUFT, J. H. (1961) J. Biophys. Biochem. Cytol. 9, 409-414. 23. VENABLE, J. H., AND COGGESTALL, R. (1965) J. Cell Biol. 25, 407-413. 24. REYNOLDS, E. S. (1963) J. CelZ Biol. 17,208-212. 25. AXEN, R., PORATH, J., AND ERNBACK, S. (1967) Nature (London) 214, 1302-1304. 26. AMSTERDAM, A., ER-EL, Z., AND SHALTIEL, S. (1974) Isr. J. Med. Sci. 10, 1580.
