Abstract
A new method for the reversible immobilization of thiol bimolecules, e.g., thiolpeptides and thiolproteins, to beaded agarose and other solid phases is reported. The method consists of an activation and a coupling step. The activation is based on oxidation of disulfides (or thiol groups via disulfides) present in a solid phase by hydrogen peroxide at moderately acidic pH. This oxidation leads to disulfide oxides (thiolsulfinate groups of which the majority are further oxidized to thiolsulfonate). The thiolsulfonate groups react easily with thiol compounds, which become immobilized via disulfide bonds. The pH range for thiol coupling is wide (pH 5-8), but for most thiols the reaction seems to proceed faster at pH>7. The stability of the reactive group to hydrolysis, especially at neutral and weakly acidic pH, is very high. The activated gel, therefore, can be stored as a suspension at pH 5 for extended periods. The method has been used to reversibly immobilize glutathione, β-galactosidase, alcohol dehydrogenase, urease, and papain, all with exposed thiol groups as well as thiolated bovine serum albumin and sweet-potato β-amylase.
Depending on the thiol content of starting thiol-agarose, thiol-sulfonate-agarose derivatives with different binding capacities can be obtained. Thus, up to 5.0 mg (16 μmol) glutathione and 15 mg thiol-protein/mL gel derivative have been immobilized.
The gel bead can be regenerated and reused at least twice. Besides agarose, cellulose, crosslinked dextran, and polyacrylamide were shown to be very suitable as supports for solid-phase thiolsulfonates.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ryéden, L. and Carlsson, J. (1989),Protein Purification, Janson J. C. and Rydén, L., eds., VCH, New York, pp. 252–274.
Brocklehurst, K., Carlsson, J., Kierstan, M., and Crook, E. (1973),Biochem. J. 133, 573.
Carlsson, J.,Axén, R., Brocklehurst, K., and Crook, E. (1974),Eur. J. Biochem. 44, 189.
Svensson, A., Carlsson, J., and Eaker, D. (1977),FEBS Lett. 73, 171.
Rydén, L. and Norder, H. (1981),J. Chromatogr. 215, 341.
Rydén, L. and Eaker, D. (1983),Eur. J. Biochem. 132, 241.
Field, L. and Khim, Y. (1972),J. Org. Chem. 37, 2710.
Isenberg, N. (1973),Int. J. Sulfur Chem. 8, 307.
Smith, D. J., Maggio, E. T., and Kenyon, G. L. (1975),Biochemistry 14, 766.
Bruice, T. W. and Kenyon, G. L. (1977),Methods in Enzymology, Colowick, S. P. and Kaplan, N. O., eds., vol. 47, Academic, New York, p. 407.
Carnevale, J. and Healey, K. (1982),Anal. Chim. Acta 140, 143.
Carlsson, J. and Batista-Viera, F. (1991),Biotechnol. Appl. Biochem.,14, 114.
Axén, R., Drevin, H., and Carlsson, J. (1975),Acta Chem. Scand. [B]29, 471.
Carlsson, J., Drevin, H., and Axén, R. (1978),Biochem. J. 173, 723.
Barnard, D. and Cole, E. R. (1959),Anal. Chim. Acta 20, 540.
Bernfeld, P. (1955),Methods in Enzymology, Colowick, S. P. and Kaplan, N. O., eds., vol. 1, Academic, New York, p. 149.
Manjón, A., Llorca, F. I., Bonete, M. J., Bastida, J., and Iborra, J. L. (1985),Process Biochem. 20, 17.
Vallee, B. L. and Hoch, F. L. (1955),Proc. Natl. Acad. Sci. USA 41, 327.
Torshinsky, Y. M. (1981),Sulfur in Proteins, Metzler, D., ed., Pergamon, Oxford, p. 53.
Brocklehurst, K. and Little, G. (1973),Biochem. J. 133, 67.
Carlsson, J., Jansson, J. C. and Sparrman, M. (1989),Protein Purification, Jansson, J. C. and Rydén, L., eds., VCH, New York, pp. 291–308.
Kluger, R. and Tsui, W.-C. (1980),Can. J. Biochem. 58, 629.
Oscarsson, S. and Porath, J. (1989),Anal. Biochem. 176, 330.
Carlsson, J. and Svensson, A. (1974),FEBS Lett. 42, 183.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Batista-Viera, F., Barbieri, M., Ovsejevi, K. et al. A new method for reversible immobilization of thiol biomolecules based on solid-phase bound thiolsulfonate groups. Appl Biochem Biotechnol 31, 175–195 (1991). https://doi.org/10.1007/BF02921788
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02921788