Fig. 11. Desorption of noncovalently immobilized protein subunits from the support in the prepared derivatives. Desorption was carried out by boiling the derivatives in the presence of SDS. Lane 1, crude extract; lane 2, fraction of purified enzyme; lane 3, enzyme immobilized on agarose-glutaraldehyde poorly activated (1 h of immobilization); lane 4, enzyme immobilized on agarose-glutaraldehyde highly activated (24 h of immobilization); lane 5, previous derivative modified with polyaldehyde-dextrans.

94,000 67,000


that at least two to three of the four subunits of the enzyme had been covalently attached to the support. However, some of the subunits were not yet covalently attached to the support and could dissociate from the whole protein. These results may explain the significant increase in the enzyme stability and the significant decrease of the dependence of the enzyme stability on the enzyme concentration found when studying this derivative.

The derivative that had been chemically cross-linked with aldehyde-dextran, after boiling in SDS, did not release any detectable protein to the supernatant. Therefore, this treatment seems to be able to fully stabilize the quaternary structure of this complex tetrameric enzyme (see Fig. 11, lane 5). This explains the nondependence of the stability of the enzyme on the enzyme concentrations when analyzing this derivative, and also explains the high stability of such enzyme derivative in the absence of any phosphate ions.

Thus, this simple two-step treatment is able to fully stabilize the quaternary structure of the enzyme.

4. Notes

1. The support should be selected as a function of the protein size: in the case of agarose, the larger the percentage of agarose, the lower the pore diameter; in the case of Sepabeads, the higher the number, the higher pore size. Other activation of the support (e.g., glioxyl, glutaraldehyde) may be utilized to fulfil this multisubunit immobilization.

2. The immobilization of multimeric enzymes may require the use of high-protein concentrations and the addition of certain compounds to keep the multimeric structure, even just during the immobilization time.

3. Magnetic stirring may act as a mill stone, when any kind of support is used. Other types of stirring devices should be used (e.g., shaker, orbital stirring, mechanical stirring)

4. Use cut pipet tips to measure the suspension.

5. Aldehyde dextran should never be exposed at a pH over 8.0 except during reduction. If the preparation takes a brown color, it should be discarded.


1. Bickerstaff, G.F. (ed). (1997) Immobilization of Enzymes and Cells, Humana Press, Totowa, NJ.

2. Guisan, J. M., Blanco, R. M., Fernandez-Lafuente, R., et al. (eds). (1993) Enzyme Stabilization by Multipoint Covalent Attachment on Activated Supports. Protein Stability and Stabilization of Enzymes, Elsevier, Amsterdam, pp. 55-62.

3. Fernandez-Lafuente, R., Rodriguez. V., Mateo, C., et al. (1999) Stabilization of multimeric enzymes via immobilization and post-immobilization techniques. J. Mol. Catal. B: Enzymatic. 7, 181-189.

4. Torchilin, V. P., Trubetskoy, V. S., Omelyananko, V. G., and Martinek, K. J. (1983) Stabilization of subunit enzyme by intersubunit crosslinking with bifunc-tional reagents: studies with glyceraldehyde-3-phosphate dehydrogenase. J. Mol. Catal. 19, 291-301.

5. Fernandez-Lafuente, R., Rodriguez, V., Mateo, C., et al. (1999) Stabilization of multimeric enzymes via immobilization and post-immobilization techniques. J. Mol. Catal B: Enzymatic. 7, 181-189.

6. Fernandez-Lafuente, R., Hernandez-Justiz, O., Mateo, C., et al. (2001) Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicil-lin acylase permits the optimization of ampicillin synthesis under dissociation conditions. Biomacromolecules 2, 95-104.

7. Pessela, B. C. C., Mateo, C., Fuentes, M., et al. (2004) Stabilization of a multimeric ß-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextan cross-linking. Biotechnol. Prog. 20, 388-392.

8. Betancor, L., Lopez-Gallego, F., Hidalgo, A., et al. (2004) Prevention of interfaction inactivation of enzymes by coating the enzyme surface with dextran-aldehyde. J. Biotechnol., 110, 201-207.

9. Guisan, J. M., Penzol, G., Armisen, P., et al. (1997) Immobilization of enzymes acting on macromolecular substrates. Reduction of steric problems. In: Immobilization of Enzymes and Cells, Methods in Biotechnology, vol. 1, (Bickerstaff, G. F., ed.), Human Press Inc., Totowa, NJ, pp. 261-275.

10. Betancor, L., Hidalgo, A., Fernandez-Lorente, G., Mateo, C., Fernandez-Lafuente, R., and Guisan J. M., (2003) Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques. Biotechnol. Prog. 19, 763-767.

11. Fuentes, M., Segura, R., Abian, O., et al. (2004) Stabilization of protein-protein interaction by specific crosslink with aldehyde-dextran. Proteomics 9, 2602-2607.

12. Hidalgo, A., Betancor, L., Lopez-Gallego, F., et al. (2003) Desing of an immobilized preparation of catalase from Thermus thermophilus to be used in a wide range of conditions. Structural stabilization of a multimeric enzyme. Enzyme Microb. Technol. 33, 278-285.

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