Compatible solutes (COMPS) are low molecular compounds that are known to stabilize macromolecular biomolecules against stress both in vivo and in vitro [1]. Their capability to protect proteins, especially enzymes, against physical denaturation, e. g. by heat, is the subject of several recent investigations. On the other hand, knowledge about the protective effect of COMPS against enzymatically catalyzed hydrolysis of native proteins is rather poor. To close this gap, we determined the influence of several COMPS on the degree of antibody proteolysis by pepsin. Using SDS-PAGE and ELISA techniques we here demonstrate that limited proteolysis of antibody heavy chains significantly could be suppressed by ectoines. In contrast, no effect could be observed if the hexapeptide Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-methyl-ester was used as the substrate to monitor the hydrolytic reaction in the presence of COMPS. For that reason, the mode of COMPS action could not be explained by a classical inhibition of the proteases catalytic activity. We rather suggest that the protection against proteolytic attack is due to the stabilization of a more compact antibody structure resulting in a less distinctive solvent exposure of the highly flexible hinge regions which prevents proteolysis via sterical hindrance. This explanation is supported by the theoretical model demanding preferential exclusion of COMPS [2] which thermodynamically induces a minimization of the protein surfaces and consequently the formation of highly compact globular protein structures. Such a universal influence on the tertiary structure would allow the application of COMPS as ingredients to avoid unwanted proteolytic digestion, e. g. during protein purification or storage. Consequently, our future work will deal with a broadly designed survey measuring the here described effect for further protein targets which are susceptible to proteolytic degradation.

[1] da Costa, M.S., Santos, H., Galinski, E.A.: in Advances in Biochemical Engineering / Biotechnology Vol. 61 (ed. Scheper T), Springer-Verlag, Berlin-Heidelberg, 117-153, 1998.

[2] Wiggins, P.M., Microbiol. Rev., 54, 432-449, 1990.