The introduction of a disulphide bond into a sensitive loop region of the thermolysin-like neutral protease from Bacillus stearothermophilus by site-directed mutagenesis resulted in an extreme stabilisation of the enzyme towards thermal inactivation [1]. Since denaturation of both the wild-type and the mutant enzymes is accompanied by rapid autoproteolytic degradation, it has not been possible to differentiate between stabilisation towards unfolding and stabilisation towards autoproteolysis, hitherto. In the present study autoproteolysis of the mutant and wild-type enzymes have been compared in the presence of guanidine hydrochloride (GdnHCl) with the aim to find conditions where conformational changes can be measured spectroscopically without the interference of autoproteolysis.

The autodegradation of the neutral proteases was analysed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as function of time and GdnHCl concentration. GdnHCl was separated from the samples before SDS-PAGE by precipitating the protein with sodium deoxycholate / trichloroacetic acid. The amount of intact proteases were quantified by densitometric evaluation of the Coomassie brillant blue-stained protein bands. As function of the GdnHCl concentration, they show typical profiles with minima, which become more pronounced with increasing time and are shifted to higher GdnHCl concentrations for the mutant protease. The decreased extent of autoproteolysis at high GdnHCl concentrations suggests that inactivation of the neutral proteases by unfolding is faster than autoproteolysis under these conditions. The kinetic studies of autoproteolysis revealed conditions where spectroscopic transition curves can be measured without significant autoproteolysis. First unfolding studies by fluorescence spectroscopy suggest that the disulphide bond effects a real conformational stabilisation of the protease molecule towards global unfolding.

[1] Mansfeld, J.Vriend, G., Dijkstra, B. W., Veltman, O. R., Van den Burg, B., Venema, G., Ulbrich-Hofmann, R., Eijsink, V. G. H., J. Biol. Chem., 272, 11152-11156, 1997.