ACTIVE-SITE TITRATION OF SERINE PROTEASES USING A FLUORIDE-ION SELECTIVE ELECTRODE AND SULFONYL FLUORIDE INHIBITORS

We report a general procedure for the determination of active enzyme c oncentrations for serine proteases. The method relies on the measureme nt of fluoride ion released from sulfonyl fluorides upon reaction with the active-site serine using an ion selective electrode. The results have been independently confirmed by amino acid analyses of subtilisin s and by spectrofluorometric and spectrophotometric titrations. The mi nimal enzyme concentration detectable is 1-10 mu M protease. The metho d is insensitive to color and turbidity of the sample and is therefore useful for measuring protease concentration in broth solutions. The a ctive enzyme concentration of subtilisin BPN' from Bacillus amylolique faciens determined by titration with phenylmethylsulfonyl fluoride is 25% higher than the concentration determined using the spectrophotomet ric burst titrant N-trans-cinnamoylimidazole. Analysis of the pre-stea dy-state burst amplitude and kinetics suggests that the extinction coe fficient for the cinnamoyl acyl-enzyme is larger than previously measu red and a significant fraction of the enzyme is present as an unproduc tive ES(2) complex. The molar extinction coefficient at 280 nm for sub tilisin BPN' is 26.5 mM(-1) cm(-1) and for subtilisin from Bacillus le ntus is 22.5 mM(-1) cm(-1). (C) 1996 Academic Press, Inc.