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Active-Site Titration of Serine Proteases Using a Fluoride Ion Selective Electrode and Sulfonyl Fluoride Inhibitors

1996; Elsevier BV; Volume: 242; Issue: 2 Linguagem: Inglês

10.1006/abio.1996.0456

1096-0309

Constance Y. Hsia, Grant Ganshaw, Christian Paech, Christopher J L Murray,

Advanced biosensing and bioanalysis techniques

We report a general procedure for the determination of active enzyme concentrations for serine proteases. The method relies on the measurement 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 subtilisins and by spectrofluorometric and spectrophotometric titrations. The minimal enzyme concentration detectable is 1–10 μMprotease. The method is insensitive to color and turbidity of the sample and is therefore useful for measuring protease concentration in broth solutions. The active enzyme concentration of subtilisin BPN′ fromBacillus amyloliquefaciensdetermined by titration with phenylmethylsulfonyl fluoride is 25% higher than the concentration determined using the spectrophotometric burst titrantN-trans-cinnamoylimidazole. Analysis of the pre-steady-state burst amplitude and kinetics suggests that the extinction coefficient for the cinnamoyl acyl-enzyme is larger than previously measured and a significant fraction of the enzyme is present as an unproductive ES2complex. The molar extinction coefficient at 280 nm for subtilisin BPN′ is 26.5 mM−1cm−1and for subtilisin fromBacillus lentusis 22.5 mM−1cm−1.