OPTIMIZATION OF THE ASSAY OF BATE AND DRUM LIQUOR FOR PROTEOLYTIC ACTIVITY

In two recent publications from this laboratory, procedures were descr ibed for measuring the proteolytic activity of bate and (bate-containi ng) drum liquor using hide powder azure (HPA) as substrate. Subsequent to that work, it was found that HPA sometimes loses much of its sensi tivity to proteolytic enzymes on storage. This led to a study of the s ubstrate and of related factors affecting the assay, which resulted in substantial revision of the assay procedure, as follows. HPA is activ ated prior to use by reduction of particle size and a mild heat treatm ent, and 200 mg are used per assay (instead of 50 mg). A clarified ext ract of the sample to Be assayed is introduced into the incubation tub e (a 25 mm x 150 mm screw-cap tube) instead of the crude material. Sin ce proteolytic activity was found to change by 10 to 15% per degrees C , good temperature control is necessary; for research purposes, a wate r-bath shaker-incubator operating at 37 degrees C and 400 rpm is recom mended. Incubation time has been reduced from 20 min to 10 min at ambi ent temperature (ca. 25 degrees C), 5 min at 37 degrees C. These modif ications have resulted in the following advantages: (1) substantially increased sensitivity, (2) a linear dependence of the color measured o n enzyme concentration, and (3) improved reproducibility.