Preparation of general proteinase substrates using 3,5-dinitrosalicylaldehyde.

To search for new proteinases in Bacillus subtilis we have developed a general method for synthesizing chromogenic proteinase substrates using 3,5-dinitrosalicylaldehyde (DNSA). Hammersten casein and soluble protein from extracts from B. subtilis cells were labeled with DNSA in the presence of NaBH4. After dialysis (pH 7.8), the resultant 3,5-dinitro-2-hydroxybenzyl-casein (DNHB-casein) and DNHB-bacterial cell protein solutions were a light orange color. A model compound, N-benzyl-3,5-dinitro-2-hydroxybenzylamine was synthesized and estimated to have a molar absorption coefficient of 14,100 M-1 cm-1 at 366 nm at pH 8, which was used to calculate dye loading on casein. Chromogenic substrates prepared in this way should retain positive charges on lysine residues. DNHB-casein and DNHB-bacterial cell protein were incubated with varying concentrations of subtilisin BPN' for varying times, precipitated with trichloroacetic acid and centrifuged. The acid-soluble supernatant fractions were made basic with NaOH and absorbances were measured at 366 nm, the absorption maximum. Color production was proportional to subtilisin concentration and times of incubation; under the assay conditions used, the limit of detection of subtilisin was about 100 ng. Five proteinase activities were detected in soluble extracts of B. subtilis using DNHB-labeled proteins as substrates.