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 usi ng 3,5-dinitrosalicylaldehyde (DNSA). Hammersten casein and soluble pr otein from extracts from B. subtilis cells were labeled with DNSA in t he presence of NaBH4. After dialysis (pH 7.8), the resultant 3,5-dinit ro-2-hydroxybenzyl-casein (DNHB-casein) and DNHB-bacterial cell protei n solutions were a light orange color. PI model compound, N-benzyl-3,5 -dinitro-2-hydroxybenzylamin was synthesized and estimated to have a m olar absorption coefficient of 14100 M(-1) cm(-1) at 366 nm at pH 8, w hich was used to calculate dye loading on casein. Chromogenic substrat es prepared in this way should retain positive charges on lysine resid ues. DNHB-casein and DNHB-bacterial cell protein were incubated with v arying concentrations of subtilisin BPN' for varying times, precipitat ed with trichloroacetic acid and centrifuged. The acid-soluble superna tant 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 c onditions used, the limit of detection of subtilisin was about 100 ng. Five proteinase activities were detected in soluble extracts of B. su btilis using DNHB-labeled proteins as substrates.