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.
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces 88003, USA.
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Gallegos
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Jun
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Hageman
D000447:Aldehydes / Q000138:chemical synthesis* / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D002364:Caseins / Q000138:chemical synthesis / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism
D000447:Aldehydes; D001426:Bacterial Proteins; D002364:Caseins; D008565:Membrane Proteins; C104308:3,5-dinitrosalicylaldehyde; D010450:Endopeptidases; D013381:Subtilisins; D008239:Lysine