Gallegos N G, Jun J, Hageman J H
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces 88003, USA.
J Biochem Biophys Methods. 1996 Oct 15;33(1):31-41. doi: 10.1016/0165-022x(96)00013-9.
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.
为了在枯草芽孢杆菌中寻找新的蛋白酶，我们开发了一种使用3,5-二硝基水杨醛（DNSA）合成生色蛋白酶底物的通用方法。在NaBH4存在的情况下，用DNSA对哈默斯坦酪蛋白和枯草芽孢杆菌细胞提取物中的可溶性蛋白质进行标记。透析（pH 7.8）后，所得的3,5-二硝基-2-羟基苄基酪蛋白（DNHB-酪蛋白）和DNHB-细菌细胞蛋白溶液呈浅橙色。合成了一种模型化合物N-苄基-3,5-二硝基-2-羟基苄胺，并估计其在pH 8时于366 nm处的摩尔吸收系数为14,100 M-1 cm-1，用于计算酪蛋白上的染料负载量。以这种方式制备的生色底物在赖氨酸残基上应保留正电荷。将DNHB-酪蛋白和DNHB-细菌细胞蛋白与不同浓度的枯草杆菌蛋白酶BPN'孵育不同时间，用三氯乙酸沉淀并离心。用NaOH将酸溶性上清液部分调至碱性，并在366 nm（最大吸收波长）处测量吸光度。颜色产生与枯草杆菌蛋白酶浓度和孵育时间成正比；在所使用的测定条件下，枯草杆菌蛋白酶的检测限约为100 ng。以DNHB标记的蛋白质为底物，在枯草芽孢杆菌的可溶性提取物中检测到了五种蛋白酶活性。
