สำนักราชบัณฑิตยสภา

199 สมศั กดิ์ ด� ำรงค์เลิ ศ และคณะ วารสารราชบัณฑิตยสถาน ปีที่ ๓๗ ฉบับที่ ๑ ม.ค.-มี.ค. ๒๕๕๕ Standard assay for determination of azo reducatase activities with cell extracts and soluble cell membrane in the presence of different redox mediators The azo reductase activity was determined anaerobically in 1.5 ml-rubber- stoppered cuvettes which were flushed before the assay with oxygen-free nitrogen gas. Azo reductase activity was routinely measured by a modification of the spectro photometric assay described previously by Kudlich et al. (29) and Russ et al. (46). For the standard assay, the anaerobically prepared reaction mixtures contained in 800 µl of 50 mM of Tris-HCl buffer (pH 7.5), 25 µM of the respective sufonated azo dyes, and 50 µM of redox mediators (FAD or AQDS). The cell extracts or solubilized cell membrane from Paenibacillus sp. strain A5 were added (200 µl, about 0.05 to 0.1 mg of protein), and the reaction mixtures were flushed again with nitrogen gas. Finally, the reaction was started by the addition of 300 µM of NADH, and the initial rate was measured at 30 0 C with a model UV WINLAB, Perkin Elmer molecular spectrophotometer for 30 min (by using 1-min measuring intervals). Reaction rates were calculated by using molar extinction coefficient of these azo dyes, which are summarized in Table 1. TABLE 1.  Properties of sulfonated azo dyes used in this study. Azo dye Generic name C.I. λ max Extinction number (nm) coefficient (mM -1 cm -1 ) Remazol Brilliant Orange 3R reactive orange 16 17757 495 15.4 Remazol Brilliant Violet 5R reactive violet 5 18097 557 6.8 Remazol Black B reactive black 5 20505 595 29.1 a The dyes were provided by Dystar Thai Ltd., and not further purified. The absorption maxima and molar extinction coefficients were determined in Na-K phosphate buffer (50 mM; pH 7.5).