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สารฮิ วมิ กช่วยเพิ่ มความสามารถการลดสี ... 202 The Journal of the Royal Institute of Thailand Vol. 37 No. 1 Jan.-Mar. 2012 started by the addition of AQDS (final concentration = 0.25 mM) form anaerobic stock solution into reaction mixture. The concentrations of reduced AQDS (AH2QDS) were de- termined spectrophotometrically at 450 nm. After 10 h of incubation, the cells of strain A5 were removed form suspension by filtration through a 0.2-µm-pore diameter filter under anaerobic atmosphere. The resulting supernatant was then transferred into 1 cm disposable plastic cuvettes. The chemical reduction of azo dyes by reduced AQDS was started by the addition of anaerobic azo dyes solutions (final concentration = 0.1 mM) into cell-free suspension. The reduction of azo dyes was determined spectrophoto-metrically at λ max of each azo dye for 30 min (using 30 sec measuring intervals). Determination of quinone component(s) in plasma membrane Paenibacillus sp. strain A5 cells were grown either aerobically or anaerobically in 100 ml. volumes. Cells were harvested by centrifugation for 10 min at 10,000 x g after the cultures reached an OD 600 ≈ 2.0. Pellets were resuspended in 6 ml chloroform:methanol (2:1, v/v) and the suspension was gently mixed overnight in complete darkness at room temperature. The suspension was filtered though Whatman no. 1 filter paper and filtrate was evaporated under reduced pressure. The residue was resuspended in ethyl acetate, and solution was spot- ted on Silica Gel 60 F254 aluminum-packed TLC plates (E. Merck, Germany). Menaquinone (MK-1) and ubiquinone (Q1) were used as standards. Samples were eluted with a mixture of n-hexane and diethyl ether (85:15, v/v). Quinones were detected on TLC plates by brief irradiation with short-wave ultraviolet light (33). Analytical techniques The concentration of sulfonated azo dyes was determined spectrophotometrically (UV WINLAB, Perkin Elmer). Sulfonated azo dyes and the corresponding reduction products were analyzed by high performance liquid chromatography (HPLC; Shimadzu model LC-3A chromatograph (Shimadzu Corp., Kyoto, Japan) equipped with Shimadzu model SPD-2A detector). A reverse-phase column was Pegasil ODS, (4.6 mm x 150 mm [inside diameter] column, Senshu Scientific Co., Ltd., Tokyo, Japan). A mobile phase composed of 50%