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    中性红褪色光度法测定白菜中过氧化氢酶活性

    Determination of Catalase Activity in Chinese Cabbage by Neutral Red Discoloration Spectrophotometry

    • 摘要: 建立了一种中性红(NR)褪色光度法测定过氧化氢酶(CAT)活性的方法。称取20 g本地大白菜样品,研碎,用磷酸盐缓冲溶液(pH 7.0)定容至1 000.0 mL,取稀释液1.00 mL于50 mL容量瓶中,再加入1 mmol·L-1 H2O2基质溶液5.00 mL,在25℃下进行酶催化反应15 min后,立即加入0.01 mol·L-1酸性Fe3+溶液(内含0.02 mol·L-1 H2SO4溶液)1.00 mL用于终止酶促反应(H2SO4)和催化褪色反应(Fe3+),再加入5×10-4mol·L-1 NR溶液3.00 mL,摇匀,在80℃水浴中进行褪色反应,25 min后加水定容。取适量样品溶液于1 cm比色皿中,以水为参比,于波长528 nm处测量其吸光度A。同时完成空白试验吸光度A0测定,根据吸光度的变化ΔA(ΔA=A0-A)确定CAT的活性(E)。结果表明:在10 min内,酶促反应符合一级动力学反应的特征,CAT活性E在0.3 U·mL-1内与ΔA呈线性关系,检出限(3S/N)为0.006 8 U·mL-1。对大白菜的叶、茎、根进行加标回收试验,CAT的含量依次为6.25,9.00,8.00 U·g-1,回收率均为104%,测定值的相对标准偏差(n=6)为1.7%~3.4%。将大白菜茎部自然放置4 d后,CAT活性降至最初的30%。

       

      Abstract: A new neutral red discoloration spectrophotometry was developed for the determination of catalase activity. 20 g of local Chinese cabbage sample was taken and grinded. The mixture was made up to 1 000.0 mL with phosphate buffer solution (pH 7.0). 1.00 mL of diluent was taken and placed into a 50 mL volumetric flask in which 5.00 mL of 1 mmol·L-1 H2O2 substrate solution was added, and the mixed solution enzymatically catalyzed at 25℃ for 15 min. After completing the reaction, 1.00 mL of acidic Fe3+ solution containing 0.02 mol·L-1 H2SO4 was added to terminate the enzymatic reaction (H2SO4) and catalyze the fading reaction (Fe3+). 3.00 mL of 5×10-4mol·L-1 NR solution was mixed with the sample solution by shaking. The fading reaction of the mixed solution was performed in a water bath at 80℃ for 25 min. After reaction, water was used to make its volume to 50.0 mL. An appropriate amount of sample solution was taken into a 1 cm cuvette with water as the reference, and its absorbance was measured at 528 nm. The determination of the absorbance A0 of the blank tested solution was measured by the above method, and ΔA(A0-A) was calculated to obtain the activity E of CAT. As shown by the results, the enzymatic reaction was consistent with the characteristics of the first order reaction within 10 minutes. Catalase activity within the range of 0.3 U·mL-1 was linearly correlated to the absorbance change ΔA, with the detection limits (3S/N) of 0.006 8 U·mL-1. The spiked recovery test was made on the leaves, stems, and roots of the Chinese cabbage, giving determined values of 6.25, 9.00, 8.00 U·g-1, and the recovery was found to be 104%, with RSDs (n=6) of the determined values in the range of 1.7%-3.4%. After leaving the stems of Chinese cabbage naturally for four days, the CAT activity dropped to 30% of the original content.

       

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