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

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.