Determination of Catalase Activity in Human Serum by Fading Spectrophotometry Based on Fenton Reagent-Methylene Blue System

Based on Fenton reagent (H₂O₂/Fe²⁺)-methylene blue (MB) system, a method for the determination of catalase (CAT) activity in human serum by fading spectrophotometry was proposed. The sample (10 μL) was taken, and added into 3.00 mL of 1 mmol·L^-1 H₂O₂ base solution. The reaction was conducted at 30 ℃ for 15 min, and 1.00 mL of 1.00 mol·L^-1 H₂SO₄ solution was added to terminate the enzymatic reaction. After oscillation well, 5.00 mL of 0.10 mmol·L^-1 MB solution and 1.00 mL of 0.01 mol·L^-1 FeSO₄ solution were added successively. The reaction was performed at room temperature for 5 min, and its volume was made up to 50 mL with water. The absorbance of the system at the wavelength of 665 nm was measured with water as reference, meanwhile the absorbance of the CAT blank system was measured, and the difference (△A) was used to calculate CAT activity. It was shown that the reductive dye MB had a maximum absorption at 665 nm with molar absorptivity of 9.5×10⁴L·mol^-1·cm^-1. The oxidation between MB and H₂O₂ could take place with Fe²⁺ as catalyst, and inhibited by CAT. Linear relationship between the values of CAT activity and △A was kept in the range of 0.01-0.06 U·mL^-1, with detection limit (3s/k) of 0.002 4 U·mL^-1. This method was applied to the analysis of 6 serum samples of healthy people at different age groups, giving results in the range of 36.3-63.2 U·mL^-1, and the CAT activity in serums of the children and elderly were significantly higher than that of the adults. RSDs (n=6) of the determined values were in the range of 1.7%-3.6%, and the spiked recoveries ranged from 92.0% to 104%.