Abstract: In pH 4.8 acetate buffer solution, o-phenylenediamine aggregated itself to particles in size of 380 nm, that exhibited five Rayleigh scattering peaks at 392, 420, 445, 484 and 507 nm. Horseradish peroxidase(HRP) has a strong catalytic effect on the H₂O₂ oxidation of o-phenylenediamine to 2,3-diaminophenazine, that caused significantly decreasing of the Rayleigh scattering intensity at 420, 445 and 484 nm. Under the optimal condition, the concentration of HRP in the range of 8.3×10^-12—4.17×10^-10 g/mL was all proportional to the decreased Rayleigh scattering intensity at 445 and 484 nm. Its regression equation, correlation coefficient and the detection limit were ΔI_{445 nm}=2.23c+11, ΔI_{484 nm}=1.47c+4.8; 0.9982, 0.9919; 3.6×10^-12 g/mL and 5.4×10^-12 g/mL HRP, respectively. A new enzyme catalytic-Rayleigh scattering spectral method was developed for assay of HRP, with a high sensitivity and selectivity, simplicity and rapidity. This assay was applied to the determination of HRP with satisfactory results.

Key words: o-Phenylenediamine, Horseradish peroxidase, Hydrogen peroxide, Rayleigh scattering assay