Figure 5. Fluorescence spectra of catalase exposed to H₂O₂.

The excitation wavelength was 340 nm. The catalase was mixed with NADPH in the ratio of 1.0 nmol of NADPH per nmol of catalase. Excess NADPH was removed by a single ultrafiltration–wash (see the Experimental section). The spectra are of the following in 0.05 M phosphate buffer, pH 6.5, after subtraction of the spectrum of the phosphate buffer: A, 1.0 μM NADPH; B, 1.0 μM bovine liver catalase without exposure to H₂O₂; C, 0.95 μM catalase after 1 h of exposure to H₂O₂ generated by glucose and glucose oxidase; D, 0.95 μM catalase after 1 h of exposure (at a catalase concentration of 7.6 μM) to H₂O₂. For both C and D, the rate of generation of H₂O₂ was 60 nmol·h⁻¹ per nmol of catalase at 37 °C.