Mechanisms of Protection of Catalase by NADPH

All incubations were with commercially available, extensively purified enzymes. Sigma was the source of the manganese superoxide dismutase from Escherichia coli. As with the studies of Kono and Fridovich (), manganese superoxide dismutase was used for most of this study because, unlike Cu,Zn superoxide dismutase, it is not inactivated by H₂O₂. Roche Molecular Biochemicals (Mannheim, Germany) was the source of catalase from bovine liver, xanthine oxidase from bovine milk, Cu,Zn superoxide dismutase from bovine erythrocytes, glucose oxidase from Apergillus niger, and glucose-6-phosphate dehydrogenase from yeast. The buffer used for most of this study was 0.1 mm EDTA, 50 mmNa₂HPO₄-KH₂PO₄ buffer, pH 7.4 (Na-K phosphate buffer). A second buffer was KR-Tes, which is a Krebs-Ringer/Tes solution containing, in the following final millimolar concentrations: NaCl 119; KCl 4.7; CaCl₂ 2.5; KH₂PO₄ 1.2; MgSO₄ 1.2; and (sodium) Tes, pH 7.4, 22.6. Crystalline bovine liver catalase was dissolved in KR-Tes, concentrated on CF-25 ultrafiltration cones (Amicon), and then washed on the cones () with the buffer to be used in each experiment. All other enzymes were both dissolved and washed with the buffer to be used in each experiment.

The solution of each enzyme was assayed for protein concentration () and activity. The activity of catalase was determined from the first-order rate constant of the rate of disappearance of H₂O₂, at an initial concentration of 10 mm, as measured by absorbance at 240 nm with a recording spectrophotometer (). By this assay, the bovine liver catalase had an activity of 23.7 s⁻¹ per micromolar catalase concentration (5.9 s⁻¹ per micromolar heme concentration). The rate of formation of H₂O₂ by glucose oxidase in the presence of 5 mm glucose was determined with the assay for H₂O₂ of Green and Hill (). The glucose oxidase catalyzed the generation of H₂O₂ at an average rate of 2,100 μmol min⁻¹ per μmol of glucose oxidase. The rate of generation of O⨪₂ by xanthine oxidase in the presence of xanthine (100 μm) was measured in 50 mm Na-K phosphate buffer, pH 7.4, by the rate of reduction of ferricytochrome c (10 μm) at 550 nm, using the extinction coefficient at 550 nm of 21,000m⁻¹ cm⁻¹, according to the method of McCord and Fridovich (). The replotting method of Sawada and Yamazaki () provided assurance that the spontaneous dismutation reaction was second-order with respect to O⨪₂ and revealed a value of 9.14 × 10⁻⁴m s fork _d/k _c², in whichk _d is the rate constant for the spontaneous dismutation reaction and k _c is the rate constant for the reduction of cytochrome c by O⨪₂. The factork _d/k _c² provided a means for correcting for the small fraction of O⨪₂ that undergoes spontaneous dismutation before the O⨪₂ can reduce cytochrome c. The rates of generation of O⨪₂, as measured by the rate of cytochrome c reduction, were similar whether catalase (2 μm) was present or absent. At pH 7.4 and 37 °C, the xanthine oxidase had an average specific activity of 115 μmol min⁻¹ per μmol of enzyme. The total rate of production of H₂O₂ by xanthine oxidase was determined from the rate of production of urate, as measured by the rate of increase in absorbance at 295 nm (), and was confirmed with the assay for H₂O₂ of Green and Hill () on incubations containing xanthine oxidase, xanthine, and superoxide dismutase. The rate of oxidation of NADPH was determined from the amount of 6-phosphogluconate formed, as described previously (). All reaction mixtures had a final volume of 1.0 ml. All incubations were at pH 7.4 and 37 °C except for one experiment in which the conditions were otherwise stated. The reaction components were at the final concentrations indicated within parentheses when the concentrations were not specified and were as follows: catalase and, when present, xanthine oxidase, xanthine (100 μm), superoxide dismutase, NADP⁺ (2 μm), glucose 6-phosphate (1 mm), glucose-6-phosphate dehydrogenase (10 μg/ml), glucose (5 mm).

For following the kinetics of reactions, readings of absorbance from the spectrophotometer were taken on each group of cuvettes at intervals of 1 min per group and were automatically stored in a computer for later statistical analysis. For obtaining spectra of catalase, absorbance readings were taken and stored at intervals of 1 nm. The absorption spectra were obtained with a Beckman DU-7 spectrophotometer at a recording speed of 1,200 nm min⁻¹. Prof. Peter Nicholls kindly provided the absorbances of equimolar concentrations of ferricatalase (the resting or free form of catalase) and compounds I, II, and III at intervals of 1 nm between 350 and 750 nm. The plots from these absorbances were similar to previously published spectra (,). The spectra for ferricatalase, compound I, and compound II were confirmed in the following manner. One ml of 100 mmpotassium Ches buffer, pH 8.6, was added to 0.5 ml of the bovine liver catalase from the bottle (20 mg/ml) to dissolve all enzyme crystals. The dissolved enzyme was further diluted in 50 mm Na-K phosphate buffer, pH 6.5, to a concentration of about 10 μm. One ml of enzyme preparation was then transferred to a cuvette. After a preincubation period of 5 min at 37 °C, the spectrum of ferricatalase was obtained. After the first spectrum was obtained, 5 μl of 3% peracetic acid were added to the cuvette, and the spectrum was immediately re-determined (compound I). One microliter of 60 mm potassium ferrocyanide was then added to the same reaction mixture, and after 10 min of incubation at 37 °C, the spectrum was determined again (compound II). The absorbances of equimolar concentrations of the four forms of catalase were used to determine the amount of each form in H₂O-treated catalase by a least squares method, as follows. The minimum was found for the sum (from 501 to 750 nm) of the squares of the difference betweenU _n andf _f A _n +f _I B _n +f _II C _n +f _III D _n, in which nis the wavelength, U _n is the absorbance of the treated catalase at wavelength n, andf _f through f _III represent the unknown fractions of the treated catalase that are in the form of ferricatalase, compound I, etc. A _n throughD _n represent the known absorbances of equimolar concentrations of ferricatalase through compound III at wavelengthn. The value for each f was obtained from the solution of the resulting simultaneous, linear equations for thefs. A visual check of the validity of the result was obtained with a spreadsheet/graphing program that allowed a comparison of the observed spectrum with the spectrum from any specified combination of the four forms of catalase. The formation of compound II is usually followed at 435 nm, the isosbestic point between ferricatalase and compound I (). The difference in extinction coefficient between ferricatalase and compound II was considered to be 32 mm⁻¹ cm⁻¹ (). As observed also by Chance (), the extinction coefficient of compound III at 435 nm was found to be similar to that of compound II. The increases in absorbance at 435 nm were therefore considered to represent the combined rate of formation of both compounds.