A Spectrophotometric Method for Measuring the Breakdown of Hydrogen Peroxide by Catalase

Several methods have been developed for following the breakdown of hydrogen peroxide catalyzed by catalase, but these either have not been sufficiently quantitative or have not proved rapid enough to yield reliable data during the critical 1st or 2nd minute of the reaction. Chemical procedures in which residual peroxide is titrated with permanganate (l-3) or an excess of permanganate is measured calorimetrically (4) are accurate except for reaction times of less than a minute, although Lemberg and Foulkes (5) developed a micromethod for obtaining data every 10 seconds (see also Ogura et al. (6)). Considerable variability is unavoidable, however, when samples must-be taken at such short intervals. The manometric method for measuring oxygen evolved from the system proved in detailed studies to be unsuited for following the rapid breakdown of peroxide in which a diffusion process across the liquid-air interface becomes limiting. This is manifested by changes in both the order of the reaction and the rate of evolution of oxygen with variations in the rate of agitation of the reaction mixture (7). Direct measurement of hydrogen peroxide by polarography provides good quantitative data during the 1st minute of the reaction which fit first order kinetics (8). However, an elaborate, special, electronic circuit is needed for such measurements. Furthermore, as pointed out by Bonmschen, Chance, and Theorell (8), this method appears to give lower values for catalase activity than do titration techniques. Preliminary experiments for following the breakdown of hydrogen peroxide by observing the decrease in light absorption of peroxide solutions in the ultraviolet were reported by Chance (9) and Chance and Herbert (10). The potentialities of this method have been investigated and a quantitative, spectrophotometric technique for following the breakdown of hydrogen peroxide has been developed for routine studies of catalase kinetics.