The reactions of catalase in the presence of the notatin system

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Abstract
With H2O2 continuously generated by the notatin system, an absorption band beyond 653 m[mu] in the spectrum of the primary catalase H2O2 complex (complex I) was found and this completed the spectroscopic analogy between catalase-peroxide and peroxi-dase-peroxide complexes in which this type of absorption band had previously been observed by visual spectroscopy to lie at 670 m[mu]. In the presence of the notatin system, the Soret band of the primary catalase-H2O2 complex was measured in the ordinary spectrophotometer and agreed substantially with that obtained previously by using the flow method and a soln. of (bottle) H2O2. The change of millimolecular extinction coeff. from catalase to catalase-H2O2 I at 405 m[mu] was 48 for horse-erythrocyte catalase which agreed to within the exptl. error with the value for a horse-liver catalase. On comparison with the value for catalase-methyl-H2O2 I, the composition of catalase-H2O2 I was 1.0 [plus or minus] 0.1 catalase hematins bound to H2O2 in the primary complex. With the notatin system, the conversion of the primary catalase-H2O2 complex into an inactive form (complex II) was observed. This complex had visible absorption bands at 536 and 572 m[mu], and was analogous to the inactive secondary catalase-alkyl-H2O2 complexes except that the number of catalase hematins bound as complex II varied with the exptl. conditions. Complex II was formed in the presence of H2O2 produced by the autoxidation of ascorbic acid or by the notatin system. The catalase-ascorbic acid compound of Lemberg and Foulkes (1948) was probably complex II. A small amt. of complex II was observed spectroscopically by direct addition of a soln. of H2O2 to dilute catalase. The rate of formation of complex II increased with an increase in the steady-state H2O2 concn. and with a decrease in pH. As determined by the rate of destruction of H2O2, the activity of catalase partially converted into complex II was proportional to the number of free hematins. At pH 6.5 about half the catalase hematins were found as complex II and at pH 3.6 nearly all were, under the particular exptl. conditions. The formation of complex II was responsible for the decrease of catalase activity during the usual Kat. f. determination and for the rapid decrease of catalase activity in acid solns. The steady-state concn. of H2O2 in the presence of catalase and the notatin system was very small. Under particular exptl. conditions, the H2O2 concn. was calculated to be of the order of 10-9 [image]. The very rapid reaction of catalase-bound methyl-H2O2 I with H2O could be studied in greater detail when H2O2 was continuously generated by the notatin system. The velocity constant for this reaction was of the same order as that of cata-lase-H2O2 I with H2O2 (3.5 x 107 [image] -1sec.-1). In this reaction H2O2 acted as an acceptor as in peroxidatic reactions of complex I with ethanol, but was about 30,000 times as active as ethanol.