Helmut SIES lnstitut ff~r Physiologische Chemie und Physikalische Biochemie, der Universittit Mftnchen, Germany and Britton CHANCE Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA Received 25 September 1970 1. Introduction The long study of the catalytic and peroxidatic ac- tivities of catalase has led to little information on its function in vivo, particularly in mammalian systems [1, 2]. In bacteria, e.g. Micrococcus lysodeikticus, spectroscopic evidence indicated that catalase is large- ly saturated with H202 in the form of the primary intermediate, compound I [3]. In mammalian tissue, the correspondence of the order of reaction and the overall flux rates for methanol metabolism in the rab- bit were suggestive of catalase's role in methanol oxi- dation [ 1 ]. Similar conclusions were reached by Tephly, Parks and Mannering [4] for methanol metab- olism in the rat. Heppel and Porterfield [5] found coupled nitrite oxidation in rat liver homogenates, and Portwich and Aebi [6] demonstrated the coupled oxidation of formate in rat liver slices. In the present investigation, spectrophotometric measurements of absorbance in the 660 nm band re- gion of catalase compound I from isolated perfused rat liver have been performed. Experimental evidence which supports an interpretation of changes of the ob- served signal to be due to the catalase system will be * Postal address: Dr. H. Sies, Institut fllr Physiologische Chemie und Physikalische Biochemie, Goethestrasse 33, 8-Miinchen- 15, Germany. presented; it includes the effects of the above-men- tioned hydrogen donors, of hydrogen peroxide gener- ating substance, of oxygen tension, and of inhibitors of the catalase system. 2. Experimental Spectrophotometry of light transmitted through a lobe of perfused liver was performed with the Rapid- spektroskop of Howaldtswerke Deutsche Werf, Kiel, as adapted by Brauser [7]. Dual wavelength absor- bance photometry by sinusoidal wavelength modula- tion [7] was used for more sensitive study of absor- bance changes as a function of time (see also [8]). Bandwidth of monochromatic light was 4 nm. Com- pensation for spectral characteristics of the apparatus was afforded by a reference beam which was reflected from a magnesia surface. In preliminary experiments, the rotating filter ap- paratus of Chance [9] as adapted for absorbancy measurements [ 10] was used with a 660 nm mea- suring filter and a 720 nm reference filter, having bandwidths of + 30 and + 7 nm, respectively. Light transmitted through a liver lobe was collected by a light pipe and was detected by an infrared-guarded silicone diode. Oxygen uptake was followed with Ag-Pt-micro- electrodes inserted into the perfusion circuit before 172 North.Holland Publishing Company - Amsterdam