Effects of Peroxide, Catalase, and Hematin in the Assay of Liver Tryptophan Pyrrolase.

Continuous assay of the soluble and inducible tryptophan pyrrolase (L-tryptophan : Hz02 oxidoreductase, EC 1.11.1.4) by direct spectrophotometry in the particle-free supcrnatant fraction of liver (1) would be a desirable alternative to t,he usual assay in whole liver homogenates (2) for studies of the adaptive variations of the concentration of this enzyme, but it is uncertain whether the total enzyme has yet been measured in these preparations. One of the two known reversibly inactive forms of the enzyme was identified in such soluble fractions. This was the apoenzyme, which was apparently supplied with hematin leached from the formed elements in homogenates, and in soluble preparations was activated by addition of hematin, without added reduction (1, 3). In whole homogenates, reduction by critical amounts of hydrogen peroxide increased the rate of the tryptophan pyrrolase reaction (4-8) by activating a second reversibly inactive form of the enzyme. Peroxide and also ascorbic acid reduce t,he inactive ferriporphyrin form of the enzyme that is present in partially purified preparations to the active ferroporphyrin form (9). The combination of added hematin plusreduction was essential to activate a highly purified apotryptophan pyrrolase from liver (lo), but the combination of these two different activators has not been tested in soluble extracts that would be suitable for measuring the total enzyme from liver. The present experiments have identified the active and both inactive forms of tryptophan pyrrolase in soluble liver preparations and have defined the conditions for their assay. The rate of the tryptophan pyrrolasc reaction in preparations from normal liver was more than doubled by either reduction with peroxide or by addition of hematin, and increased more than the sum of these separate effects with the two activators in combination. -1scorbic acid was a less effective reductant. The critical steady state concentration of hydrogen peroxide for optimal reduction was a calculat,ed concentration of lo-r0 M, maintained by generating it at a particular rate relative to the catalase content. of the reaction mixture. Thus, endogenous or added peroxide, hematin, and catalase were identified as the important variables determining whether all or some of the three known