Abstract
The enzyme uroporphyrinogen III synthase catalyzes a remarkable reaction in which preuroporphyrinogen is rearranged and cyclized to yield uroporphyrinogen III. Uroporphyrinogen III is the common precursor for hemes, chlorophylls, vitamin B12, and all other tetrapyrroles. This chapter presents coupled assay for uroporphyrinogen III synthase. In the method presented in this chapter, preuroporphyrinogen is generated in situ from porphobilinogen by the action of purified porphobilinogen deaminase and the amount of uroporphyrinogen III formed by the synthase from the preuroporphyrinogen is then quantified, against the unavoidable formation of uroporphyrinogen. This is achieved by spectrophotometric or fluorometric analysis of uroporphyrins I and III, the formation of which is validated by high-performance liquid chromatography separation of the isomers. The method is particularly useful during enzyme purifications in which large numbers of samples need to be assayed rapidly. The assay method has also been adapted to measure enzyme levels in blood and other tissues.
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