A rapid direct assay for uroporphyrinogen III cosynthetase

Abstract

Uroporphyrinogen III (3) the precursor of heme, chlorophyll and vitamin Brz is formed from porphobilinogen by the action of two enzymes, porphobilinogen deaminase and uroporphyrinogen III cosynthetase [4]. In the absence of cosynthetase only the uroporphyrinogen I isomer (4) is the formed. Until recently little was known about the individual role played by the two enzymes which were generally thought to function in a complex without the liberation of intermediates between porphobilinogen and uroporphyrinogen III. Consequently uroporphyrinogen III cosynthetase has traditionally been assayed indirectly by the estimation of the isomer ratio between uroporphyrinogen I and III formed, a complex procedure which has greatly hampered the purification and study of the enzyme. Originally uroporphyrin I and III methyl esters were separated by paper chromatography or cellulose thin-layer chromatography (TLC) [5] with unreliable results because of the physical interaction between the two isomers [6]. A more reliable but time consuming separation was accomplished by decarboxylating the uroporphyrins to coproporphyrins which could then be separated and quantified as methyl esters by TLC [7] or high pressure liquid chromatography (HPLC) [8]. The direct separation method of uroporphyrin I and III dimethyl esters [3,9] was a recent important advance which has made the practical routine analysis of cosynthetase more feasible. This latter method has been used by us in the purification and examination of uroporphyrinogen III cosynthetase from Rhodopseudomonus spheroides [lo]. Although this procedure is a major improvement in both speed and accuracy, it still involves esterification, extraction, TLC and HPLC, leaving a requirement for a rapid