51] Purification of porphobilinogen synthase from bovine liver

Abstract

This chapter describes the Purification of porphobilinogen synthase from bovine liver. Porphobilinogen synthase (5-aminolevulinic acid dehydratase, EC 4.2.1.24), the second enzyme of the tetrapyrrole biosynthesis pathway, catalyzes the condensation between two molecules of 5-aminolevulinic acid to form the pyrrole porphobilinogen. The relatively high level of 5- aminolevulinic acid dehydratase in bovine liver has made this source particularly convenient for the isolation of enzyme for the large scale enzymatic preparation of porphobilinogen. Also, the near neutral pH optimum of the bovine enzyme makes it more attractive than the bacterial dehydratase (pH optimum 8.4) for coupling with 5-aminolevulinic acid synthetase in the preparation of stereospecifically labeled porphobilinogen. For the assay of the enzyme, the product of the enzymic reaction, porphobilinogen, is estimated using modified Ehrlich's reagent. Since the reaction of pyrroles with Ehrlich's reagent is affected by the presence of thiols, dithioerythritol, used to stabilize the enzyme, is removed prior to the addition of the reagent by precipitation with HgCl2. 5-Aminolevulinic acid dehydratase (500 units) was incubated at 37° for 15 min in 5 ml 0.1 M potassium phosphate buffer (pH 6.8) containing 10 mM dithioerythritol and added to 2 litres 10 mM potassium phosphate buffer containing 10 mM dithioerythritol. The enzyme showed a single but broad protein band (mobility 0.38) on staining with Coomassie Brilliant Blue after electrophoresis on 5% polyacrylamide gels. Assay of a duplicate gel revealed enzyme activity coincident with the protein band. Enzyme which had been S-carboxymethylated to mask all thiol groups showed a single sharp protein band on gels suggesting that during electrophoresis of the native enzyme several closely related partially oxidized species are present.