Purification and characterization of animal porphobilinogen synthases. I. Bovine liver porphobilinogen synthase

Article abstract of DOI:10.1515/bchm2.1977.358.2.1081

Porphobilinogen synthase was purified from ox liver by ammonium sulfate fractionation, heat denaturation and column chromatography (purification: 400-fold; specific activity 4.72 nkat). The molecular weight of the native enzyme obtained by thin-layer gel filtration is about 280 000. Using 8M urea in the presence of dithiothreitol as reducing agent, the molecule breaks down into 8 subunits of molecular weight 36 000 (dodecyl-sulfate gel electrophoresis); the preparation of aminoethylated subunit is described. According to the above-mentioned molecular weight and to the quantitative amino acid analysis after total hydrolysis, the following composition of the enzyme sbunit was calculated Asx_23-24 Thr₇ Ser_23-24 Glx_29-31 Pro_22-23 Gly_22-24 Ala_36-37 Val_23-26 Met₇ Ile₉ Leu_34-35 Tyr₁₀ Phe_11-12 Lys_11-12 Cys_6-7 His_6-8 Arg₂₂ Trp_1-2. The subunits, having two free sulfhydryl groups, therefore consist of a chain of about 306 amino acids. The Dansyl-Edman procedure did not enable identification of any free N-terminal amino acid. The acyl group blocking the N-terminus is an acetyl group. It was identified, after hydrazinolysis of the enzyme, by means of chromatographic comparison with 1-formyl-2-dansyl-hydrazine and 1-acetyl-2-dansylhydrazine, whose syntheses and UV spectra are described.

Full text of DOI:10.1515/bchm2.1977.358.2.1081

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