Purification and characterization of animal porphobilinogen synthases. I. Bovine liver porphobilinogen synthase.
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 (dodecylsulfate gel electrophoresis); the preparation of aminoethylated subunit is described. According to the above-mentioned molecular weight and to the above-mentioned molecular weight and to the quantitative amino acid analysis after total hydrolysis, the following compositon of the enzymes subunit was calculated ASX23-25 Thr7 Ser23-24 Glx29-31 Pro22-23 Gly22-24 Ala36-37 Val23-26 Met7 Ile9 Leu34-35 Tyr10 Phe11-12 Lys11-12 Cys6-7 His6-8 Arg22 Trp1-2. The subunits, having two free sulfhydryl groups, therefore consists 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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003619:Dansyl Compounds; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D000623:Porphobilinogen Synthase
D000596:Amino Acids; D003619:Dansyl Compounds; D046911:Macromolecular Substances; D010446:Peptide Fragments; D000623:Porphobilinogen Synthase