Purification and characterization of animal porphobilinogen synthases—II. Dog liver porphobilinogen synthase

Abstract

1. 1. A method is described for the purification of porphobilinogen synthase (PBG-S) from dog liver (acetone dry powder; ammonium sulfate fractionation: controlled heat denaturation; ion exchange chromatography; gel filtration). The 911-fold purified enzyme with a specific activity of 372 in U/mg and 6.2 nkat/mg enzyme protein, respectively, appears as a single band in disc electrophoresis. 2. 2. The optimum pH of the enzyme is 6.8; the Michaelis constant K m = 1.77 · 10 −4M with 5-aminolevulinic acid as substrate. 3. 3. The determination of the molecular weight of the native multisubunit enzyme (268,000 dalton) and of the subunit (33,500 dalton) are in favour of an octameric structure. 4. 4. The acyi group blocking the N-terminus has been identified as an acetyl group (hydrazinolysis; dansylation;TLC: HPLC). 5. 5. The amino acid composition of the PBG-S subunit is as follows CH 3CO-NH-(Asx 22Thr 9–10Ser 18Glx 28–29Pro 20–21Gly 22Ala 35–37Val 23–25Met 5 Ile 8–9Leu 32–33Tyr 10Phe 12Lys 12Cys 3HiS 7Arg 22Trp 3)-Ala-Leu-COOH. 6. 6. In the presence of ganidinium chloride only one free sulfhydryl group per subunit (3 cysteine residues) could be detected. 7. 7. The purified enzyme contains 0.56 gram atoms of zinc per octamer (neutron activating analysis).