UROPORPHYRINOGEN-I SYNTHASE FROM HUMAN-ERYTHROCYTES - SEPARATION, PURIFICATION, AND PROPERTIES OF ISOENZYMES

Uroporphyrinogen I synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] from human erythrocytes was separated into two active protein peaks (A and B) on DEAE-cellulose, by ammonium sulfate fractionation, on Sephadex G-100, and on DEAE-Sephadex A-50 with a NaCl gradient. The final purification was 613 and 743 times for A and B, respectively. The corresponding yields were 2.2 and 3.4%. Fraction A was separated further into two (A 1 and A 2) active protein bands and fraction B into three (B 1, B 2, and B 3) on analytical polyacrylamide disc gel electrophoresis. Bands A 1 and A 2 were identical with B 1 and B 2; B 3 represented a third isoenzyme. Molecular weights (mean ± SEM), measured by gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, were 38,000 ± 1000 for B 1 and 40,000 ± 1000 for B 2 and B 3. Isoelectric focusing on 4% polyacrylamide gel separated both fractions A and B into three active protein bands. Maximal activity of the enzyme was found in gel cuts (5-mm) at pH 5.6 for both fractions A and B.