Uroporphyrinogen I synthase from human erythrocytes: separation, purification, and properties of isoenzymes.

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Abstract
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 (A1 and A2) active protein bands and fraction B into three (B1, B2, and B3) on analytical polyacrylamide disc gel electrophoresis. Bands A1 and A2 were identical with B1 and B2; B3 represented a third isoenzyme. Molecular weights (mean +/- SEM), measured by gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, were 38,000 +/- 1000 for B1 and 40,000 +/- 1000 for B2 and B3. 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.

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