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δ-Aminolevulinate dehyratase(EC 4.2.1.24) was purified from bovine liver by employing affinity chromatographic methods. Sequential chromatography on DEAE-cellulose, octyl-Sepharose, phenyl-Sepharose, and Sephadex G-200 gel filtration, after treating the homogenate at 64℃ for 10 minutes and precipitating the active substances by 35∼45% saturation with aminoium sulfate resulted in 620 fold purification with 20% yield. Purification by the chromatography on octyl- and phenyl-Sepharose exploiting unique hydrophobic properties of the enzyme increased the specific activity further. The final purified enzyme had a specifid activity 21.1 unit/㎎ of protein. It appeared homogeneous by polyacrylamide disc gel electrophoresis which showed single antigenicity by two-dimensional immunoelectrophresis on agarose gel plate containing rabbit antiserum. The molecular weight of the native enzyme was estimated to be 281,800 by method of polyacrylamide disc gel electrophoresis. The enzyme was dissociated into a single sulunit of approximately Mr: 35,000 by treatment of sodium dodecyl sulfate polyacrylamide disc gel electrophoresis indicating that the enzyme was composed of eight apparently identical subunits. The enzyme had a optimum pH of 6.5∼7.0 for the activity, an isoelectric point of 5.85, and Km of 0.73 mM and V_max of 23.8 unit/㎎ of protein. The enzyme was quite resistant to heat showing the 50% inactivation at 15 minutes when treated at 65 and 70℃ and 3 mintues when treated at 75℃. The purified enzyme did not show any activity unless thiol reagents were added in sufficient quantities. 1,4-dithiothreitol in a concentration as low as 0.5 mM wasa more potent than β-mercaptoethanol in activating the purified enzyme. β-mercaptoethanol, however, induced the greater activation by higher concentrations within a range of 10 to 200 mM than that by 1,4-dithiothreitol.
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