Purification by affinity chromatography and properties of uroporphyrinogen I synthetase from Chlorella regularis.
Uroporphyrinogen I synthetase (porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8) from Chlorella regularis was purified to homogeneity by affinity chromatography on porphobilinogen-AH-Sepharose 4B, which was prepared by reacting carbodiimide with substrate, porphobilinogen. The enzyme was purified 232-fold from the initial crude extract and specific activity was 348 nmol porphyrinogen I formed (mg protein)-1 . h-1 at pH 7.4. The molecular weight of the enzyme was 35 000-36 000 as determined by Sephadex G-100 gel filtration. This enzyme was acidic protein having an isoelectric point of 4.2. The enzyme exhibited a single pH optimum at a pH value of 7.4 both in phosphate and Tris-HCl buffer. The Km value for porphobilinogen was 89 microM as measured by its consumption and 85 microM when uroporphyrin formation was used. The Arrhenius plot obtained from the enzyme activity measurements appeared triphasic with breaks occurring at 35 and 46 degrees C and activation energy was calculated to be 21 700 (10-35 degrees C), 12 700 (35-46 degrees C) and 1800 cal . mol-1 (46-65 degrees C). This enzyme was heat stable and the enzyme still retained 87% of activity, even after 1 h incubation at 75 degrees C.
D000596:Amino Acids / Q000032:analysis; D000642:Ammonia-Lyases / Q000302:isolation & purification*; D002708:Chlorella / Q000201:enzymology*; D002846:Chromatography, Affinity; D006358:Hot Temperature; D011163:Hydroxymethylbilane Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000596:Amino Acids; D013439:Sulfhydryl Reagents; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases