Human red cell porphobilinogen deaminase. A simpler method of purification and some unusual properties.
A simpler method for purifying human red cell deaminase, using a mixture of n-butanol and chloroform, which denatures hemoglobin, followed by ammonium sulphate fractionation, heat treatment, Sephadex G-100 and DEAE-cellulose chromatography, yielding a 3400 fold purified enzyme is described. Some properties of purified deaminase were studied. The enzyme seems to have a strict requirement for oxygen, neither PBG consumption nor uroporphyrinogens formation were measured under anaerobiosis. Uroporphyrinogens formation was linear with both protein and time over a wide range of enzyme concentration and up to 2 h. The optimum pH was 7.4 and the mol. wt was 40,000 +/- 4000. The enzyme was heat-stable and increased its activity by heating. Ammonium and hydroxylamine ions inhibited the reaction. K+ and Na+ ions did not greatly affect activity, while most divalent cations tested significantly diminished uroporphyrinogen formation and to a lesser degree PBG consumption. Direct plots of velocity against PBG concentration were hyperbolic, however double-reciprocal plots were non-linear, Hill plots gave an n value of 2 and Eadie plots were bell-shaped, indicating the existence of weakly positive cooperative effect between 2 binding sites for PBG per molecule of deaminase.
D020001:1-Butanol; D000642:Ammonia-Lyases / Q000097:blood*; D000645:Ammonium Sulfate; D000440:Butanols; D002725:Chloroform; D002845:Chromatography; D004355:Drug Stability; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011163:Hydroxymethylbilane Synthase / Q000037:antagonists & inhibitors / Q000097:blood*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008970:Molecular Weight; D010100:Oxygen / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014577:Uroporphyrinogens / Q000378:metabolism
D000440:Butanols; D014577:Uroporphyrinogens; D015636:Magnesium Chloride; D012965:Sodium Chloride; D002725:Chloroform; D020001:1-Butanol; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases; D008274:Magnesium; D010100:Oxygen; D000645:Ammonium Sulfate