Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties
1. 1. 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. 2....
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| Autores principales: | , , , |
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| Formato: | JOUR |
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| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_0020711X_v17_n4_p485_Fumagalli |
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| Sumario: | 1. 1. 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. 2. 2. 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. 3. 3. Uroporphyrinogens formation was linear with both protein and time over a wide range of enzyme concentration and up to 2 h. 4. 4. 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. 5. 5. 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. 6. 6. 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. © 1985. |
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