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: Fumagalli, S.A., Kotler, M.L., Rossetti, M.V., Del C. Batlle, A.M.
Formato: JOUR
Materias:
porphobilinogen deaminase
uroporphyrinogen
blood and hemopoietic system
erythrocyte
human
human cell
1-Butanol
Ammonia-Lyases
Ammonium Sulfate
Butanols
Chloroform
Chromatography
Drug Stability
Erythrocytes
Human
Hydrogen-Ion Concentration
Hydroxymethylbilane Synthase
Kinetics
Magnesium
Magnesium Chloride
Molecular Weight
Oxygen
Sodium Chloride
Support, Non-U.S. Gov't
Temperature
Uroporphyrinogens
Acceso en línea:http://hdl.handle.net/20.500.12110/paper_0020711X_v17_n4_p485_Fumagalli
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Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires
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spelling todo:paper_0020711X_v17_n4_p485_Fumagalli2023-10-03T14:17:49Z Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties Fumagalli, S.A. Kotler, M.L. Rossetti, M.V. Del C. Batlle, A.M. porphobilinogen deaminase uroporphyrinogen blood and hemopoietic system erythrocyte human human cell 1-Butanol Ammonia-Lyases Ammonium Sulfate Butanols Chloroform Chromatography Drug Stability Erythrocytes Human Hydrogen-Ion Concentration Hydroxymethylbilane Synthase Kinetics Magnesium Magnesium Chloride Molecular Weight Oxygen Sodium Chloride Support, Non-U.S. Gov't Temperature Uroporphyrinogens 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. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_0020711X_v17_n4_p485_Fumagalli
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-134
collection Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic porphobilinogen deaminase
uroporphyrinogen
blood and hemopoietic system
erythrocyte
human
human cell
1-Butanol
Ammonia-Lyases
Ammonium Sulfate
Butanols
Chloroform
Chromatography
Drug Stability
Erythrocytes
Human
Hydrogen-Ion Concentration
Hydroxymethylbilane Synthase
Kinetics
Magnesium
Magnesium Chloride
Molecular Weight
Oxygen
Sodium Chloride
Support, Non-U.S. Gov't
Temperature
Uroporphyrinogens
spellingShingle porphobilinogen deaminase
uroporphyrinogen
blood and hemopoietic system
erythrocyte
human
human cell
1-Butanol
Ammonia-Lyases
Ammonium Sulfate
Butanols
Chloroform
Chromatography
Drug Stability
Erythrocytes
Human
Hydrogen-Ion Concentration
Hydroxymethylbilane Synthase
Kinetics
Magnesium
Magnesium Chloride
Molecular Weight
Oxygen
Sodium Chloride
Support, Non-U.S. Gov't
Temperature
Uroporphyrinogens
Fumagalli, S.A.
Kotler, M.L.
Rossetti, M.V.
Del C. Batlle, A.M.
Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties
topic_facet porphobilinogen deaminase
uroporphyrinogen
blood and hemopoietic system
erythrocyte
human
human cell
1-Butanol
Ammonia-Lyases
Ammonium Sulfate
Butanols
Chloroform
Chromatography
Drug Stability
Erythrocytes
Human
Hydrogen-Ion Concentration
Hydroxymethylbilane Synthase
Kinetics
Magnesium
Magnesium Chloride
Molecular Weight
Oxygen
Sodium Chloride
Support, Non-U.S. Gov't
Temperature
Uroporphyrinogens
description 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.
format JOUR
author Fumagalli, S.A.
Kotler, M.L.
Rossetti, M.V.
Del C. Batlle, A.M.
author_facet Fumagalli, S.A.
Kotler, M.L.
Rossetti, M.V.
Del C. Batlle, A.M.
author_sort Fumagalli, S.A.
title Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties
title_short Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties
title_full Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties
title_fullStr Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties
title_full_unstemmed Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties
title_sort human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties
url http://hdl.handle.net/20.500.12110/paper_0020711X_v17_n4_p485_Fumagalli
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