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ORIGINAL ARTICLE
Year : 2018  |  Volume : 15  |  Issue : 4  |  Page : 349-356

Human carbonic anhydrase: Purification and characterization study in thalassemia major patients compared to healthy subjects


Department of Chemistry, College of Science, Kirkuk University, Kirkuk, Iraq

Correspondence Address:
Israa Ghassan Zainal
Department of Chemistry, College of Science, Kirkuk University, Kirkuk
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/MJBL.MJBL_81_18

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Background: Carbonic anhydrase(CA) catalyzes the reversible reaction of converting carbon dioxide to bicarbonate. Objective: This study was aimed to isolate and purify human erythrocytes CA and study its physicochemical properties of the enzyme reaction for ß-thalassemia major patients. Materials and Methods: The blood samples included 61samples of blood(31males and 30females) from ß-thalassemia patients visited Azadi Hospital/Kirkuk city. Healthy individuals as control group included 40 participants. The separated fractions were obtained using four steps: extraction by ethanol and chloroform, ammonium sulfate precipitation, dialysis, and gel filtration chromatography; finally, the CA was analyzed by polyacrylamide gel electrophoresis. Results: The CA activity showed significant(P≤0.05) decrease, total protein showed nonsignificant(P≥0.05) increase, and specific activity significantly(P≤0.05) increased in patients group compared to healthy individuals. CA was partially purified with a factor of 22.5 and 18 by extraction with ethanol and chloroform and 1.5,1.4 for Fraction I and 1,2 for Fraction II using gel filtration chromatography. The optimum conditions for the CA reaction in patients group were enzyme concentration(6 μl), substrate concentration(6 Mm), pH=7.4, and temperature 37°C. The electrophoresis study indicated that the bands of CA in patients group showed bands with less intensity than the bands in healthy individuals. Conclusion: The best method to purify CA from human erythrocytes with high recovery and fold of purification was ethanol–chloroform extraction.


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