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

Abstract EN

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 61 samples of blood (31 males and 30 females) from ßhalassemia 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.