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

• Main Authors: Salwa Saleh Hussein, Israa Ghassan Zainal
• Format: Article
• Language: English
• Published: Wolters Kluwer Medknow Publications 2018-01-01
• Series: Medical Journal of Babylon
• Subjects: Carbonic anhydrase; electrophoresis; gel filtration; purification; ß-thalassemia
• Online Access: http://www.medjbabylon.org/article.asp?issn=1812-156X;year=2018;volume=15;issue=4;spage=349;epage=356;aulast=Hussein

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 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.