Purification and some properties of carbonic anhydrase from Elephas trogontherii (Steppe elephant) bone

Abstract

Four isoenzymes of carbonic anhydrase (CA) were purified from Elephas trogontherii (steppe elephant) bone (approx 0.3-0.5 million years old) from different locations (outer peripheral, cytosolic, inner peripheral and integral) using Sepharose 4B-L-tyrosine sulphanilamide affinity chromatography and their kinetics properties were investigated and compared with known CA isoenzymes. The purification degree of CAs was monitored by SDS-PAGE. Purification fold for outer peripheral, inner peripheral, cytosolic and integral CA was 395.6, 652.8, 1091 and 429.3 and the molecular mass (as determined by gel filtration chromatography) was 37, 36, 35, and 39 kDa, respectively. The optimal temperature for isozymes was 10-20, 30, 30 and 60ºC and optimal pH was between 7.5-11, 7.5-10, 7.5-10 and 7.5 respec-tively. Km values (at optimum pH and 20ºC) for p-nitrophenyl acetate as substrate were 4.83, 6.80, 4.525 and 3.86 mM and the Vmax values for the same substrate were 0.00097, 0.0149, 0.00249 and 0.00072 μmol/L*min, respectively. I₅₀ values of isoenzymes for the inhibitors of CA ̶ sulphanilamide, KSCN, acetazolamide and NaN₃ were also determined.