Carbonic Anhydrase: Affinity Purification and Kinetic Properties from Rainbow Trout Lens

Kinetic behaviour and some properties of carbonic anhydrase (CA) enzyme purified from lenses of rainbow trout (Oncorhynchus mykiss) were described. The purification procedure was composed of homogenate preparation and Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. Yield and specific activity of enzyme were approximately 60% and 17 EU/mg proteins respectively. The overall purification was approximately 900-fold. To check the purity of enzyme, SDS polyacrylamide gel electrophoresis was performed, which showed a single band. The molecular mass of native enzyme was estimated to be 27 kDa by gel filtration column chromatography. pH optimum, stable pH and optimum temperature of the enzyme were 9.5 in 0.025 M boric acid buffer, 7.2 in 0.025 M Tris-SO₄ buffer and 22.5°C, respectively. K_m, which is Michaelis constant, and V_max, which is the maximum rate of the enzyme, values were determined as 5.10 mM and 1.31 μmol/mg protein x minute. The inhibitor effect of sulfanilamide was also examined. K_i, which is the inhibition constant, value was also calculated as 0.757±0.01x10⁻⁵ mM for sulfanilamide and determined that sulfanilamide inhibits the enzyme in a noncompetitively manner.