Effects of Some Metals on Carbonic Anhydrase from Brains of Rainbow Trout

Abstract

Carbonic anhydrase (CA) enzyme was purified from rainbow trout brain by Sepharose-4B-L: -tyrosine-sulfanilamide affinity chromatography. The enzyme was obtained with a specific activity of 2,275 EU mg(-1) and a yield of 22.5%. The sample obtained from the affinity column was used for kinetic properties and inhibition studies. Both optimum and stable pH were found as 9.0 in 1 M Tris-SO(4) at 4 degrees C, respectively. To check the purity and subunit molecular weight of enzyme, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed, and MW was found as approximately 29.0 kDa. The molecular weight of native enzyme was estimated to be approximately 27.3 kDa by gel filtration chromatography. The purified enzyme had apparent K (m),V (max), and k (cat) as follows: 0.92 mM, 0.207 micromol.min(-1) and 43.6 s(-1) for p-nitrophenylacetate. The inhibitory effects of Co(II), Cu(II), Zn(II), Ag(I), and Cd(II) on CA enzyme activity were determined using the esterase method under in vitro conditions at low concentrations of the corresponding metals. The obtained IC(50) values, which cause 50% inhibition on in vitro enzyme activity, were 0.05, 30, 0.31, 159, and 82.5 mM for cobalt, copper, zinc, silver, and cadmium, respectively. K ( i ) values were also calculated from Linewaever-Burk plots for these substances as 0.014, 27.68, 2.15, 193.86, and 94.18 for cobalt, copper, zinc, silver, and cadmium, respectively; it was determined that cobalt, silver and cadmium inhibited the enzyme competitively, copper inhibited noncompetitively while zinc inhibited the enzyme uncompetitively.

Purification of carbonic anhydrase-II from sheep liver and inhibitory effects of some heavy metals on enzyme activity

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