Purification and some kinetic properties of carbonic anhydrase from rainbow trout (Oncorhynchus mykiss) liver and metal inhibition.
In the present study, carbonic anhydrase (CA) enzyme was purified from rainbow trout (RT) liver with a specific activity of 4318 EUxmg(-1) and a yield of 38% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 2260-fold. To check the purity and determine subunit molecular weight of enzyme, SDS-polyacrylamide gel electrophoresis was performed, which showed a single band and MW of approx. 29.4 kDa. The molecular weight of native enzyme was estimated to be approx. 31 kDa by Sephadex-G 200 gel filtration chromatography. Optimum and stable pH were determined as 9.0 in 1 M Tris-SO(4) buffer and 8.5 in 1 M Tris-SO(4) buffer at 4 degrees C, respectively. The optimum temperature, activation energy (E(a)), activation enthalpy ((DeltaH) and Q(10) from Arrhenius plot for the RT liver CA were 40 degrees C, 2.88 kcal/mol, 2.288 kcal/mol and 1.53, respectively. The purified enzyme had an apparent K(m) and V(max) of 0.66 mM and 0.126 micromol x min(-1) for 4-nitrophenylacetate, respectively. K(cat) of the CA was found to be 32.8 s(-1). The inhibitory effects of low concentrations of different metals (Co(II), Cu(II), Zn(II) and Ag(I)) on CA activity were determined using the esterase method under in vitro conditions. The obtained IC(50) values, 50% inhibition of in vitro enzyme activity, were 0.03 mM for cobalt, 30 mM for copper, 47.1 mM for zinc and 0.01 mM for silver. K(i) values for these substances were also calculated from Linewaever-Burk plots as 0.050 mM for cobalt, 1.950 mM for copper, 7.035 mM for zinc and 2.190 mM for silver respectively and determined that cobalt and zinc inhibit the enzyme a competitive manner and copper and silver inhibit the enzyme in an uncompetitive manner.
Atatürk University, Faculty of Science and Arts, Department of Chemistry, Turkey.
Hakan
H
Soyut
Sükrü
S
Beydemir
D000818:Animals; D002257:Carbonic Anhydrase Inhibitors / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008670:Metals / Q000378:metabolism*; D008970:Molecular Weight; D017686:Oncorhynchus mykiss / Q000378:metabolism*
D002257:Carbonic Anhydrase Inhibitors; D008670:Metals; D002256:Carbonic Anhydrases