Purification and some kinetic properties of carbonic anhydrase from rainbow trout (<i>Oncorhynchus mykiss</i>) liver and metal inhibition

SÖYÜT H., Beydemir Ş.

PROTEIN AND PEPTIDE LETTERS, vol.15, no.5, pp.528-535, 2008 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 15 Issue: 5
  • Publication Date: 2008
  • Doi Number: 10.2174/092986608784567627
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.528-535
  • Anadolu University Affiliated: No


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-SO4 buffer and 8.5 in 1 M Tris-SO4 buffer at 4 degrees C, respectively. The optimum temperature, activation energy (E-a), activation enthalpy (Delta H) and Q(10) Q 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 mu mol 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 IC50 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.