Purification and characterization of superoxide dismutase from chicken liver


Ozturk-Urek R., Tarhan L.

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, vol.128, no.2, pp.205-212, 2001 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 128 Issue: 2
  • Publication Date: 2001
  • Doi Number: 10.1016/s1096-4959(00)00300-6
  • Journal Name: COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.205-212
  • Keywords: superoxide dismutase, metaloenzyme, chicken liver, purification, pH activity, pH stability, thermal stability, inhibition effect, ionic strength activity, COPPER, ENZYME
  • Dokuz Eylül University Affiliated: Yes

Abstract

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme has been isolated, purified and partially characterized from chicken liver. The following steps were carried out in order to purify chicken liver SOD. Initially, the liver was homogenized and hemoglobin was removed. Subsequently protein precipitation was effected with (NH4)(2)SO4, methanol, (NH4)(2)SO4-methanol and polyethylene glycol methods. The product from polyethylene glycol-3350 precipitation was found to have the highest SOD activity. Polyethylene glycol was removed by chromatography using a PD-10 column. After passing through an ultrafilter, the superoxide dismutase was fractionated by DEAE-ion chromatography and then Sephadex G-75 gel filtration chromatography. During this purification procedure, a specific activity of 4818.2 IU/mg was reached, corresponding to 285.8-fold purification. The purified enzyme, which was characterized as cyanide-sensitive SOD, contained two subunits having Cu and Zn elements with a molecular weight of 16000 +/- 500 for each. The optimum pH of purified CuZnSOD was determined to be 8.9. The enzyme was found to have good pH stability in the pH range 6.0-7.5 at 25 degreesC over a 2-h incubation period and displayed good thermal stability up to 45 degreesC at pH 7.4 over a 1-h incubation period. The SOD enzyme was not inhibited by DTT and beta -mercaptoethanol, but inhibited by CN- and H2O2. In the presence of 2 mM iodoacetamide, the enzyme showed an approximately 40% activity loss. Finally, the inhibitory effect of ionic strength on SOD was also investigated. (C) 2001 Elsevier Science Inc. All rights reserved.