Dismutation properties of purified and GDA modified CuZnSOD from chicken heart.
Superoxide dismutase (SOD, 1.15.1.1) from chicken heart has been purified 139-fold with specific activity of 2130 IU/mg. Purified SOD has a molecular weight 31.0 +/- 1.0 kDa and is composed of two equally sized subunits each having 1.1 +/- 0.03 and 0.97 +/- 0.02 atoms of Cu and Zn elements, respectively. Purified CuZnSOD modified by covalent attachment of the glutaraldehyde (GDA) in presence and absence of bovine serum albumin (BSA). The optimum conditions were obtained with a series of modification reactions as 0.25 mg/mL CuZnSOD in 50 mM phosphate buffer, pH 7.5 containing 3% GDA in presence and absence of 0.25 mg/mL BSA. The highest recovery activity of modified SODs was determined as 23.4 and 18.5% for the designated SOD-I and SOD-II derivatives, respectively. The recovery activity of SOD-I reached 28.6% while SOD-II didn't change significantly and determined as 19% after the reaction with 1% ethylendiamine. The activity variations of native and modified CuZnSODs were investigated depending on the pH and temperature. Optimum pH values for native and modified SOD-I, -II were determined as 8.8, 8.3, and 8.2, respectively. The native and modified SODs have the same optimum temperatures approximately as 35 degrees C. The pH- and thermal-stability properties of modified SODs were found to be better than native SOD, in the pH range of 6.5-8.5 at 25 degrees C after 6 h, and up to 40 degrees C at pH 7.4 after 3 h incubation period. Inhibitory effects of ditiothreitol (DTT), beta-mercaptoethanol, and iodoacetamide were not observed on the native and modified SODs activities after 5 h incubation period. Phenylmethylsulfonylfloride (PMSF), H20O2, and EDTA were caused by slight inhibition on the enzyme activities.
Demirel
L Arzu Bozkaya
LA
Faculty of Education, Department of Chemistry, University of Dokuz Eylül, Buca, Izmir, Turkey.
Tarhan
Leman
L
D000818:Animals; D002417:Cattle; D002645:Chickens; D003300:Copper / Q000737:chemistry; D004795:Enzyme Stability; D005976:Glutaral / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D009206:Myocardium / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000302:isolation & purification
D008345:Manganese; D003300:Copper; D013482:Superoxide Dismutase; D005976:Glutaral