Two-step purification of Cu,Zn-superoxide dismutase from pumpkin (Cucurbita moschata) pulp

Copper and zinc containing superoxide dismutase (Cu,Zn-SOD) from pumpkin (Cucurbita moschata) stringy pulp was purified in two steps. The process included the combination of heat treatment (55 degrees C, 45 min) with ammonium sulfate (10%) precipitation and immobilized metal affinity chromatography, yielding a 87-fold purification (activity recovery of 42%) and a single band upon SDS-PAGE. The enzyme had a molecular mass of 35.0 kDa, as determined by gel filtration chromatography and 2884 U mg(-1) protein-specific activity. It was a dimeric enzyme with two identical subunits of 17,477 Da, as measured using MicrOTOF-Q mass spectrometer and each subunit had a Cu and Zn element. Pumpkin pulp Cu,Zn-SOD is a novel glycosylated enzyme, which possesses higher molecular mass and wider range of pH dependence of the activity than other plant Cu,Zn-SOD. This naturally glycoprotein remained its activity at pH 4-12 at 25 degrees C for 2 h and the enzyme remained its activity lower than 50 degrees C at pH 7.8 for 45 min. (C) 2011 Elsevier B.V. All rights reserved.