A preparative size-exclusion chromatography (SEC) column (500 X 10 mm) packed with regenerated cellulose gel particles was used for the separation of superoxide dismutase (SOD) from pig blood. The fraction F-5, with an M-w of 12.4 X 10(4), composed of more aspartic acid (Asp), glutamine (Glu), and tyrosine (Tyr) but less cysteine (Cys), has the highest specific activity (204 units per mg protein), which was higher than that of the commercial product (175 units per mg protein). The change of fluorescence intensities at zero concentration of the fractions was identical to that of the activities. The results from amino acid sequence analysis and size-exclusion chromatography combined with static laser light scattering (SEC-LLS) analysis indicated that the separation mechanism of SOD is based on both size exclusion and ion affinity. They also showed that the activity of SOD is determined mainly by its composition. At a flow rate of 0.62 mL min(-1), 42 mg of a high specific activity product can be obtained from crude SOD in 1 day. (C) 2003 Wiley Periodicals, Inc.