Purification and characterization of the Cu,Zn SOD from Escherichia coli

• Published in: Free radical biology & medicine 1996, Vol.21 (1), p.117-121
• Main Authors: Benov, Ludmil T., Beyer, Wayne F., Stevens, Robert D., Fridovich, Irwin
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Bacteriocuprein; Chromatography, DEAE-Cellulose; Chromatography, High Pressure Liquid; Copper - analysis; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - enzymology; Free radicals; from Escherichia coli; Humans; Kinetics; Molecular Sequence Data; Periplasmic Cu; Spectrophotometry, Atomic; Spectrophotometry, Ultraviolet; Superoxide Dismutase - chemistry; Superoxide Dismutase - isolation & purification; Superoxide Dismutase - metabolism; Zn SOD
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/0891-5849(95)02217-1

The periplasmic Cu,Zn superoxide dismutase has been purified to homogeneity by a procedure, which depended upon osmotic shock followed by two chromatographic columns. Its subunit weight, determined by electrospray ionization mass spectrometry, was found to be 15,737 ± 1.6. The second derivative ultraviolet spectrum indicated a lack of tryptophan. The amino acid composition as well as a partial N-terminal amino acid sequence is reported. The specific activity was 3700 U/mg and the corresponding copper content was 0.77 atoms Cu/subunit. The enzyme was quite unstable and overnight dialysis against EDTA or even prolonged dialysis against neutral phosphate buffer caused partial loss of activity and of copper and visible precipitation. It is likely that some losses occurred during the isolation procedure, and if these could have been prevented the copper content would have been 1.0 Cu/subunit and the specific activity would have been 4800 U/mg. It now appears likely that gram negative bacteria will commonly be found to contain a periplasmic Cu,ZnSOD.