Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes.
Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (pIs) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component. Due to the fact that this enzyme is expressed in E. coli, N-terminal acetylation or glycosylation should be ruled out. When purified by small-scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms. Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material. Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2-SOD, as reported in the literature. Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190-240 nm range, but marked differences in the 250-350 nm region. Treatment with EDTA produces 1-2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms. It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer.
Wenisch
E
E
Institute of Applied Microbiology, School of Food and Biotechnology, University of Agriculture and Forestry, Vienna.
Vorauer
K
K
Jungbauer
A
A
Katinger
H
H
Righetti
P G
PG
D002942:Circular Dichroism; D004572:Electrolysis / Q000295:instrumentation*; D005782:Gels; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007525:Isoelectric Focusing / Q000295:instrumentation*; D008567:Membranes, Artificial; D011994:Recombinant Proteins / Q000302:isolation & purification; D013482:Superoxide Dismutase / Q000302:isolation & purification*
D005782:Gels; D007477:Ions; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D013482:Superoxide Dismutase