Fig. 4.

Effect of reducing agents on stability of pollen multimeric SOD enzymes. a Total protein 1-D profiles of pollen (cv. ‘Picual’) extracts treated with DTT or tributylphosphine (TBP) at different concentrations prior to SDS-PAGE. Thirty micrograms of total protein were loaded per lane. Protein markers (kDa) are displayed on the left. b Western blot as in A probed with a customized anti-olive pollen Cu,Zn-SOD Ab (sequence accession no. EU250769.1). The antibody was able to recognized a ~ 16 kDa chloroplastic SOD (CSD2) and a 23.4 kDa peroxisomal (CSD3) enzyme. A protein band of 15.2 kDa grouping cytosolic CSD1.1A, CSD1.2 and CSD1.3 forms, and a 14.5 kDa corresponding to the cytosolic CSD1.1B enzyme, respectively, were also visible. Arrows indicate monomeric enzymes while black arrowheads point out putative multimeric forms as in Fig. 3. White arrowheads show new putative multimeric SOD forms not detected by the anti-CSD2 antibody