Figure 3.

Heme staining of pmPOX fractions after modified SDS-PAGE. Electrophoresis was performed using a low concentrated SDS-PAGE, i.e. 0.1% (w/v) SDS in all solutions and gels without dithiothreitol or mercaptoethanol. Thus, the oligomers were not separated into their subunits. Heme-containing protein bands were visualized by their reaction with the peroxidase substrates tetramethylbenzidine (TMB) and H₂O₂ as described in “Materials and Methods.” Left, pmPOX1 (a) and pmPOX2 (b) are shown after cation exchange chromatography. Further purification of these fractions by size exclusion is presented on the right: pmPOX1 (c), pmPOX2a (d), and pmPOX2b (e). In addition, f shows pmPOX2b treated with 25 mm dithiothreitol. Bars indicate the corresponding molecular masses. After size exclusion chromatography, the PM-bound enzymes had apparent molecular masses of 70 and 40 kD for pmPOX1 and pmPOX2b, whereas pmPOX2a exhibited a broad protein band between 100 and 170 kD.