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. 2014 Jan 22;9(1):e86435. doi: 10.1371/journal.pone.0086435

Figure 2. D86G substitution renders recombinant CYB5D2 incapable of binding heme.

Figure 2

A) Images of GST-CYB5D2 (CYB5D2)-bound and GST-CYB5D2(D86G)-bound glutathione-agarose. B) 10 µg of purified recombinant GST-CYB5D2 (CYB5D2) and GST-CYB5D2(D86G) (CYB5D2(D86G)) was separated by SDS-PAGE, followed by Coomassie blue staining. C) Recombinant GST-CYB5D2 and GST-CYB5D2(D86G) protein, referred to as CYB5D2 and CYB5D2(D86G) respectively, were scanned for their absorbance peaks within the indicated wavelength range. D) Recombinant GST-CYB5D2 (CYB5D2) and GST-CYB5D2(D86G) (10 μg) were incubated with hemin and analyzed by in-gel peroxidase reaction staining to detect heme binding (see Materials and Methods for technical details). Presence of free (unbound) heme was also detected (middle panel). Equal loading of recombinant proteins was verified by Coomassie blue staining (bottom panel).