Heme binding assay

Hemin agarose was prepared according to Busch et al. (2017). Briefly, 2 ml of aminoethyl-agarose beads (aminoethyl 6% agarose beads cross-linked 50 % v/v, Goldbio, MO) were transferred to a column and washed with 7 ml water, 7 ml 33% dimethylformamide (DMF), 7 ml 66% DMF and finally with 14 ml of 100% DMF. A 50 mM hemin solution (in DMF) was mixed with an equal volume of 1,1′-carbonyldiimidazole (CDI, 26.6 mg/ml DMF, Sigma-Aldrich, Munich, Germany) and incubated for 15 min at 80°C (dark). To remove undissolved hemin, the hemin:CDI mixture was centrifuged for 30 min at RT (16,000 g). The supernatant was mixed with the DMF-washed agarose beads and incubated for 18 hr on a rotary incubator in the dark. The next day, the matrix was washed with approximately 70 column volumes (CV) of 25% pyridine (Sigma-Aldrich, Munich, Ger) until the flow-through was transparent. After washing with 10 CV of double-distilled water, the matrix was equilibrated with 10 CV PBS. Finally, the matrix was resuspended in PBS, resulting in 33% hemin-agarose solution. Aliquots of the matrix were stored at 4°C until further use. To assay for hemin binding, 10 µg of recombinant proteins were incubated with 75 µl of the hemin-agarose or PBS equilibrated agarose beads (control) in a final volume of 500 µl PBSplus (PBS supplemented with 10% glycerol, 0.2% Tween 20). After 30 min of incubation on a rotary incubator at RT, samples were transferred to centrifuge columns and centrifuged at 1500 rpm (RT) for 1 min. Columns were washed 10 times with PBSplus supplemented with 500 mM NaCl. Bound proteins were eluted using 30 µl of PAGE loading buffer and were analyzed by SDS-PAGE.