FIG. 4.

HasA_yp is a heme-binding protein. (A) Isolation of HasA_yp by heme affinity chromatography. Coomassie blue staining is shown. Concentrated supernatants were incubated with hemin-agarose as described in Materials and Methods. (Left) HasA_sm, NA, E. coli MC4100(pSyc150, pHasA_sm) concentrated supernatant not adsorbed (flowthrough material in excess) on hemin-agarose; A, E. coli MC4100(pSyc150, pHasA_sm) concentrated supernatant adsorbed on hemin-agarose. (Right) HasA_yp, NA, E. coli MC4100(pSyc150, pHasA_yp) concentrated supernatant not adsorbed on hemin-agarose; A, E. coli MC4100(pSyc150, pHasA_yp) concentrated supernatant adsorbed on hemin-agarose. The presence of HasA_sm and HasA_yp in NA lanes indicates that the capacity of the hemin-agarose columns was saturated by the supernatant concentrates used here. (B) Heme acquisition and formation of holoHasA_sm and holoHasA_yp after incubation of HasA_sm or HasA_yp with heme or hemoglobin. Four microliters (1 μg/ml) of HasA_sm prepared from ammonium sulfate-concentrated supernatants of E. coli MC4100(pSyc150, pHasA_sm) and 2 μl of HasA_yp (2 μg/ml) prepared from ammonium sulfate-concentrated supernatant of E. coli MC4100(pSyc150, pHasA_yp) were incubated with equal volumes of heme or hemoglobin (10⁻³ M) for 45 min at room temperature. The mixtures were loaded onto a 15% polyacrylamide gel and electrophoresed in the absence of SDS. The gel was stained with Coomassie blue. Protein bands complexed with heme (holoproteins) have a faster migration rate than uncomplexed protein (apoproteins).