Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein.
Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. (We refer to this interaction as a "crosslink," without intending to imply knowledge as to its chemical nature.) Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. [14C]Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H2O2 is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.
Lin
J J
JJ
Department of Biology, Washington University, St. Louis, MO 63130.
Patino
M M
MM
Gaffield
L
L
Walden
W E
WE
Smith
A
A
Thach
R E
RE
D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents; D005293:Ferritins / Q000378:metabolism; D006427:Hemin / Q000378:metabolism*; D035941:Iron Regulatory Protein 1; D035925:Iron-Regulatory Proteins; D007700:Kinetics; D008970:Molecular Weight
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D035925:Iron-Regulatory Proteins; D006427:Hemin; D005293:Ferritins; D035941:Iron Regulatory Protein 1