A protein with multiple heme-binding sites from rabbit serum.

A 93,000 molecular weight protein (HBP.93) which binds hemin and protoporphyrin IX with high affinity has been isolated from rabbit serum using affinity chromatography on hemin-conjugated agarose. The amino acid composition of this protein is unique in that the proline and histidine contents are remarkably high (16.6 and 9.9 mol %, respectively). A large increase in the absorbance of the Soret region arises from the heme-protein interaction. The spectrophotometric titration showed that the protein can bind 25-35 mol of hemin/mol of protein. The apparent dissociation constant was estimated to be 1-4 X 10(-7) M for hemin at pH 7.4 and approximately 10(-6) M for protoporphyrin IX at pH 9.2. The similarity of the difference spectrum of heme-HBP.93 complex to that of heme-hemopexin complex suggests that a bisimidazol-type coordination of heme iron is involved in the binding. The extremely high capacity of HBP.93 to bind heme is also demonstrated by a large increase in the sedimentation velocity of the protein upon heme binding. The native heme-protein complex migrates faster than the heme-free protein in a polyacrylamide gel at pH 8.8; the increased mobility appears to be due to the charge on the carboxyl groups of the bound heme. Although the use of a hemin-agarose column has failed to reveal a protein of similar size and heme affinity in the sera of a number of other species, including man, the heme-binding properties and high histidine level of the human alpha 2-histidine-rich glycoprotein raise the possibility that the two proteins are related.