Preferred heme binding sites of histidine-rich glycoprotein.
The heme binding sites of rabbit histidine-rich glycoprotein (HRG), 94 kDa, were studied with rose bengal (RB), a fluorescein derivative that associates with histidine residues. Difference absorbance spectra indicate that HRG binds RB at two thermodynamically preferred sites (Kd approximately 2 microM) that are spectroscopically equivalent. Up to 18-22 equiv of RB can also be bound by a set of lower affinity sites. Mesoheme is capable of displacing RB from the two preferred sites (Kd = 0.6 microM) and provides evidence that the two sites are not identical. Two peptides isolated from plasmin-digested HRG, one 35-kDa peptide rich in histidine (approximately 30 mol %) and one 15-kDa peptide relatively poor in histidine (approximately 4 mol %), also bind RB and mesoheme. The two preferred RB binding sites of HRG are located on the 15-kDa histidine-poor peptide and the lower affinity "class" of sites on the 35-kDa histidine-rich peptide. Mesoheme or RB quenches the tryptophan fluorescence of HRG and the histidine-poor peptide with an apparent binding stoichiometry near 2. Fluorescence quenching also indicates that 1-2 equiv of Cu(II) binds to the 15-kDa peptide, and absorbance spectroscopy provides evidence that Cu(II) is capable of displacing heme from the peptide. The fluorescence lifetimes of RB, determined by phase-modulation fluorometry, indicate that the two preferred sites in the histidine-poor domain are more apolar than the more numerous sites located in the histidine-rich region of the protein.
D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins; D006023:Glycoproteins / Q000097:blood*; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D011506:Proteins; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D000099053:Histidine-Rich Glycoprotein
D001798:Blood Proteins; D006023:Glycoproteins; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D006418:Heme