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 micr...

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Bibliographic Details
Published in:Biochemistry (Easton) 1985-10, Vol.24 (21), p.5919-5924
Main Authors: Burch, Mary Kappel, Morgan, William T
Format: Article
Language:English
Subjects:
Animals
Binding Sites
Biological and medical sciences
Blood Proteins
Fundamental and applied biological sciences. Psychology
Glycoproteins - blood
Heme - metabolism
Interactions. Associations
Intermolecular phenomena
Kinetics
Molecular biophysics
Molecular Weight
Protein Binding
Proteins
Rabbits
Spectrometry, Fluorescence
Spectrophotometry
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00342a034