Further characterization of the interaction of histidine-rich glycoprotein with heparin: evidence for the binding of two molecules of histidine-rich glycoprotein by high molecular weight heparin and for the involvement of histidine residues in heparin binding.
Rabbit histidine-rich glycoprotein (HRG, 94 kDa) binds heparin with high affinity (apparent Kd 60-110 nM). Eosin Y (1 equiv) bound to HRG was used as a reporter group to monitor associations of HRG with heparins of molecular mass 10, 17.5, and 30 kDa. The stoichiometries of the heparin-HRG complexes were determined by fluorescence and absorbance measurements as well as by analytical ultracentrifugation. Two types of complex form: complexes of 1 heparin:1 HRG and of 1 heparin:2 HRG. The 1:2 complex formation requires a minimum heparin chain length since 17.5-kDa but not 10-kDa heparin binds two HRG molecules. The formation of the 1:2 complexes of the larger heparin fractions is enhanced by divalent copper or zinc (1-10 equiv) bound to HRG. However, metal is not required for complex formation since all sizes of heparin examined interact tightly with HRG in the presence of ethylenediaminetetraacetic acid. Between 0.1 and 0.3 M ionic strength, both 1:1 and 1:2 complexes of heparin with HRG are progressively destabilized. No heparin-HRG complex is found at ionic strengths of 0.5 M. Between pH 8.5 and pH 6.5 both 1:2 and 1:1 complexes are found with 17.5-kDa heparin, but at pH 5.5 only 1:1 complexes are formed. The heparin-HRG interaction is progressively decreased by modification of the histidine residues of HRG, whereas modification of 22 of the 33 lysine residues of HRG has little effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Burch
M K
MK
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Blackburn
M N
MN
Morgan
W T
WT
D000818:Animals; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D000099053:Histidine-Rich Glycoprotein
D006023:Glycoproteins; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D006493:Heparin; D004047:Diethyl Pyrocarbonate