Paramagnetic probes of the domain structure of histidine-rich glycoprotein.
The interaction of Cu2+ and Fe3+-mesoporphyrin with histidine-rich glycoprotein (HRG) from rabbit serum was examined spectroscopically. The first equivalent of Cu2+ binds to HRG producing a type II electron paramagnetic resonance (EPR) spectrum with g[[ = 2.25, gm = 2.05, A[[ = 0.019 cm-1 (180 G), and superhyperfine along gm. These spectral parameters suggest moderately covalent coordination of Cu2+ to the protein by nitrogens. With increasing Cu2+ the superhyperfine disappears; however, the g and A values change only marginally. The increase in EPR signal amplitude throughout the addition of 1-15 equiv of Cu2+ is linear and thereafter maximizes, suggesting 18-22 equiv are bound. In contrast, changes in the circular dichroism spectrum at 280 nm appear sigmoidal and can be interpreted as the binding of Cu2+ to two structurally distinct regions of the protein. Evidence for two structurally distinct binding domains is found by comparing EPR spectra of Cu2+ complexes of HRG with spectra from complexes of two of its major proteolysis products (peptides). After binding 1 equiv of Cu2+, both the 30-kDa histidine-rich peptide and the native protein exhibit identical spectra including the pronounced superhyperfine. In contrast, the spectrum of the histidine-normal 45-kDa peptide with 1 equiv of Cu2+ bound lacks superhyperfine and parallels closely that of the native protein with 20 equiv bound. Finally, Fe3+-mesoporphyrin binds to HRG exhibiting both high-spin (g = 6.05) and low-spin (gz = 2.94, gy = 2.25, gx = 1.50) EPR resonances, and the latter imply bis(histidine) coordination.(ABSTRACT TRUNCATED AT 250 WORDS)
Muhoberac
B B
BB
Department of Chemistry, Purdue University School of Science, Indiana University-Purdue University, Indianapolis 46223.
Burch
M K
MK
Morgan
W T
WT
D000818:Animals; D001798:Blood Proteins; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006023:Glycoproteins / Q000097:blood*; D008652:Mesoporphyrins / Q000378:metabolism*; D011166:Porphyrins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins; D011817:Rabbits; D000099053:Histidine-Rich Glycoprotein
D001798:Blood Proteins; D006023:Glycoproteins; D008652:Mesoporphyrins; D011166:Porphyrins; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; C031468:mesoporphyrin IX; D003300:Copper