Heteronuclear NMR studies of cobalamins. 31P NMR observations of cobalamins bound to a haptocorrin from chicken serum.
A vitamin B12-binding protein (haptocorrin) from chicken serum has been purified to homogeneity by photodissociative affinity chromatography and characterized by gel electrophoresis and UV-visible spectrophotometry of its aquocobalamin, hydroxocobalamin, and cyanocobalamin complexes. The haptocorrin is a glycoprotein with a molecular mass of about 70 kDa and a protein moiety of about 40 kDa. 31P NMR resonances of the haptocorrin-cobalamin complexes are relatively broad singlets (with or without proton decoupling) shifted downfield by 0.7-1.0 ppm from the position of the free cobalamin resonances. From the line width data, the relaxation of the phosphorus nucleus is found to be dominated by chemical shift anisotropy with a very minor (13%) component from dipolar interaction with the two nearest neighbor protons. The rotational correlation time of the haptocorrin at 25 degrees C is estimated to be 85 ns and the activation energy for rotational correlation 3.9 +/- 0.3 kcal mol-1. The downfield shift of the 31P resonances of cobalamins upon binding to the haptocorrin cannot be due to hydrogen bonding phosphodiester moiety or displacement of the axial base by a group on the protein. Calculations also show that the downfield shift is very unlikely to be due to dipolar deshielding of the phosphorus nucleus by the ring current of an aromatic residue of the protein. It is concluded that the downfield shift of the 31P resonance must be due to sterically induced changes in phosphodiester conformation which may, or may not, involve steric compression of the axial Co-N bond.
Brown
K L
KL
Department of Chemistry, University of Texas, Arlington 76019.
Marques
H M
HM
Jacobsen
D W
DW
D000465:Algorithms; D000818:Animals; D002645:Chickens; D009682:Magnetic Resonance Spectroscopy; D013056:Spectrophotometry, Ultraviolet; D014155:Transcobalamins / Q000097:blood*; D014805:Vitamin B 12
D014155:Transcobalamins; D014805:Vitamin B 12