C-13-NMR relaxation study of heparin-disaccharide interactions with tripeptides GRG and GKG

Heparin is a polydisperse sulphated copolymer consisting mostly of 1 --> 4 linked glucosamine and uronic acid residues, i.e. 2-de oxy-2-sulphamido-D-glucopyranose 6-sulphate and L-idopyranosyluronic acid 2-sulphate. C-13 NMR has been used to study the interactions of heparinase-derived and purified heparin disaccharide with N- and C-terminally-blocked tripeptides GRG and GKG. Titration of the disaccharide with peptide indicates that GRG binds the disaccharide more strongly than does GKG, with interactions in either case being stronger at uronate ring positions. In the presence of GRG, a carboxylate pK(a) depression suggests electrostatic interactions between the arginine guanidinium group and the uronate carboxylate group. C-13 relaxation data have been acquired for all disaccharide and peptide carbons in the presence and absence of GRG and GKG. C-13 relaxation rates for the disaccharide are significantly faster in the presence of peptide, especially with GRG. Analysis of these relaxation data has been done in terms of molecular diffusion constants, D-perpendicular to and D-parallel to and an angle alpha between D-parallel to and a molecular frame defined by the moment of inertia tensor calculated for an internally rigid disaccharide. Disaccharide conformational space in these calculations has been sampled for both uronate half-chair forms (H-2(1) and H-1(2)) and over a range of glycosidic bond angles defined by motional order parameters and inter-residue nuclear Overhauser effects (+/-30 degrees from the average). In the absence of peptide, the ratio D-perpendicular to/D-parallel to falls between 0.4 and 0.7; therefore molecular diffusion occurs preferentially about D-parallel to, which runs through both disaccharide rings. In the presence of peptide, D-perpendicular to/D-parallel to is decreased, indicating that GRG is oriented along D-parallel to and proximal to the uronic acid ring A model for this is shown.