Mikhailov D, Mayo K H, Pervin A, Linhardt R J
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455, USA.
Biochem J. 1996 Apr 15;315 ( Pt 2)(Pt 2):447-54. doi: 10.1042/bj3150447.
Heparin is a polydisperse sulphated copolymer consisting mostly of 1-->4 linked glucosamine and uronic acid residues, i.e. 2-deoxy-2-sulphamido-D-glucopyranose 6-sulphate and L-idopyranosyluronic acid 2-sulphate. 13C 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 pKa depression suggests electrostatic interactions between the arginine guanidinium group and the uronate carboxylate group. 13C relaxation data have been acquired for all disaccharide and peptide carbons in the presence and absence of GRG and GKG. 13C 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 [symbol: see text] and D parallel, and an angle alpha between D parallel 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 (2H1 and 1H2) and over a range of glycosidic bond angles defined by motional order parameters and inter-residue nuclear Overhauser effects (+/- 30 degree from the average). In the absence of peptide, the ratio D [symbol: see text] /D parallel falls between 0.4 and 0.7; therefore molecular diffusion occurs preferentially about D parallel, which runs through both disaccharide rings. In the presence of peptide, D [symbol: see text] /D parallel is decreased, indicating that GRG is oriented along D parallel and proximal to the uronic acid ring. A model for this is shown.
肝素是一种多分散的硫酸化共聚物，主要由1→4连接的葡糖胺和糖醛酸残基组成，即2-脱氧-2-氨基磺酰基-D-吡喃葡萄糖6-硫酸盐和L-艾杜糖醛酸2-硫酸盐。13C核磁共振已被用于研究肝素酶衍生和纯化的肝素二糖与N端和C端封闭的三肽GRG和GKG的相互作用。用肽对二糖进行滴定表明，GRG比GKG更强烈地结合二糖，在这两种情况下，在糖醛酸环位置的相互作用更强。在GRG存在的情况下，羧酸盐pKa降低表明精氨酸胍基与糖醛酸羧酸盐基团之间存在静电相互作用。已获取了在有和没有GRG和GKG的情况下所有二糖和肽碳的13C弛豫数据。在肽存在的情况下，尤其是与GRG一起时，二糖的13C弛豫速率明显更快。已根据分子扩散常数D[符号：见正文]和平行扩散常数D以及D平行与由为内部刚性二糖计算的惯性张量矩定义的分子框架之间的夹角α对这些弛豫数据进行了分析。在这些计算中，对糖醛酸半椅形式（2H1和1H2）以及由运动序参量和残基间核Overhauser效应定义的一系列糖苷键角（相对于平均值±30度）对二糖构象空间进行了采样。在没有肽的情况下，D[符号：见正文]/D平行的值在0.4到0.7之间；因此分子扩散优先围绕贯穿两个二糖环的D平行发生。在肽存在的情况下，D[符号：见正文]/D平行降低，表明GRG沿D平行方向取向且靠近糖醛酸环。展示了一个对此的模型。
