MOLECULAR-WEIGHT OF LOW-MOLECULAR-WEIGHT HEPARINS BY C-13 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Heparin and low molecular weight heparins are polydisperse polysacchar ides with a degree of polymerization ranging from 4 to similar to 40. The determination of their average molecular weights has traditionally relied on size exclusion chromatography involving the use of oligosac charides of known size and molecular weight as standards. C-13 NMR spe ctroscopy is applied for the first time to obtain the molecular weight s of low molecular weight heparins. The signal intensities of the redu cing end and internal anomeric carbons, having distinctive chemical sh ifts in the C-13 NMR spectrum, are measured to determine the molecular weight. Compared to techniques utilizing broad band decoupling or sel ective decoupling of anomeric protons, distortionless enhancement pola rization transfer pulse sequence gave better quantitation of signal in tensities of anomeric carbons. Molecular weight was calculated from th e calibrated ratio of signal intensities of the anomeric carbons of re ducing end groups and internal residues, and the disaccharide composit ional analysis. The calibrated signal intensity ratio is determined us ing the T-1 relaxation rates of anomeric carbons of model oligosacchar ides. The disaccharide composition of low molecular weight-heparins is obtained using capillary electrophoresis. Signal averaging over 40000 -90000 transients, requiring a total of 12-18 h on a 360-MHz NMR spect rometer was adequate to measure molecular weights in the range of 3000 -7000. The measured molecular weights of twelve low molecular weight h eparins, analyzed by this C-13 NMR spectroscopic technique, correlated well with the number average molecular weights obtained using high pe rformance-gel permeation chromatography and gradient polyacrylamide ge l electrophoresis. In addition to establishing the number average mole cular weight, the C-13 NMR spectra helped distinguish the structural p roperties of different commercially prepared low molecular weight hepa rins.