Molecular Weight of Low Molecular Heparins by 13C NMR Spectroscopy

Abstract

Heparin and low molecular weight heparins are polydisperse polysaccharides with a degree of polymerization ranging from 4 to approximately 40. The determination of their average molecular weights has traditionally relied on size exclusion chromatography involving the use of oligosaccharides of known size and molecular weight as standards. 13C NMR spectroscopy is applied for the first time to obtain the molecular weights of low molecular weight heparins. The signal intensities of the reducing end and internal anomeric carbons, having distinctive chemical shifts in the 13C NMR spectrum, are measured to determine the molecular weight. Compared to techniques utilizing broad band decoupling or selective decoupling of anomeric protons, distortionless enhancement polarization transfer pulse sequence gave better quantitation of signal intensities of anomeric carbons. Molecular weight was calculated from the calibrated ratio of signal intensities of the anomeric carbons of reducing end groups and internal residues, and the disaccharide compositional analysis. The calibrated signal intensity ratio is determined using the T1 relaxation rates of anomeric carbons of model oligosaccharides. The disaccharide composition of low molecular weight-heparins is obtained using capillary electrophoresis. Signal averaging over 40,000-90,000 transients, requiring a total of 12-18 h on a 360-MHz NMR spectrometer was adequate to measure molecular weights in the range of 3000-7000. The measured molecular weights of twelve low molecular weight heparins, analyzed by this 13C NMR spectroscopic technique, correlated well with the number average molecular weights obtained using high performance-gel permeation chromatography and gradient polyacrylamide gel electrophoresis. In addition to establishing the number average molecular weight, the 13C NMR spectra helped distinguish the structural properties of different commercially prepared low molecular weight heparins.