Haydn G. Morris

Skeletal keratan sulfate chain molecular weight calibration by high-performance gel-permeation chromatography

A method has been developed for the molecular sizing of skeletal keratan sulfate chains using an HPLC gel-permeation chromatography system. Keratan sulfate chains and keratanase-derived oligosaccharides were prepared from the nucleus pulposus of bovine intervertebral disc (6-year-old animals). A Bio-Gel TSK 30 XL column eluted in 0.2 M NaCl and at 30 degrees C was calibrated with keratan sulfate oligosaccharides of known size as well as 3H-end-labeled keratan sulfate chains to yield the relationship.

Structural studies of two populations of keratan sulphate chains from mature bovine articular cartilage

Two discrete peptido-keratan sulphate fragments were isolatedvia chondroitinase ABC and trypsin digestion of a proteoglycan aggregate fraction prepared from bovine femoral head cartilage (six year old animals). The larger fragments (Kav=0.07, CL-6B) contained peptides substituted with several keratan sulphate (KS) chains from the KS-rich region of the proteoglycan and the smaller fragments (Kav=0.5, CL-6B) contained peptides with, perhaps, only one KS chain and the stubs of post-chondroitinase-treated chondroitin sulphate chains.The two peptido-KS samples and the KS chains derived from these by alkaline borohydride reduction were characterised by13C-NMR spectroscopy. The two populations of KS chains were also examined by chromatography (Sephadex G-75), and keratanase digestion followed by chromatography on Bio-Gel P-10. From the results it was concluded that the KS chains from the two major trypsin-derived peptido-KS fragments had similar sulphation levels, distributions of hydrodynamic sizes and susceptibilities to keratanase.

An investigation of a maximum entropy method for the processing of 1H and 13C nmr spectra from glycosaminoglycan oligo- and poly-saccharides

Abstract Maximum Entropy (MEM) processing of 1 H and 13 C spectra from representative keratan sulphate, heparan sulphate and dermatan sulphate glycosaminoglycan polymers or oligomers is considered. The advantages and disadvantages of one specific approach, the MEM procedure, with regard to the accurate determinations of spin-spin couplings, chemical shifts and for resolution enhancements, are discussed. It is concluded that, providing the original spectrum has adequate signal-to-noise (S/N) ratio, useful resolution enhancement can be achieved without degradation of spectral noise, for the more accurate determinations of spin-spin couplings, chemical shifts and the unravelling of incompletely resolved structure. In situations of poor S/N ratio, noise uncertainties persist and artificial noise reduction by extra exponential smoothing followed by more severe MEM enhancement can lead to loss of spectral information.