This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.As NMR analysis requires highly pure sample and our assays show substantial impurity in this sample, we have attempted further purification by ion exchange and size exclusion chromatography. We have also done preliminary analysis by NMR. Methods: Size Exclusion ChromatographyThe sample was first dialyzed through 1kD MWCO dialysis tubing then lyophilized, loaded onto a 1.5x30cm Superose 6 size exclusion column and eluted at 0.5ml/min in 10mM ammonium formate pH 5.0. Detection was accomplished by means of a Knauer refractive index detector and by phenol sulfuric acid assay. Retention times were compared to dextran standards of 10kD, and 511kD molecular weight.CM ChromatographyThe sample was loaded onto a Waters CM Sep Pak in 10mM ammonium formate pH 5.0 in an attempt to clean up the sample prior to SEC. Essentially all material was observed to elute from the column without binding.Phenol/Sulfuric Acid Assay for Carbohydrate QuantitationThe method of Dubois et al. (Dubois et al. (1956) Anal. Chem. 28 350-356) was used for carbohydrate quantitation in the column eluate. Briefly, 10ul aliquots of the desired fractions were added to test tubes with 20ul 5% phenol. 0.1ml concentrated sulfuric acid was then added and absorbance at 490nm was read on a Molecular Devices Spectra Max plate reader.NMR SpectroscopyThe samples were deuterium-exchanged by lyophilization from D2O and dissolved in 0.7 mL D2O. Proton and COSY NMR spectra were acquired on a Varian 300 MHz spectrometer at 293 K (20 C) using standard Varian pulse sequences.
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