title: A source of error in the chromatographic study of 35S-sulfate labeled mucous glycoproteins secreted by the gill epithelium of Mytilus edulis.
abstract: HPLC combined with [35S]-sulfate/[3H]-glucosamine radiolabeling were employed to study the synthesis and secretion of mucous glycoproteins. The secreted radiolabeled glycoproteins were separated from the medium by precipitation with a mixture of trichloroacetic-phosphotungstic acids (TCA/PTA). The redissolved glycoproteins were chromatographed on an anion exchange protein column at varying pH of the mobile phase and fractions were collected for liquid scintillation counting. Varying the pH of the mobile phase from pH 3 to 7 resulted in a decrease of glycoprotein bound [35S] from 69.5 to 0.5% of the total recovered [35S]-sulfate with the remainder recovered as free [35S]-sulfate. The [3H]-labeled glycoprotein recovered under the uV peaks at this pH range was 99.5%. When high performance size exclusion chromatography was performed the change in mobile phase pH did not affect the 100% recovery of either [35S]-or [3H]-labels under the uV peaks. No free [35S]-sulfate was obtained when [35S]-labeled glycoproteins were separated from the medium using dialysis. These data suggest that the standard method of TCA/PTA precipitation of [35S]-labeled glycoproteins may cleave the [35S]-sulfate ester linkages to the oligosaccharide chains. The [35S]-sulfate may then rebind to the macromolecule by a relatively strong noncovalent bond. This may prove critical in anion exchange protein HPLC studies.
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004848:Epithelium / Q000378:metabolism; D005880:Gills / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D007553:Isotope Labeling; D009093:Mucus / Q000378:metabolism*; D010772:Phosphotungstic Acid; D013462:Sulfur Radioisotopes; D014238:Trichloroacetic Acid
chemical_list: D006023:Glycoproteins; D013462:Sulfur Radioisotopes; D010772:Phosphotungstic Acid; D014238:Trichloroacetic Acid