High-pressure, anion-exchange chromatography of proteoglycans

Although high-performance liquid chromatography has been used extensively to characterize the glycosaminoglycan chains of proteoglycans, very few researchers have reported the use of this technology for the separation of intact proteoglycan species. The high molarity denaturing buffers required for proteoglycan disaggregation and separation are often not compatible with the low back-pressure limitations imposed by many of the HPLC systems designed for the separation of biological macromolecules. In this study, heparan sulfate and dermatan sulfate proteoglycans, obtained by the metabolic labeling of cultured corneal endothelial cells, were rapidly and completely separated in less than an hour in a high-pressure liquid chromatography system. The separation, which used a Dionex BioLC system equipped with a Pharmacia Superloop and a ProPac PA1 column, also effected a greater than 10-fold concentration of the proteoglycans during the separation procedure. All buffers were 8 M in urea, and the back-pressures generated during the separation were well below the limit of the system. The pooled fractions from the ion-exchange column were subsequently analyzed for glycosaminoglycan composition and molecular size. The system was able to resolve dermatan sulfate-substituted species from heparan sulfate-substituted species in a single chromatographic step. The proteoglycan nature of the recovered products was established by Sepharose CL-4B chromatography and gel electrophoresis.