ISOLATION AND PURIFICATION OF PROTEOGLYCANS

Purification of a protein typically involves development of a quantita tive assay to track protein integrity (e.g. enzyme activity) during su bsequent isolation steps. The generalized procedure involves choosing the source of the protein, defining extraction conditions, developing bulk purification methods followed by refined, more selective methods. The purification of proteoglycans is often complicated by a) limited source quantities, b) necessity of chaotropic solvents for efficient e xtraction, c) their large molecular size and d) lack of defined functi ons to enable purity (i.e. activity, conformation) to be assessed. Bec ause the usual goal of proteoglycan purification is physical character ization (intact molecular weight, core protein and glycosaminoglycan c lass and size), the problems of a suitable assay and/or native conform ation are avoided. The 'assay' for tracking proteoglycan isolation typ ically utilizes uronic acid content or radiolabel incorporation as a m arker. Once extracted from their cellular/extracellular environment, p roteoglycans can be isolated by density gradient centrifugation and/or column chromatography techniques. Recent advances in the composition of chromatographic supports have enabled the application of ion-exchan ge. gel permeation, hydrophobic interaction and affinity chromatograph y resins using efficient high-pressure liquid chromatography to proteo glycan purification.