Microscale characterization of the structure-activity relationship of a heparin-binding glycopeptide using affinity capillary electrophoresis and immobilized enzymes.

A heparin-binding glycopeptide (T3) from human serum amyloid P component was characterized by taking advantage of two important features of capillary electrophoresis: the low sample consumption and the possibility of doing on-line binding studies. Incubations with neuraminidase and proteolytic enzymes were carried out with enzymes immobilized on paramagnetic microbeads. Affinity capillary electrophoresis subsequently was used to characterize T3 and its fragments with respect to heparin binding. We find that an intact glycan moiety makes the C-terminal part of T3 relatively resistant to chymotryptic clevage. This protection is lost upon desialylation. Also, the C-terminus of T3 is involved in heparin binding while the N-terminal part of the molecule has no appreciable binding activity. The micromethods presented here make it feasible to perform structure-function studies even on the small amounts of analytes that are typically available when working with glycopeptides from natural sources.