Binding of proline- and hydroxyproline-containing peptides and proteins to the capillary wall

Abstract

Sticking of peptides (as demonstrated by peak distortion) containing a high proportion of glycine and proline residues to the capillary wall was investigated. At acid pH where the carboxy terminal proline (e.g. Gly–Pro) is protonated, distinct sticking of these peptides occurred. On the contrary, the Pro–Gly peptide yielded a well shaped peak. At alkaline pH, where the situation was reversed, the peptide sticking most to the wall was Gly–Gly, while the behaviour of the dipeptides possessing N- or C-terminal proline did not suffer from sticking to the wall. It was concluded that the separation of simple proline- and glycine-containing dipeptides can be partly optimized by manipulating the pH of the background electrolyte; particularly if cetyltrimethylammonium bromide, methanol or acetonitrile is added to the run buffer (reversed polarity mode with cetyltrimethylammonium bromide). However, all attempts to resolve more complex mixtures of proline- (and hydroxyproline-) containing di- and tripeptides failed; addition of an organic modifier, i.e. methanol and acetonitrile, hexylamine, triethylamine and cetyltrimethylammonium bromide was unsuccessful. Such separations can be materialized by using simple 25 mmol/l phosphate buffer at pH 10.5 provided that the sample is dissolved in (aqueous) 17.5 mmol/l Brij or 33 mmol/l sodium dodecyl sulfate. These systems are also applicable to large polypeptides rich in proline, hydroxyproline and glycine: typically parent collagen α-chains, their dimers, trimers and higher polymers were successfully separated.