High performance capillary electrophoresis of casein phosphopeptides containing 2-5 phosphoseryl residues: relationship between absolute electrophoretic mobility and peptide charge and size.

Abstract

Multiple phosphoseryl-containing peptides from casein containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- stabilize amorphous calcium phosphate at neutral and alkaline pH and have been shown to be anticariogenic in various in vitro, animal and human experiments. In an approach to obtain insight into the structure and function of these peptides, we previously developed a method for their analysis using high-performance capillary electrophoresis (HPCE). A linear relationship was obtained between absolute electrophoretic mobility and q/M2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M is the molecular mass. M2/3 is a measure of the surface area of a sphere that has a volume proportional to the M of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. To examine this relationship further, we have analyzed a range of high-performance liquid chromatography (HPLC)-purified cluster, di- and triphosphorylated peptides from casein enzymic (trypsin, alcalase and pancreatin) digests using HPCE and attempted to relate their absolute electrophoretic mobility to various models incorporating peptide size and charge. Absolute electrophoretic mobility of casein phosphopeptides containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- was most closely correlated with q/M2/3. Di- and triphosphorylated peptide absolute electrophoretic mobility correlated with both q/M2/3 and ln(q+l)/n0.43. However, for both the q/M2/3 and In(q+l)/n0.43 relationships with absolute electrophoretic mobility, the di- and triphosphorylated peptides formed a separate linear relationship to that of the cluster peptides. From these relationships, a di- or triphosphorylated peptide exhibited a greater absolute electrophoretic mobility than a corresponding cluster peptide with sam q/M2/3 or In(q+l)/n0.43 value.(ABSTRACT TRUNCATED AT 250 WORDS)