Investigation of the applications of reverse-phase high performance liquid chromatography in the structural studies of collagen-like model peptide

Abstract

Various characterization techniques, such as circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, have been reported and widely used for the structural studies of the triple helical twists of collagen. In this study, the potential applications of reverse-phase high performance liquid chromatography (RP-HPLC) in the structural studies of collagen triple helix were investigated. RP-HPLC analyses of (Pro-Hyp-Gly)10 and (Pro-Pro-Gly)10 resulted in numerous peaks indicative of triple helical conformations (denoted as THP) and a single peak representative of the unfolded single strand peptide (denoted as SSP). This characterization technique by RP-HPLC is based on the different hydrophobicities possessed by the folded and unfolded conformations. The triple helical characteristic of the THP peak was confirmed by several independent control experiments, including temperature dependency analyses. A new approach to study the cooperative thermal transition between the folded and unfolded conformations as well as determination of melting point temperatures (Tm) was also introduced in this study. HPLC denaturation experiments were performed based on the interconversion between the THP and SSP peaks at different temperatures. The Tm values of (Pro-Pro-Gly)10 and (Pro-Hyp-Gly)10 dissolved in HPLC buffer were obtained from the midpoints of the cooperative transition curves as 26.0 °C and 42.0 °C respectively.