A cautionary note when using pepsin as a probe for the formation of a collagen triple helix

The ability of the collagen triple helix to resist digestion with proteases has been used as a conformational probe to ascertain whether a collagenous molecule has formed a correctly aligned helix during biosynthesis or during refolding of the protein in vitro. During our studies into the synthesis and folding of a variety of engineered procollagen polypeptide chains, we noted that resistance to digestion with proteases, in particular pepsin, could be misleading and does not necessarily indicate the formation of a collagen triple helix. These results clearly show that resistance to pepsin digestion alone should not be used to indicate correct folding and that preferably an alternative assay should be used to unequivocally demonstrate the formation of a correctly aligned triple helix.