Characterization of helical structures in gelatin networks and model polypeptides by circular dichroism

Reversible physical crosslinks (junction zones) in gelatin gels and films are thought to consist of triple helical collagen-like structures, however other structures may also occur. In an attempt to understand the relation between the material properties and the various junction structures and their abundance, we characterized these structures using circular dichroism (CD) as a function of the conditions applied. It appeared that the commonly used reference CD spectra for random coil and 3(1) helix could not be used to deconvolute the spectra observed for gelatin. Most probably, the commonly-used random coil reference spectra do not really reflect unordered structure, while the random coil reference based on the cationic form of polylysine actually reflects a clear helicity. Denatured gelatin or collagen were found to be better approximations of an unordered polypeptide structure. The prolyl chromophore was shown to have a pronounced influence on the shape and position of the CD spectra. Thus, it was concluded that only polypeptides with the appropriate prolyl content can be used as references, while analysing the relative secondary structure contents by CD. By using temperature-dependent CD, we were able to discern multiple and single helical structures at high and low gelatin concentrations, respectively, by the observed cooperativity of helix-to-coil transition.