Analysis of the binding of commercial stains to protein materials used in artworks

In order to contribute to the optimisation of a reliable protocol for localising proteins in art samples, we have analysed (in water solution) the interaction between some of the fluorescent stains commonly used in gel electrophoresis (Figure 1) and proteinaceous paint binders. Of these, dyes 1 and 3 have also been used to detect proteins in paint cross sections. Dyes 1 and 2 bind to the proteins in a non-covalent way; dyes 3, 4 and 5 undergo covalent binding. As for biosubstrates dried egg white (EW) based on ovalbumin, casein (CAS) from dried cow milk, rabbit skin glue (RSG) based on partially hydrolysed collagen and, as a standard reference, purified chicken egg ovalbumin (OVA) were taken into account. The affinity constants for binding of the fluorescent stains to different protein matrices were obtained by fluorescence titrations in aqueous solution. Moreover, the circularly polarized luminescence (CPL) technique [3,4] was used to analyse the binding features. The results obtained contribute to our understanding of the different affinity of the dyes for selected proteins and to shed light on the binding mechanism in these complex systems. Moreover, it is demonstrated that CPL spectroscopy can be applied in the analysis of complex matrices of practical interest and that it can give information about the occurrence of an interaction when fluorescence or ECD methods fail.