Tagging of collagen with red emissive Ru(II) complex for biological application

Labelling of biomolecules have attracted more attention due to its ability to study the biological events that occurs inside the cell. The labelling of the biomolecules such as proteins and DNA have often been limited with organic fluorophore/dyes. The understanding of protein function varies from seconds to days and organic fluorophores such as fluorescein, rhodamine and cyanine dyes that undergoes photobleaching and might not be a suitable candidate for the studies. On the other hand, phosphorescent metal complexes have been used as probe for labelling the globular proteins such as bovine serum albumin, carbonic anhydrase, IgG and lysozyme, etc. In this study, we have explored the use of red emitting Ru(II) polypyridyl complex as probe for tagging the fibrillar protein i.e. collagen. The conjugation of Ru(II) complex with collagen was carried out using EDC/NHS procedure. By conjugating the Ru(II) complex, collagen did not lose its triple helical nature as evidenced from the CD spectra. Digestion of Ru-collagen using collagenase increased the emission intensity in similar line with commercially available FITC-collagen. Using microscale thermophoresis, the binding affinity (K_d) of polyphenols to Ru modified collagen was determined and found to be in the order: Catechin>Quercetin>Gallic acid and is similar to the binding affinity of native collagen which clearly indicates the Ru conjugation did not impact the binding of the polyphenol. Further, the binding affinity of collagenase to Ru-collagen was determined and exhibit the strong binding compared to native collagen. The present data clearly demonstrates that red emissive nature of Ru-collagen offers a potential solution for imaging as well as substrate for determining the binding affinity of small molecule directly either through fluorescence or microscale thermophoresis technique.