Highly fluorescent protein labeling using dendritic peptide derivatives

Non-specific fluorescent dyes and photosensitizers are routinely used in cl inical practice for the photodetection and photoablation of superficial les ions. Future applications in photomedicine are likely to rely on the select ive delivery of photoactive compounds to diseased areas, using specific tar geting agents such as antibodies. This fact underlines the need for methods that allow the chemically defined conjugation of several photoactive molec ules to a single protein 'vehicle', with full retention of binding affinity . Here, we present methods for the site-specific fluorescent labeling of pr oteins using dendritic peptides, which had been chemically modified with mu ltiple molecules of fluorescein. Branched peptide derivatives can be stably conjugated to proteins either by reaction with suitable free reactive grou ps or by using the high-affinity non-covalent interaction between calmoduli n and a specific binding peptide. Chemical modification of proteins with on e, two or four molecules of fluorescein resulted in a proportional increase in protein fluorescence.