Allophycocyanin 1 as a near-infrared fluorescent tracer:: Isolation, characterization, chemical modification, and use in a homogeneous fluorescence resonance energy transfer system

Allophycocyanin 1 (APC 1), isolated from Mastigocladus laminosus, retains the same (alpha-beta)(3) trimeric structure as allophycocyanin (APC), but incorporates a peptide linker in its core leading to a 28% increase in its fluorescence quantum yield compared to APC. Moreover, APC 1 exhibits an unexpectedly good stability at very low concentrations, at extreme pHs, or diluted in a low ionic strength medium whereas, under the same conditions, APC dissociates into an (alpha-beta) monomer, indicating that the peptide linker acts as a stabilizer of its trimeric structure. APC1 crosslinking experiments performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide gave a high reaction yield (95%) and showed a similar crosslinking process as previously described for APC. Fluorescence quantum yields of crosslinked APC1 or APC decrease by 20% after labeling on antibody or streptavidin. However, quantum yields of the crosslinked APC1 conjugates remain 25% higher than those of crosslinked APC conjugates. Associated with a europium trisbipyridine cryptate as donor, crosslinked APC1 was compared with crosslinked APC as acceptor in homogeneous time resolved fluorescence technology based on a fluorescence resonance energy transfer process. Using crosslinked APC1, assay performances were increased by 20%, showing that APC1 could be considered as a very promising near infrared fluorescent probe to replace APC in its biological applications. (C) 2001 Academic Press.