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 t he same (alpha-beta)(3) trimeric structure as allophycocyanin (APC), but in corporates a peptide linker in its core leading to a 28% increase in its fl uorescence quantum yield compared to APC. Moreover, APC 1 exhibits an unexp ectedly good stability at very low concentrations, at extreme pHs, or dilut ed in a low ionic strength medium whereas, under the same conditions, APC d issociates into an (alpha-beta) monomer, indicating that the peptide linker acts as a stabilizer of its trimeric structure. APC1 crosslinking experime nts performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide gave a hi gh reaction yield (95%) and showed a similar crosslinking process as previo usly described for APC. Fluorescence quantum yields of crosslinked APC1 or APC decrease by 20% after labeling on antibody or streptavidin. However, qu antum yields of the crosslinked APC1 conjugates remain 25% higher than thos e of crosslinked APC conjugates. Associated with a europium trisbipyridine cryptate as donor, crosslinked APC1 was compared with crosslinked APC as ac ceptor in homogeneous time resolved fluorescence technology based on a fluo rescence resonance energy transfer process. Using crosslinked APC1, assay p erformances were increased by 20%, showing that APC1 could be considered as a very promising near infrared fluorescent probe to replace APC in its bio logical applications. (C) 2001 Academic Press.