Allophycocyanin 1 as a Near-Infrared Fluorescent Tracer: Isolation, Characterization, Chemical Modification, and Use in a Homogeneous Fluorescence Resonance Energy Transfer System

✍ Scribed by E. Trinquet; F. Maurin; M. Préaudat; G. Mathis

• Book ID: 102560192
• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 178 KB
• Volume: 296
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5298

✦ Synopsis

Allophycocyanin 1 (APC1), isolated from Mastigocladus laminosus, retains the same (␣-␤) 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, APC1 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 (␣-␤) 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.