Allophycocyanin 1 as a near-infrared fluorescent tracer: isolation, characterization, chemical modification, and use in a homogeneous fluorescence resonance energy transfer system.
Allophycocyanin 1 (APC1), 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, 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 (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.
Trinquet
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Division In Vitro Technologies, Cis Bio International, Bagnols sur Cèze Cedex, F-30204, France. etrinquet@cisbiointernational.fr
Maurin
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Préaudat
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Mathis
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D002850:Chromatography, Gel / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D004355:Drug Stability; D056890:Eukaryota / Q000737:chemistry*; D007259:Infrared Rays; D010798:Phycocyanin / Q000737:chemistry / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D003432:Cross-Linking Reagents; C002497:allophycocyanin; D010798:Phycocyanin