Allophycocyanin 1 as a near-infrared fluorescent tracer: Isolation, characterization, chemical modification, and use in a homogeneous fluorescence resonance energy transfer system
E. Trinquet et al., Allophycocyanin 1 as a near-infrared fluorescent tracer: Isolation, characterization, chemical modification, and use in a homogeneous fluorescence resonance energy transfer system, ANALYT BIOC, 296(2), 2001, pp. 232-244
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.