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Journal of the Korean Chemical Society (JKCS)

ISSN 1017-2548(Print)
ISSN 2234-8530(Online)
Volume 55, Number 5
JKCSEZ 55(5)
October 20, 2011 

Specific Binding of Nile Red to Apomyoglobin
Salina A Chowdhury, Manho Lim*
Fluorescence correlation spectroscopy, Fluorescence lifetime, pH denaturation, Folding/unfolding dynamics
Fluorescence correlation spectroscopy (FCS) is an emerging fluorescence technique used to study the dynamics of proteins on a millisecond to microsecond time scale at the single-molecule level. Solution pH-modulated protein conformational changes can be manifested by binding rate, fluorescence lifetime, and binding specificity of a probe molecule. The fluorescence lifetime of Nile red (NR) bound to apomyoglobin (apoMb) was measured to be 6 ± 0.3 ns, much longer than that in water solution (2.9 ± 0.2 ns). As the unfolding population of apoMb increased by lowering pH of solution, the fraction for the longer lifetime of NR decreased with an increasing fraction for the shorter lifetime of NR in water. Unlike 1-anilino-8-naphthalene sulfonic acid, which has many lifetimes due to nonspecific binding to the unfolded apoMb, NR bound to apoMb possesses only a single lifetime. These results suggest that NR binds specifically to native apoMb and thus can be utilized to probe the folding/unfolding dynamics of apoMb using FCS.
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