Abstract ▼ AI-Helper

The dynamics of the binding reaction of nile red (NR) to horse apomyoglobin (apoMb) has been investigated by fluorescence correlation spectroscopy (FCS). FCS is useful in measuring chemical reaction rates and diffusion coefficients of molecules in equilibrium in the range of 10 μs – 10 ms. Whe...

The dynamics of the binding reaction of nile red (NR) to horse apomyoglobin (apoMb) has been investigated by fluorescence correlation spectroscopy (FCS). FCS is useful in measuring chemical reaction rates and diffusion coefficients of molecules in equilibrium in the range of 10 μs – 10 ms. When the ligand is small enough that the diffusion of the macromolecule is unaffected by the ligand binding and if we assume pseudo-first-order conditions with an abundance of ligand, the on-off rate coefficients can be determined directly. To take pseudo-first-order conditions, it is necessary to separate the autocorrelation function (ACF) of individual components of bound and free ligand. Since NR strongly fluoresces when it is within the hydrophobic heme pocket of the protein but is weakly fluorescent in solution. This weakly fluorescent contribution may be significant. The FLCS method, a fusion of time-correlated single photon counting (TCSPC) and FCS, makes possible to separate bound and free NR and we can extract the on and off rate constants from the fit of the ACF. Specifically, the on rate is 30μM-1s-1 and the off rate is 730s-1 at pH7 and 20℃. The on rate appears to be activation controlled reaction, when we compared with 8-anilno-1-naphthalene sulfate (ANS).

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