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2-Cy3 reported in ref 2e is different from common biotin-fluorophore conjugates. The emission of this compound increases at a [Cy3]:[avidin] ratio (n) between 0 and 2. At n = 1, the enhancement factor is about 1.5. However, emission quenching occurs at n > 2. At n = 4, the emission intensity of the solution is about one-half that of the control solutions.
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2. These different observations have been discussed in the text.
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note
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All of the adducts showed no or very small blue shifts (≤5 nm) in emission wavelength as compared to the corresponding free complexes. Nevertheless, the hydrophobicity associated with a protein molecule in aqueous solution cannot compare to pure dichloromethane. Thus, we suppose that the enhancement of emission intensity and lifetimes of the complexes upon binding to avidin is closely related to an increase in local hydrophobicity.
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The rate constants and selected linear plots are included in the Supporting Information.
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A lower enhancement factor (1.5) in pure water than in 50 mM phosphate buffer (2.27, Table 3) is probably due to ionic-strength effects. However, a similar enhancement factor (ca. 2.28) was achieved when a more concentrated phosphate buffer (200 mM) was used in the avidin-binding studies. It is unlikely that the enhancement factor would increase further in more concentrated buffers.
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