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The peptide concentration was 22 μM in 25 mM Mes and 10 mM β-mercaptoethanol. Excitation and emission wavelengths were 295 and 350 nm respectively. The solid line corresponds to a fit of the experimental points with eqn (3). The broken line corresponds to a fit with an equation assuming either binding of a single proton or non-cooperative binding of two protons.
