The binding of biotin to tetrameric avidin changes the environment of tryptophan residues. Binding reduces the total tryptophan fluorescence by 34%, shifts the emission peak from 337 to 324 nm, and reduces the fluorescence bandwidth from 61 to 46 nm. These changes are consistent with the movement of tryptophans to a nonpolar, internal environment. In the absence of biotin, iodide readily quenches the fluorescence of 20-29% of the initial fluorescence, which likely corresponds to one tryptophan located in a positively charged environment. Iodide may have weak access to additional fluorescence, corresponding to perhaps one additional tryptophan. Acrylamide, in the absence of biotin, has good access to three-fourths or more of the fluorescence, but the remainder, due to one or two tryptophans, is well shielded. The binding of biotin completely prevents iodide quenching and decreases acrylamide access dramatically. The data indicate that biotin binding shifts two or three tryptophans to an internal, hydrophobic, shielded environment.
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