10. A dye that is non-fluorescent in water becomes fluorescent with an emission
maximum at 430 nm when bound to a globular protein. Its absorption maximum is 400
nm. Given this information, you can conclude that the dye binding to the protein causes:
a. an increase in Stokes shift
b. a decrease in intersystem crossing efficiency
c. a decrease in excited state radiative rate
d. a decrease in excited state non-radiative rate
The dye transitions from non-fluorescent in water to fluorescent with 430 nm emission and 400 nm absorption upon globular protein binding, indicating enhanced fluorescence quantum yield. This occurs because protein binding restricts non-radiative decay pathways active in aqueous solution. The correct answer is option a. an increase in Stokes shift.
Question Breakdown
Stokes shift measures the difference between absorption (λabs) and emission (λem) maxima, here 430−400=30 nm. In water, the dye likely shows minimal or no observable emission due to efficient non-radiative relaxation via solvent interactions or twisted intramolecular charge transfer (TICT). Protein binding rigidifies the environment, enabling observable emission and a defined Stokes shift of 30 nm, thus an increase from effectively zero.
Option Analysis
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a. an increase in Stokes shift: Correct. Non-fluorescent dyes in water have quenched emission, implying no measurable λem; protein-induced fluorescence defines a 30 nm shift via reduced solvent relaxation.
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b. a decrease in intersystem crossing efficiency: Incorrect. Intersystem crossing (ISC) promotes triplet states and phosphorescence, but aqueous quenching typically involves vibrational or TICT paths, not ISC; no data suggests ISC change.
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c. a decrease in excited state radiative rate: Incorrect. Radiative rate (kr) governs emission speed; a decrease would lower quantum yield (ϕ=kr/(kr+knr)), contradicting fluorescence enhancement.
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d. a decrease in excited state non-radiative rate: Plausible but not concluded from given data. Protein binding often reduces knr (e.g., via rigidity), boosting ϕ, yet the query emphasizes spectral shift, directly evidencing Stokes shift change.
Fluorescence Mechanism
Fluorescent dyes like ANS exhibit environment-sensitive quantum yields: polar water promotes TICT/non-radiative decay, quenching emission; hydrophobic protein pockets inhibit this, enhancing fluorescence and defining Stokes shift via excited-state relaxation. This “protein-induced fluorescence enhancement” is common in biophysical probes.
