Site-specific recognition of fluorescein by human serum albumin: A steady-state and time-resolved spectroscopic study

• Fluorescein bound to human serum albumin (HSA) is widely used in medical applications.
• In this work, we investigated the binding mechanism of fluorescein with HSA.
• We assigned a location for fluorescein inside HSA by performing a series of quenching experiments.
• Fluorescein is found to be chemically stable inside HSA with moderate binding strength.
• We found the local environment in the binding site to resemble that of water-DMSO mixture.

The increased interest in using fluorescein as a fluorescent probe in biology and medicine is associated with its distinct absorption and fluorescence signals that are transparent to biological samples. Herein, we characterize the binding mechanism of fluorescein inside human serum albumin which is used as a carrier and protector for fluorescein in medical applications. Binding of fluorescein in human serum albumin causes partial fluorescence quenching of the sole tryptophan residue in the protein (W214). The estimated W214-fluorescein distance (2.42 nm) and the calculated quenching rate constant (kq = 5.13 × 1012 M−1 s−1), both indicate binding of fluorescein in subdomain IIA. A site-competitive experiment shows that fluorescein is located in the warfarin binding pocket. The estimated binding constant (K = 10,000 M−1) points to a moderate binding strength of the fluorescein–human serum albumin complex that should not affect the fluorescein release to the target when used as a probe.

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