Noncovalent Association Thermodynamics of Turn‐On Fluorescent Probes with Human Serum Albumin: Dual‐Concentration Ratio Method

A dual-concentration ratio method is developed for quantifying the binding affinity of turn-on fluorescent probes (L) and a given protein (P). Only two samples at different [L]₀/[protein]₀ are required. It is an easy way to greatly reduce the amounts of fluorescent probes and proteins, as well as the acquisition time.

Abstract

Efficient quantification of the affinity of a drug and the targeted protein is critical for strategic drug design. Among the various molecules, turn-on fluorescent probes are the most promising signal transducers to reveal the binding strength and site-specificity of designed drugs. However, the conventional method of measuring the binding ability of turn-on fluorescent probes by using the fractional occupancy under the law of mass action is time-consuming and a massive sample is required. Here, we report a new method, called dual-concentration ratio method, for quantifying the binding affinity of fluorescent probes and human serum albumin (HSA). Temperature-dependent fluorescence intensity ratios of a one-to-one complex (L ⋅ HSA) for a turn-on fluorescent probe (L), e. g., ThT (thioflavin T) or DG (dansylglycine), with HSA at two different values of [L]₀/[HSA]₀ under the constraint [HSA]₀>[L]₀ were collected. The van't Hoff analysis on these association constants further resulted in the thermodynamic properties. Since only two samples at different [L]₀/[HSA]₀ are required without the need of [L]₀/[HSA]₀ at a wide range, the dual-concentration ratio method is an easy way to greatly reduce the amounts of fluorescent probes and proteins, as well as the acquisition time.

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