Chen Han-Yu, Teng Chung-Siang, Lin Pin-Han, Liu Ching-Ping, Liu Wei-Min, Chu Li-Kang
Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Rd, Hsinchu, 300044, Taiwan.
Department of Chemistry, Fu Jen Catholic University, 510, Zhongzheng Rd., New Taipei City, 242062, Taiwan.
Chembiochem. 2023 Oct 4;24(19):e202300370. doi: 10.1002/cbic.202300370. Epub 2023 Aug 16.
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] /[HSA] under the constraint [HSA] >[L] were collected. The van't Hoff analysis on these association constants further resulted in the thermodynamic properties. Since only two samples at different [L] /[HSA] are required without the need of [L] /[HSA] 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.
