Label‐Free and Immobilization‐Free Protein‐Binding Assays by Ultraviolet Transient Absorption Microscopy

ABSTRACT Protein–ligand interactions are central to understanding biological mechanisms and drug discovery, yet conventional assays often rely on labeling or immobilization that can alter natural binding. Here, we introduce ultraviolet transient absorption microscopy (UV‐TAM), which directly detects binding through ligand‐induced changes in the excited‐state dynamics of tryptophan residues. Using a femtosecond deep‐UV pump and a near‐UV probe, UV‐TAM enables label‐free, in‐solution measurements with only microliter sample volumes. We demonstrate its capability using plasma proteins—bovine serum albumin and hemoglobin—with alkaloid ligands, berberine and palmatine. Binding events are clearly identified through time‐resolved spectral changes. Quantitative analysis of hemoglobin–alkaloid interactions yields dissociation constants in close agreement with isothermal titration calorimetry. UV‐TAM thus provides a robust, calibration‐free platform for studying protein interactions in solution, with significant potential for biochemical research and high‐throughput drug discovery.