The 1-anilino-8-naphthalenesulfonate (ANS) fluorescent dye is widely used in protein folding studies due to the significant increase in its fluorescence quantum yield upon binding to protein hydrophobic regions that become accessible during protein unfolding. However, when modeling cellular macromolecular crowding conditions in protein folding experiments in vitro using crowding agents with guanidine hydrochloride (GdnHCl) as the denaturant, the observed changes in ANS spectral characteristics require careful consideration. This study demonstrates that crowding agents can form clusters that interact differently with ANS. Furthermore, GdnHCl can disrupt these clusters and directly affect the ANS spectral characteristics. A model for the interaction between GdnHCl, crowders, and ANS is proposed. Using bovine serum albumin (BSA) as a model protein, the limitations of using ANS for studying conformational transitions induced by GdnHCl in the presence of crowding agents are demonstrated.
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