A partially folded intermediate conformation is induced in pectate lyase Cby the addition of 8-anilino-1-naphthalenesulfonate (ANS)

Addition of 8-anilino-1-naphthalenesulfonate (ANS) to acid-denatured pectat e lyase C (pelC) leads to a large increase in the fluorescence quantum yiel d near 480 mn. The conventional interpretation of such an observation is th at the ANS is binding to a partially folded intermediate such as a molten g lobule. Far-ultraviolet circular dichroism demonstrates that the enhanced f luorescence results from the induction of a partially folded protein specie s that adopts a large fraction of native-like secondary structure on bindin g ANS. Thus, ANS does not act as a probe to detect a partially folded speci es, but induces such a species. Near-ultraviolet circular dichroism suggest s that ANS is bound to the protein in a specific conformation. The mechanis m of ANS binding and structure induction was probed. The interaction of aci d-unfolded pelC with several ANS analogs was investigated. The results stro ngly indicate that the combined effects of hydrophobic and electrostatic in teractions account for the relatively high binding affinity of ANS for acid -unfolded pelC. These results demonstrate the need for caution in interpret ing enhancement of ANS fluorescence as evidence for the presence of molten globule or other partially folded protein intermediates.