Monitoring the Tanford transition in beta-lactoglobulin by 8-anilino-1-naphthalene sulfonate and mass spectrometry.
The fluorescent dye 8-anilino-1-naphthalene sulfonate (ANS) is known to interact with proteins by conformation-specific hydrophobic interactions and rather nonspecific electrostatic interactions. To which category the complexes detectable by mass spectrometry (MS) belong is still the subject of debate. Here, the Tanford transition in beta-lactoglobulin (BLG) is exploited as an experimental device to expose hydrophobic binding sites by an increase in pH, rather than, as usually done, by lowering the pH. Complex formation is monitored by electrospray ionization (ESI)-MS and fluorescence spectroscopy. Both techniques reveal stronger ANS binding to BLG at pH 7.9 than at pH 5.9, suggesting that dye binding inside the calyx, which is known to be hydrophobically driven in solution, can contribute to the complexes detected by ESI-MS. Electrostatic interactions between the protein and the ANS sulfonate group can only be weaker at pH 7.9 than at pH 5.9, supporting the interpretation of the results by the protein conformational change. Lysozyme is used as a negative control, which shows no variation in the interaction with ANS in the same range of pH, in the absence of conformational changes. However, comparison of MS and fluorescence data at variable pH for BLG and myoglobin (Mb) suggests that conformation-specific ANS binding to proteins is detectable by ESI-MS only inside well-structured cavities of folded structures, like the BLG calyx and apoMb heme pocket. Indeed, ANS interactions with highly dynamic structures or molten globules, although detectable in solution, are easily lost in the gas phase.
Santambrogio
Carlo
C
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy.
Grandori
Rita
R
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D007782:Lactoglobulins / Q000737:chemistry*; D008832:Microchemistry; D036103:Nanotechnology; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D007782:Lactoglobulins