Structural changes of α-chymotrypsin in reverse micelles of AOT studied by steady state and transient state fluorescence spectroscopy

Time-resolved fluorescence of a-chymotrypsin (alpha -chym) solubilised in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles was studied as a function of the amount of encapsulated water and initial pH. In reverse micelles the anionic interface leads to some conformational rearrangements at "pH(ext)" = 10 due to electrostatic interactions with the ionised groups of alpha -chym. The data point to the protein's location in a bulk water environment, which accounts for its high stability in these reverse micelles and allows the distinction of three emitting classes of tryptophan residues within the protein's matrix. Fluorescence quenching results show that these residues are differently accessed to the quencher molecules used (acrylamide, succinimide and iodide) and are consistent with a mechanism of penetration for the interaction with the protein. (C) 2001 Elsevier Science B.V. All rights reserved.