Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

We utilized electrospray ionization mass spectrometry (ESI-MS) and hydrogen -deuterium exchange (HX) to detect unfolding of hen egg white lysozyme duri ng salt-induced precipitation. Deuterated lysozyme was dissolved in protona ted buffer at pH 2.16 and precipitated with ammonium sulfate, sodium chlori de, and potassium thiocyanate, ESI-MS was used to detect mass differences i n lysozyme due to the loss of deuterons for solvent protons, providing insi ght on the conformational history of the protein during the labeling experi ment. Precipitation with ammonium sulfate and sodium chloride did not unfol d lysozyme, consistent with the known stabilizing effects of kosmotropic sa lts. Potassium thiocyanate, an aggressive chaotrope, was an effective preci pitant at 0.2 M, but also disrupted lysozyme structure and caused the forma tion of precipitate fractions that did not readily redissolve into aqueous solution without the use of a chemical denaturant. Precipitation with 1.0 M thiocyanate resulted in faster rates of unfolding and larger amounts of th e insoluble precipitate. The unfolding kinetics were biphasic, exhibiting a slow phase after a few hours that presumably reflected a smaller propensit y for lysozyme to unfold in the precipitated state. Bimodal mass distributi ons in the ESI-MS spectra for the thiocyanate precipitates indicate two sta tes for lysozyme in this system, a native and a molten globule-like partial ly unfolded state. ESI-MS analysis of the insoluble precipitates indicated that they consisted primarily of protein molecules that had unfolded. inves tigation of the HX behavior of lysozyme in a KSCN solution at low protein c oncentrations confirmed the destabilizing effect of the salt on the protein structure, even when there was almost no solid phase present. The HX/ESI-M S results provide insight into the mechanism combining precipitation and de naturation for such a system, both in terms of obtaining quantitative kinet ic and stability information and the identification of the conformers prese nt. (C) 2001 John Wiley & Sons, Inc.