Conformational behavior of alpha-2-macroglobulin: Aggregation and inhibition induced by TFE.

Alpha-2-macroglobulin (αM), a pan-proteinase inhibitor, inhibits a variety of endogenous and exogenous proteinases and constitutes an important part of body's innate defense system. In the present study, we explored how trifluoroethanol (TFE) may modulate the structure, antiproteinase activity and aggregation of αM. TFE was sequentially added over a range of 0-20% (v/v) and the effects induced were studied by activity assay, intrinsic fluorescence, ANS fluorescence, circular dichroism, turbidity assay, Rayleigh scattering measurement and ThT fluorescence measurement. Decrease in activity and increase in fluorescence intensity of αM upon addition of TFE shows structural deviation from the native structure and suggests aggregation of protein upon solvent addition. Increase in turbidity and Rayleigh scattering of modified αM confirms the formation of aggregates. Insignificant ThT fluorescence intensity of TFE treated αM is indicative of amorphous or non-amyloid aggregation. Further, circular dichroism results indicate the changes in secondary structure of native αM as negative ellipticity decreased on addition of the polar solvent to the inhibitor. The turbidometric analysis, Rayleigh scattering, ThT fluorescence intensity of modified αM suggests that the protein might be driven towards non-amyloid or amorphous aggregation. Our studies provide important mechanistic insight how αM undergoes conformational and functional changes when exposed to TFE.