Protection of bovine serum albumin from aggregation by Tween 80.

In an attempt to explain the mechanism of protein stabilization conferred by detergents, we investigated the effect of Tween 80 on aggregation of bovine serum albumin (BSA) using circular dichroism (CD) and native gel electrophoresis. CD thermal scans showed that BSA denatures at about 54 degrees C in 20 mM Tris, pH 7.2, forming soluble aggregates. Because of this aggregation, thermal unfolding of BSA under these conditions was only partially reversible, as indicated by reduced signal changes in the second scan. On the basis of this observation, BSA was thermally stressed by incubating at 50, 60, or 70 degrees C for 15 min and then analyzed by native gel electrophoresis. There was no change at 50 degrees C before and after heating, whereas at 60 degrees C the intensity of the original monomer and dimer bands decreased and that of aggregate bands increased, with much greater changes at 70 degrees C. Addition of Tween 80 before heating reduced aggregation and increased the monomer content. These effects of Tween 80 were greater as its concentration was increased from 0.001 to 1%. There was no correlation between the protective effects and the critical micelle concentration (CMC) of Tween 80. Addition of Tween 80 after 15 min incubation at 70 degrees C, or after 70 degrees C heating followed by cooling to room temperature, had no effect, demonstrating that Tween 80 must be present during the 70 degrees C heating step to be protective. Native gel electrophoresis run at 60 degrees C showed multiple aggregate bands and new bands migrating around the dimer and monomer positions, which may correspond to precursors of aggregates. Tween 80 reduced formation of these new bands and aggregates, further demonstrating that it must be present during heating. Finally, CD thermal scans showed that 0.1% Tween 80 only slightly increased the apparent melting temperature. The observed stabilization of BSA against heat treatment is, therefore, due to Tween 80 altering aggregation behavior rather than inducing significant stabilization of the native state. Copyright 2000 Wiley-Liss, Inc.