Differentiation of proteins based on characteristic patterns of association and denaturation in solutions of SDS

Abstract

This paper shows that proteins display an unexpectedly wide range of behaviors in buffers containing moderate (0.1-10 mM) concentrations of SDS (complete unfolding, formation of stable intermediate states, specific association with SDS, and various kinetic phenomena); capillary electrophoresis provides a convenient method of examining these behaviors. Examination of the dynamics of the response of proteins to SDS offers a way to differentiate and characterize proteins. Based on a survey of 18 different proteins, we demonstrate that proteins differ in the concentrations of SDS at which they denature, in the rates of unfolding in SDS, and in the profiles of the denaturation pathways. We also demonstrate that these differences can be exploited in the analysis of mixtures.

Fig. 1.

Electropherograms showing the denaturation of six representative proteins. (A) Albumin from human serum. (B) Ubiquitin. (C) α-Lactalbumin. (D) β-LgbB. (E) BCA. (F) SOD. The concentration of SDS in the buffer is labeled on each electropherogram, and the concentration of SDS at which the protein denatures on the capillary is boxed and listed in Table 1. These electropherograms were measured in Tris-Gly buffer (25 mM Tris/192 mM glycine, pH 8.4) at 15°C.

Fig. 2.

A mixture of myoglobin, BCA, SOD, and α-LgbB in increasing concentrations of SDS in the separation buffer in the CE. The order of denaturation in increasing concentrations of SDS is myoglobin, β-LgbB, BCA, and SOD, which does not denature at the concentrations of SDS run in this experiment.