Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: Measurement of Delta G and its variation along the pH scale

Free-solution capillary zone electrophoresis (CZE) can be used to monitor f olding/ unfolding transitions of proteins and to construct the classical si gmoidal transition curve describing this isomerization process. By performi ng a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea](1/2), which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an id ea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneo usly unfold even in the absence of a denaturant. The equation describing th e sigmoidal folding/ unfolding transition can be used for deriving the term DeltaG degrees, which refers to the intrinsic difference in the Gibb's fre e energy between the (total or partial) denatured state and the reference s tate, taken usually as the native configuration of a protein. The variation of DeltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically cal culated) to be of the order of 7-10 kcal/mol and is here interpreted by ass uming that at pH 2.5 and below there is an additionally stretching of the p olypeptide coil due to coulombic repulsion, as the unfolded chain looses it s zwitterionic character and assumes a pure (or very nearly so) cationic su rface. Given the minute amounts of sample required, the fully automated sta te of the analysis, the rapidity and ease of operation, it is hoped that th e CZE technique will become more and more popular in the years to come for monitoring folding/ unfolding transitions of proteins.