Chevron plot

In fitting to a two-state model, the logarithm of the folding and unfolding rates is assumed to depend linearly on the denaturant concentration, thus resulting in the slopes mf and mu, called the folding and unfolding m-values, respectively (also called the kinetic m-values).

The relaxation to the new equilibrium is monitored by spectroscopic probes such as fluorescence or less frequently by circular dichroism (CD).

The final protein concentration in the mixture is usually 1-20 μM, depending on the constraints imposed by the amplitude of relaxation and the signal-to-noise ratio.

The unfolding limb is generated in a similar fashion by mixing denaturant-free protein with a concentrated denaturant solution in buffer.

In many cases the folding limb roll-overs are ignored as they occur at low denaturant concentrations, and the data is fit to a two-state model with a linear dependence of the rates.