A pulse-chase-competition experiment to determine if a folding intermediate is on or off-pathway: application to ribonuclease A.

A modified pulse-chase experiment is applied to determine if the native-like intermediate IN of ribonuclease A is on or off-pathway. The 1H label retained in the native protein is compared when separate samples of 1H-labeled IN and unfolded protein are allowed to fold to native in identical conditions. The solvent is 2H2O and the pH* is such that the unfolded protein rapidly exchanges its peptide NH protons with solvent, and IN does not. If IN is on-pathway, more 1H-label will be retained in the test sample starting with IN than in the control sample starting with unfolded protein. The results show that IN is a productive (on-pathway) intermediate. Application of the modified pulse-chase experiment to the study of rapidly formed folding intermediates may be possible when a rapid mixing device is used.