Determination of cis-trans proline isomerization by trypsin proteolysis. Application to a model pentapeptide and to oxidized ribonuclease A.

It is shown, by examination of a model pentapeptide, that trypsin will only cleave substrate bonds in a polypeptide chain when the peptide bond following the active bond is in the trans isomeric state. The cis form must isomerize to trans before it can be cleaved. Taking advantage of this isomeric specificity, the sequence-Lys91-Tyr92-Pro93- is examined in oxidized RNase A. It is shown that the Tyr-Pro bond exists 33% in the cis form at equilibrium and that the cis-to-trans relaxation time for isomerization is 5.0 min at 10 degrees C. The fragment 92-98 has about the same cis content (35%) as does oxidized RNase A but has a much slower relaxation time (11 min). This suggests that overall chain dynamics may exert some effect on the kinetics of isomerization.