Comparison of NH exchange and circular dichroism as techniques for measuring the parameters of the helix-coil transition in peptides.

Circular dichroism and NH exchange are compared directly as techniques for measuring helix content in peptides and the parameters of the helix-coil transition. To cover a broad range of helix contents, alanine-based peptides with chain lengths varying from 12 to 22 residues are examined over the temperature range from 0.6 to 26.9 degrees C in 1 M sodium chloride, 2H2O. Helix-coil transition theory independently fits both circular dichroism and exchange data, but the helix contents measured by exchange are larger than those measured by circular dichroism. The two techniques are brought into agreement by removing the assumption that the intrinsic chemical exchange rate in the helix is the same as the exchange rate measured for short unstructured model peptides. This modification allows the circular dichroism and NH exchange data to be described by the same set of helix parameters and indicates that the intrinsic exchange rate in the presence of helical structure is reduced approximately 17% relative to the rates measured in unstructured models. To test the possibility that this effect is electrostatic in origin, the sensitivity of the exchange reaction to ionic strength is determined. A substantial dependence of exchange rate on ionic strength is found, but the form of the dependence is complex. In studies of the exchange rates of native proteins, the exchange-competent form of the protein is assumed to exchange with the same rate constant as a blocked dipeptide with the identical amino acid sequences. Our result suggests that this assumption will be seriously in error in some cases because of charge effects in the protein.