Conformational Studies by 1H Nuclear Magnetic Resonance of the Basic Pancreatic Trypsin Inhibitor after Reduction of the Disulfide Bond between Cys‐14 and Cys‐38

The disulfide bond Cys‐14–Cys‐38 in the basic pancreatic trypsin inhibitor was reduced and the cysteinyl side chains were protected with iodoacetamide, iodoacetic acid or ethyleneimine. At pH values near 5, the first of these protecting groups is neutral, the second negatively charged and the third positively charged. High‐resolution 1H nuclear magnetic resonance showed that the average spatial structures of the three modified proteins are essentially identical. The influence of the different electrostatic charges on the stability and the internal flexibility of the proteins could thus be studied in a fixed spatial arrangement of the polypeptide chain. In 2H2O solution at p2H 5.3, the denaturation temperatures are 79°C for the carboxymethylated, 76°C for the carboxyamidomethylated and 70°C for the aminoethylated reduced inhibitor. Comparison of the three species indicates that stabilizing and destabilizing effects of the charged groups are mainly due to local interactions with nearby charged amino acid side chains, rather than to the variations of the overall charge of the proteins. The stabilizing or destabilizing effects per charged group were estimated to be of the order of 1.0–2.0 kJ mol‐1. The different stabilities of the three proteins are also manifested in the amide proton exchange rates, whereas the internal mobilities of corresponding aromatic rings were found to be identical in the three species.