Tritium-hydrogen exchange experiments, using a new rapid dialysis technique, have been performed on the cyclic decapeptide gramicidin S-A in aqueous solution. The technique, countercurrent dialysis, was found to give highly accurate results and should have general applicability to exchange measurements. The molecule has eight exchangeable peptide hydrogens, all of which may be followed in the pHmin region. Slowly exchanging peptide hydrogens are clearly present. In the base-catalyzed region, there are four rapidly exchanging hydrogens and two groups of two slow hydrogens each, the slowest of which exchange more than an order of magnitude more slowly than those of poly-DL-alanine. In the acid-catalyzed region, there are again four slow hydrogens, but they are found in one kinetic class. The results for the pH dependence of the exchange rate are consistent with an EX2 mechanism of exchange. This fact constitutes important evidence for the validity of this mechanism since gramicidin S-A, with respect to exchange, is a much simpler system than a protein. The free energies of the opening reactions are calculated to be about 0.483 and 1.73 kcal per mole for the two slow classes in the base-catalyzed region. Since the energy of the molecule has been estimated to beof the order of —100 kcal/mole, it is clear that the conformational changes needed to allow the slow hydrogens to exchange are not major ones and perhaps involve only small rotations about bonds. Our data for exchange in aqueous solution are found to agree quite well with nuclear magnetic resonance data obtained in methanol and dimethyl sulfoxide, thus implying that the primary conformational determinants of this molecule are steric effects and backbone energetics. Finally, the results are shown to be consistent with one of the possible models of gramicidin S-A proposedearlier byHodgkin and Oughton (1957) (Hodgkin, D. C., and Oughton, B. M. (1957), Biochem. J. 65, 752) or by Schwyzer and Sieber (1957) (Schwyzer, R., and Sieber, P. (1957), Hele. Chem. Acta 15, 624)and the refinement of this model by Stern et al. (1968) (Stern, A., Gibbons, W., and Craig, L. C. (1968), Proc. Natl. Acad. Sci. 61, 734. © 1969, American Chemical Society. All rights reserved.
