MASS-SPECTROMETRIC DETERMINATION OF ISOTOPIC EXCHANGE-RATES OF AMIDE HYDROGENS LOCATED ON THE SURFACES OF PROTEINS

The rates at which peptide amide hydrogens in folded proteins undergo isotopic exchange are reduced by factors of 10(0)-10(-8) relative to e xchange rates at the same peptide linkages in unfolded proteins. To me asure the isotopic exchange rates of the most rapidly exchanging pepti de amide hydrogens in proteins, a now-quench deuterium exchange-in ste p has been added to the protein fragmentation/mass spectrometry method (Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531). Isotopic exc hange rates in eight short segments spanning the entire backbone of cy tochrome c have been determined for exchange-in times of 0.2-120 s. Th ese results show that the isotopic exchange rates of 10 of the peptide amide hydrogens in cytochrome c are similar to those expected for unf olded cyt c, while the exchange rates for 33 other non-hydrogen-bonded amide hydrogens are much less than expected for unfolded cyt c. Since the isotopic exchange rates of the most rapidly exchanging amide hydr ogens in folded proteins are a direct measure of their access to the a queous solvent, the ability to determine these isotopic exchange rates points to the possibility of using quenched-now amide hydrogen exchan ge and mass spectrometry as a tool for identifying protein surfaces in volved with binding.