HYDROGEN-EXCHANGE AND PROTEIN HYDRATION - THE DEUTERON SPIN RELAXATION DISPERSIONS OF BOVINE PANCREATIC TRYPSIN-INHIBITOR AND UBIQUITIN

Water deuteron (H-2) spin relaxation was used to study hydrogen exchange, hydration, and protein dynamics in aqueous solutions of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin. The frequency dispersion of the longitudinal H-2 relaxation rate was measured in the pD range 2 to 11 at 27 degrees C. In contrast to the previously reported water O-17 relaxation dispersion from the same samples, the H-2 dispersion depends strongly on pD. This pD dependence is due to labile protein deuterons in acidic side-chains and surface peptide groups, which exchange rapidly with water deuterons. The pD dependence of the H-2 relaxation in BPTI solutions could be quantitatively accounted for in terms of known pK values and hydrogen exchange rate constants. For ubiquitin, labile protein deuterons contribute importantly to the H-2 relaxation dispersion even in the neutral pD range. The H-2 relaxation data also provided information about the orientational order and internal motion of OD and ND bonds in side-chains and surface peptides. A comparison of the water contribution to the H-2 dispersion with the O-17 dispersion indicates that one of the four internal water molecules of BPTI, presumably the deeply buried water molecule W122, exchanges more slowly (10(-6) to 10(-4) second) than the other three (10(-8) to 10(-6) second).