Studies of Hydrogen Exchange in Proteins: I. The Exchange Kinetics of Bovine Carbonic Anhydrase

Abstract

Bovine carbonic anhydrase form B studied by hydrogen exchange kinetics shows slower rates than measured in model compounds for 50% of its hydrogen atoms; 18% of the hydrogen atoms remain unchanged after 24 hours. In this respect the enzyme is similar to ribonuclease and chymotrypsinogen. The pH and ionic strength dependence favors out-exchange with different rate constants for a large number of hydrogen sites both in native and in acid-unfolded states. The pH dependence further indicates that the protein molecule expands or shows loosening of structure with pH changing from 9 to 5. The activation energy for the exchange rate in the unfolded state is 21 ± 4 kcal. In the native state, the exchange of the 80 slowest hydrogen atoms shows an activation energy varying with temperature from 37 ± 4 kcal at 37° to 48 ± 4 kcal at 40°. The present observations are best explained by the existence of two exchange mechanisms; one proceeding through cooperative reversible thermal unfolding, the dominating process at higher temperatures, and another proceeding through noncooperative local rearrangements of low activation energy, dominating the exchange at low temperature.