Hydrogen exchange in proteins as a measure of solvent exclusion due to ligands. Nuclease and myoglobin.

The kinetic distribution of hydrogen atoms back-exchanging from tritium-labeled proteins has been estimated from the results of interrupted flow and slow, continuousflow gel filtration. Staphylococcal nuclease exchanges almost all of it bound tritium atoms in a few hours at pH 8.1, but in the presence of calcium ions and deoxythymidine 3’,5’-diphosphate about 34 hydrogen atoms remain associated with the protein for periods longer than 8 hours. The effect of temperature on the binding of tritium, in the presence and in the absence of these ligands, has been studied. Sperm whale myoglobin shows an analogous trapping of tritium atoms upon the addition of hematin to apomyoglobin. The hematin causes the retention of about 25 hydrogen atoms for periods longer than 20 hours at pH 7.0; protoporphyrin IX traps fewer hydrogen atoms. These results indicate that the addition of the ligands to these two proteins causes the exclusion of solvent water from otherwise exchangeable protein hydrogen atoms. The mechanism of this effect probably involves some combination of direct shielding of the protein from the solvent by the ligand, and changes in conformation and motility.