Heats of binding protons to globular proteins

The heats of binding of protons Δ H b to three globular proteins, lysozyme, chymotrysinogen A, and oxidized cytochrome c, from pH 11‐2 or lower at 25°C were determined by flow microcalorimetry. In addition, the acid–base titrations of chymotrypsinogen A and oxidized cytochrome c were investigated under conditions similar to those used in the calorimetric experiments. The results of the calorimetric experiments and the acid–base titrations for lysozyme and chymotrypsinogen A in the neutral and alkaline pH regions are in accord with the Linderström‐Lang model with a pH‐independent electrostatic factor. However, in order to interpret the results on oxidized cytochrome c in the same pH region, it is necessary to assume the protonation of two unidentified groups; one of these groups, with Δ H b = −18 kcal/mol and p K a = 9.4 was detected in previous work [Watt, G. D. & Sturtevant, J. M. (1969) Biochemistry 8, 4567]. The normal heats of protonation for carboxyl, ϵ‐amino, phenolic, α‐amino, and imidazole groups on globular proteins as deduced from the study are 0, −10.5, −6.3, −10.0, and −6.3 kcal/mol, respectively. Δ H b of chymotrypsinogen A between pH 4.5 and 1.3 is +30 kcal/mol. This value, which is undoubtedly too large to be accounted for by the protonation of normal carboxyl groups, leads to the conclusion that this protein undergoes a pH‐induced conformational change in this pH region. The same idea can be applied to explain the abnormal Δ H b observed for oxidized cytochrome c in the alkaline pH region.