Heats of binding protons to globular proteins

Abstract

AbstractThe heats of binding of protons ΔHb 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 ΔHb = −18 kcal/mol and pKa = 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. ΔHb 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 ΔHb observed for oxidized cytochrome c in the alkaline pH region.