Electrostatic interactions in the acid denaturation of α‐lactalbumin determined by nmr

α‐Lactalbumin (α‐LA) undergoes a pH‐dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pigα‐LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation inα‐LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation ofα‐LA are concentrated in the calcium binding region of the protein.