The protonation state and activity of enzymes in low-water media are affected by the aqueous pH before drying (''pH memory''). However, both protonation and activity will change if buffer ions can be removed as volatile or organic-extractable weak acids or bases. With NH 4 OOCH buffers, in which both ions can be removed, pH memory disappears completely for subtilisin-catalyzed transesterification in hexane. Only weak pH memory is found with buffers having one volatile component, NH 4 -phosphate and NaOOCH. The changes in ionization state result from proton exchanges like Protein-COO ؊ NH 4 ؉ 3 Protein-COOH ؉ NH 3 (g) and Protein-NH 3 ؉ HCOO ؊ 3 Protein-NH 2 ؉ HOOCH (g). An equivalent, complementary picture is that net charges on the protein and buffer ions must remain equal and opposite. With NaOOCH buffers, loss of some HCOO ؊ ions gives a more negative net charge on the protein, balanced by the excess Na ؉ . With NH 4 -phosphate buffers, loss of NH 3 gives protein with a more positive net charge. The resulting catalytic activities were high and low, respectively, similar to those after drying from Na-phosphate buffers of optimal (8.5) and acid pH. All of the above effects have been demonstrated for both covalently immobilized subtilisin and the lyophilized free enzyme. Subtilisin lyophilized from NH 4 OOCH buffers gave pH Ϸ4 after redissolution in water, probably because removal of HCOO ؊ counterions remains incomplete. The resulting catalytic activity was low. The effects are discussed in relation to the possible locations, in low-dielectric media, of the positive charge that balances the net negative catalytic triad in active subtilisin.A central theme of enzymology is that protein ionization, which is pH-dependent, is a key determinant of enzyme activity. In low-water media (1, 2, 3), the initial demonstration of a ''pH memory'' effect (4) caused great interest. Enzymic activity was found to be critically dependent on the aqueous pH before drying, even when there is no true aqueous phase under the assay conditions. Another manifestation of this phenomenon is the observed effect on the solid state stability of proteins of ionization of labile side chains (5).pH memory has been attributed to a fixation of protein catalytic group ionizations after drying of the biocatalyst preparation. The standard model for this fixation process is the maintenance of all of the ionization states present (protein and buffer species) before the freezing of the preparation (6). The various effects of additives on the ionization state of low-water proteins has been reviewed recently (7).The most systematic studies of pH memory have all used Na or K phosphate buffers (4,8,9). Many buffers that might be used are derived from weak acids or bases that are volatile enough to be removed during drying (or hydrophobic enough to be extracted into an organic solvent). Removal of buffer ions in this way will irreversibly change the protonation state of the enzyme. We show here how this leads to the disappearance of most pH memory an...