Volatile buffers can override the "pH memory" of subtilisin catalysis in organic media.
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 NH4OOCH 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, NH4-phosphate and NaOOCH. The changes in ionization state result from proton exchanges like Protein-COO-NH4+ --> Protein-COOH + NH3 (g) and Protein-NH3+HCOO- --> Protein-NH2 + 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 NH4-phosphate buffers, loss of NH3 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 NH4OOCH buffers gave pH approximately 4 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.
Zacharis
E
E
Departments of Bioscience and Biotechnology and Pure and Applied Chemistry, University of Strathclyde, Glasgow, G1 1XW, United Kingdom.
Halling
P J
PJ
Rees
D G
DG
D000818:Animals; D002021:Buffers; D002384:Catalysis; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005561:Formates; D005612:Freeze Drying; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism; D009211:Myoglobin / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism*