Systems for Acid-Base Buffering

It should be dear that there are several possible mechanisms by which the protonation state of an enzyme may be altered in low-water media. It will often be desirable to try to maintain the optimal state by controlling acid-base conditions, rather than just relying on pH memory. This can be done by the addition to the reaction system of add-base buffers, as in aqueous media. However, the details of these buffer systems and how they work is usually somewhat different. 

The equilibria represented by Eqs. (4) and (5) can be employed to set up buffering based on agents dissolved in the bulk non-aqueous phase. As the equilibria indicate, the state of ionizable groups in the enzyme will depend on the ratio of buffer forms added to the bulk phase the acid and its ion-paired salt with Na+ (or another cation) the base and its ion-paired hydrochloride salt (or similar). Also in analogy to aqueous buffers, a given pair will only be usable over a given range of acidity/basicity. The 

Identification of buffers that can be dissolved in the bulk phase is restricted by solubility, usually of the ionized form. An alternative approach is to choose buffers expected to be almost completely insoluble in the reaction medium, which will remain as suspended crystals. Convenient choices are zwitterionic solids and their salts, which will give rise to equilibria as shown in Eqs. (8) and (9). 

Of course, if such equilibria are to be established, a mechanism is required for the transfer of H+ and counter-ions between the solid buffers and the enzyme molecules. Quite surprisingly, this usually does not seem to be a limitation. Only quite small quantities of ions must be exchanged, which will make equilibration easier. Probably traces of acids and bases soluble in the bulk phase can catalyze the transfers by equilibria such as Eqs. (4) and (5). If rates of equilibration are inadequate, deliberate addition of such transfer agents should help. 

Vulfson, P.J. Hailing and H.L. Holland, (eds.) Methods in Biotechnology Enzymes in Nonaqueous Solvents. Humana Press, Totowa, NJ, USA, 2001. 

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