K2 k12 k21

and shifts in pKa values between native and denatured states or between bound and free forms of a complex can cause changes in binding constants or stability of the protein due to pH effects. For example, the acid denat-uration of proteins may be a consequence of anomalous shifts of the pKa values of a small number of amino acids in the native protein.19 Several possible causes of such shifts have been proposed. They may arise from interactions among ionisable groups on the protein molecule; for example, an acidic group will have its pKa lowered by interactions with basic groups. Other suggested cases of shifts of pKa include hydrogen-bonding interactions with nonionisable groups and the degree of exposure to the bulk solvent; for example, an acidic group will have its pKa increased if it is fully or partially removed from solvent, but the effect may be reversed by strong hydrogen-bonding interactions with other groups.

The calculation of pKa values of protein molecules thus requires a detailed consideration of the environment of each ionisable group, and is consequently highly complex. An additional complication is that a protein with N ionisable residues has 2N possible ionisation states; the extent of the problem is apparent when it is realised that a moderately sized protein may contain as many as 50 ionisable groups.

Various methods have been proposed whereby the microdissociation constants for the morphine system may be evaluated.14 Other drugs for which microdissociation constants have been derived include the tetracyclines,15 doxorubicin,16 cephalosporin,17 dopamine18 and the group of drugs shown in Box 3.9.13

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