An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.

The burial of charged residues in proteins is rare as it is thermodynamically strongly disfavored. However, in "standard mechanism" protein inhibitors of serine proteinases, the P 1 residue, which is highly exposed, becomes buried in the S 1 specificity pocket of the enzyme. In many enzymes, such as Streptomyces griseus proteinase B (SGPB) the S 1 pocket is hydrophobic. We measured the pH dependence of the association equilibrium constant for the interaction of SGPB with turkey ovomucoid third domain P 1 mutants,