Rational drug design is based on understanding the principles of molecular recognition which govern receptor-ligand interactions. Recently, investigations of the mechanism of action and design of selective inhibitors for dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3; DHFR) have been greatly facilitated by advances in X-ray crystallography, recombinant-DNA technology, and molecular modeling techniques.1-3 The enzyme, by catalyzing the NADPH-dependent reduction of 7,8-dihydrofolate (H2F) to tetrahydrofolate (H4F), is primarily responsible for the maintenance of essential intracellular cofactor pools of key importance for biosynthetic reactions requiring one-carbon unit transfer. Consequently, the synthesis of highly specific anti-folates has been spectacularly successful in producing powerful therapeutic agents.4 For example, pyrimethamine is 1400-fold more active against the malaria enzyme isolated from Plasmodium berghei than the rat liver enzyme.5 The basis for this specificity has been postulated to result from species variation along the active-site surfaces.5 Unfortunately, shortly after implementation, the therapeutic efficacy of pyrimethamine was jeopardized by the emergence of resistant strains of Plasmodium falciparum. The resistance was not caused by dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene amplification or via a reduction in the drug permeability of the cells.6 Resistant strains from various sources showed a 400-1000-fold increase in the ID50 for pyrimethamine.7 The cDNAs for DHFR from these strains were isolated, and sequence analysis revealed an asparagine for serine substitution at residue 108 in all cases.8,9 Sequence alignment of P. falciparum DHFR with Escherichia coli and vertebrate DHFRs revealed that serine-108 corresponds to strictly conserved threonine-46 (Figure 1). Since an X-ray structure of the malarial DHFR does not exist, structures of the bacterial and vertebrate DHFR-ligand complexes have been used to analyze the structural basis for resistance9 owing to the remarkable conservation observed in DHFR tertiary structures despite sequence homologies as low as 30%. Consequently, we sought to quantify the ability of E. coli DHFR to serve as a useful model for the malarial enzyme, substituting by oligonucleotide mutagenesis position 46 with serine, asparagine, and alanine, and evaluating the respective kinetic parameters for these mutant enzymes. This paper outlines the use of affinity capillary electrophoresis2,3 (ACE) as a technique for measuring binding constants of proteins for ligands. We illustrate this use with a model system comprising carbonic anhydrase B (CAB, EC 4.2.1.1, from bovine erythrocytes) and 4-alkylbenzenesulfonamides.4 The principle of the method is illustrated schematically in eq 1. The electrophoretic mobility μ of a protein is related to its mass (M) and net charge (Z) by a relationship of the approximate form μ - Z/M2/3.5 If the protein binds a charged ligand of relatively small mass, the change in μ due to the change in mass [from M2/3 to (M + m)2/3] is small relative to the change in μ due to the change in charge (from Z to Z ± z). Thus, the protein-ligand complex will migrate at a different rate than the uncomplexed protein.6 By measuring migration times (t) as a function of the concentration of charged ligand present in the buffer, it is possible to estimate Kd. These measurements are best carried out by measuring changes in t relative to another protein having a similar value of migration time