The K-i of carbonic anhydrase (CA) inhibitors is often determined by the stopped-flow CO2 hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO2 and the inability to determine the K-i below several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the K-d below several nM. In contrast, the thermal shift assay can accurately determine picomolar affinities. New equations estimating CO2 concentration were developed for the determination of k(cat) and KM of CA I and CA II. The inhibitor dose-response curves were analyzed using Hill and Morrison equations demonstrating that only the Morrison model is applicable for the determination of tight-binding inhibitor K-i. The measurements of interactions between ten inhibitors and seven CA isoforms showed the limitations and advantages of all three techniques. Inhibitor 6 exhibited the K-d of 50 pM and was highly selective towards human CA IX, an isoform which is nearly absent in healthy human, but highly over expressed in numerous cancers. Combination of inhibition and binding techniques was necessary for precise determination of CA-high-affinity inhibitor interactions and future drug design. (C) 2017 Elsevier Inc. All rights reserved.