Evaluating the affinity and binding kinetics between small molecules or fragments and proteins is a key part of developing a specific and potent small molecule pharmaceutical. Fragments typically range in size from 100 Da to 300 Da and therefore require a technique that is capable of accurately and reliably measuring the binding of compounds as small as 100 Da. Biosensors allow for measuring kinetics and affinities in a label-free manner. In this application note, we demonstrate the sensitivity of the BiOptix 404pi biosensor by analyzing the affinity and kinetics of two very low molecular weight compounds (95 and 157 Da) as they interact with carbonic anhydrase isozyme II.
Carbonic anhydrase II (CAII) from bovine erythrocytes (Sigma) was amine-coupled to a BiOptix CMD200m sensor chip using standard EDC/NHS coupling chemistry with blocking of the remaining active ester groups by ethanolamine. Reference flow cells were activated and blocked without CAII immobilization. For benzenesulfonamide (Sigma, 157 Da) the running buffer was PBS + 3% DMSO. For methylsulfonamide, (Acros Organics, 95 Da) the running buffer was PBS. Table 1 summarizes the parameters of the kinetic assay. All samples were randomly injected in triplicate with at least five buffer blank injections initially as well as buffer blanks every fourth injection for double referencing. All injections were performed at 25ºC at a 100 μL/min flow rate.
For both compounds, the data were processed and evaluated with Scrubber 2.0 (Biologic Software) using a steady-state interaction model. Figures 1 and 2 depict the sensorgrams as well as the corresponding steady state titrations for benzenesulfonamide and methylsulfonamide, respectively. Additionally, benzenesulfonamide was fit to a 1:1 kinetic interaction model using Scrubber 2.0 as shown in Figure 1.