Human alpha thrombin solution

The value of dissociation constant (Kd) for chosen RE31 variants was measured using a Biacore TM X100 system (GE Healthcare Life Science), human alpha-thrombin solution (Haematologic Technologies, Inc.) and commercially available Biotin CAPture KIT (GE Healthcare Life Science). The 5′-biotinylated oligonucleotides were dissolved in a running buffer -10 mM phosphate buffered saline (138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, 0.05% Tween-20, pH 7.4) to achieve a sample concentration of 1 µM. The samples were denatured at 95 °C for 6 min and then cooled to room temperature overnight. The oligonucleotides were immobilized on a chip surface via the streptavidin-biotin coupling method. The flow rate was set to 30 µl/min and the unbound oligonucleotide was removed by the treatment with 50 mM aqueous NaOH. Next, serially diluted thrombin solution in the concentration range of 12.5 -200 nM along with a bovine serum albumin (BSA, Sigma-Aldrich ® ) and sample with only running buffer were injected. The measurements were performed at 25 ºC. After each run, the system was regenerated by treatment with 50 mM aqueous NaOH. Data analysis was performed using the Biacore X100 Evaluation software and a 1:1 binding mode.