Faster interaction measurements using MP-SPR KineticTitration

Application Note #155

Figure 2.
Small molecular weight drug tolcapone (7 concentrations) binding to human serum albumin (HSA) measured using KineticTitration. Measured data was fitted using TraceDrawer software to determine affinity and kinetic constants. Dissociation rate was measured after the last sample. The biomolecular interaction is a two state

binding event.

KineticTitration significantly reduces time required to run an assay with different concentrations. It is also useful for interactions that are difficult to regenerate or when regeneration damages the ligand on the surface. In the measurement, analyte samples are flown over the surface in a series from low to high concentration, without dissociation and regeneration between the samples as required in other methods. The dissociation rate is measured
after the last analyte sample.

Here, KineticTitration was utilized to measure binding of small molecular weight drug (tolcapone, 273 Da) to an immobilized protein (human serum albumin, HSA). The results revealed two different binding sites, first with KD = 9.7 × 10-7 M, ka = 1.99 × 103 (M*s)-1 and kd = 1.93 × 10-3 s-1 and second with KD2 = 1.9 × 10-6 M, ka2 = 1.41 × 102 (M*s)-1, kd 2 = 2.67 × 10-4 s-1. The dissociation of Tolcapone from HSA is slow, so the typical multi-step measurement cycle would have taken 3 times more time than using KineticTitration.

Recommended instrument for this application:

200_otso_small.jpg 400_KONTIO_LOGO_24012019_cropped.jpg 210_vasa_small.jpg 410A_KAURIS_instrument.jpg  220A_naali_small.jpg 420_ilves_small.jpg
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Further reading

  • If you are interested in molecular interaction, we recommend to have a look at AN#144 about small molecule binding kinetics, AN#147 about PureKinetics™ and AN#138 about interaction of antibody and antigen.
  • Do you want to learn more about PureKinetics™? Click here.

  • Do you want to see how MP-SPR instruments work? Click here.

  • Do you want to know more about MP-SPR 420A ILVES instrument? Click here.


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