Ligand Binding

Figure 1 Normalized fluorescence lifetime decay spectra of free (blue) and phenytoin bound (magenta) HSA. Even small waveform changes like this yield excellent results.

Ligand binding is a important class of assays where one measures the degree to which a small molecule, the ligand, interacts with a large molecule, generally a protein. Examples of ligand binding include enzyme inhibitor assays, assyas of allosteric modulators, and many others.

Here we show the application of Direct Waveform Recording™ to the measurement of free versus phenytoin bound human serum albumin and compare the result to fluorescence intensity measurement of the same samples. The figure on the right shows the lifetime decay curves for both the free and the completely bound protein. This relatively small change still yields very useful results when properly analysed.

In figures 2 and 3 below, readings of fluorescence intensity are on the left. One can see that it is difficult to say much about which wells contain hits. On the right is data generated by automatically comparing fluorescence lifetime waveforms captured by Direct Waveform Recording™ with our proprietary software. Positive numbers on the Y-axis correspond to increased population of conformation 1 (the free protein) and negative values correspond to increase population of conformation 2 (the bound form).

Figure 2 Fluorescence Intensity

The data are clearly interpretable without any further mathematical or statistical machinations. Three wells (25, 30, and 44) are in the activation class. It appears that seven wells (11, 14, 47, 51, 56, 60, and 63) correspond to the same (increased) ligand concentration, three wells (18, 27, and 62) correspond to yet a higher ligand concentration, and one well (57) to the highest ligand concentration. This corresponds very well with the actual situation. Fifty-one wells had 10 uM concentration, three had 0 ligand concentration, seven had 20 uM concentration, three had 30 uM concentration, and one had 40 uM concentration.

Figure 3 Direct Waveform Recording™

This clearly demonstrates the power of measuring fluorescence lifetime waveforms instead of fluorescence intensity to determine ligand-protein binding in HTS assays. Using our Direct Waveform Recording™ and analysis software to capture and automatically compare these lifetime spectra represents a new paradigm and provides unprecedented data quality and speed for these and other fluorescence-based screening applications. This technology is embodied only in the NovaFluor PR Fluorescence Lifetime Plate Reader. Contact us to learn more.