Ligand Binding

In the graphs above, 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). 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. 51 wells
had 10 uM concentration, 3 had 0 ligand concentration, 7 had 20 uM concentration, 3 had 30 uM concentration, and one had 40 uM
concentration.

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.

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.