text: At low concentrations of TNP-ATP (≤1 μM), fluorescent intensity is proportional to the concentration of TNP added. However, at concentrations exceeding 1 μM, inner filter effects cause this relationship to no longer be linear. To correct this, researchers must determine the ratio of the predicted theoretical fluorescence intensity (assuming linearity) to the observed fluorescence intensity and then apply this correction factor. However, in most cases, researchers will try to keep the concentration of TNP to lower than 1 μM.
  To determine binding affinities, TNP-ATP is added to a solution and then titrated with protein. This produces a saturation curve from which the binding affinity can be determined. The number of binding sites may also be determined through this saturation curve by looking to see if there are sudden changes in slope. One can also titrate a fixed amount of protein with increasing additions of TNP-ATP to obtain a saturation curve. To do so, however, may get complicated due to the inner filter effects that will need to be corrected for.
  To determine dissociation constants, TNP-ATP can be competed off of a protein with ATP.  The value of the dissociation constant K for a single-site binding can then be obtained by applying the Langmuir equation for a curve fit:
  where RFU is relative fluorescent units, RFU is the fluorescence observed, RFU is the fluorescence of free TNP-ATP, and RFU is the fluorescence of TNP-ATP when completely bound to a protein.
  To measure an ATP competitor, one can add competitor to pre-incubated samples of protein:TNP-ATP. The fraction of TNP-ATP bound to the protein can be calculated via:
  where θ is that fraction, and RFU is the value of fluorescence intensity at saturation, meaning when 100% of TNP-ATP is bound.
  The dissociation constants for TNP and competitor can then be calculated through the equation:
  For reasons not yet fully understood, TNP-ATP typically binds the ATP binding sites of proteins and enzymes anywhere from one to three times tighter than regular ATP. The dissociation constants are usually around 0.3–50 μM.