Hello,everyone!
I had a problem when I was doing fluorescence spectrum, and I hope someone can give me some advice on how to solve it.
I want to explore the interaction between synthetic peptide and thrombin by fluorescence spectrum, but my peptide has tryptophan, which will interfere with the fluorescence result, so the accurate result cannot be measured. Could anyone please provide some directions to solve the problem?
THANKS YOU!
I suggest attaching a fluorophore with excitation in the visible region of the spectrum to your peptide, somewhere in the sequence where it will not interfere with binding of the peptide to thrombin. Then you can use the fluorescence polarization of this fluorophore to measure binding of the peptide to thrombin. In this experiment, you would use a fixed concentration of the fluorescent peptide and vary the concentration of thrombin.
Is it convincing that I only used UV spectrum and circular dichroism to characterize the binding of peptide to thrombin? Because the use of introducing a new fluorescence group to do fluorescence spectrum may need to re-examine whether the peptide after the introduction of fluorescence group will cause changes in pharmacodynamics and other changes.@Adam B Shapiro
That is true. A way to get around that objection is to use the fluorescent peptide as an indicator, but measure the displacement of the fluorescent peptide by the unlabeled peptide. The experiment goes like this. First, using a single concentration of the fluorescently tagged peptide, titrate the thrombin concentration until the fluorescence polarization signal is sufficient to clearly show binding. Then, using that pairing of peptide and thrombin concentrations, titrate the unlabeled peptide concentration and observe the decrease of polarization. This is a fluorescence polarization competition assay.
Alternatively, there are plenty of other methods of measuring the equilibrium binding of a peptide to a protein. You could immobilize one or the other on an surface plasmon resonance (SPR) chip and flow the partner over the chip.
If the peptide is not too large or sticky, you could use equilibrium dialysis or equilibrium ultrafiltration.
You could use isothermal titration calorimetry.
You could use a thrombin enzyme activity assay with a synthetic chromogenic or fluorogenic substrate to measure inhibition of that activity by the peptide.
All of this assumes that the peptide is not a substrate for cleavage by thrombin.
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