Most of the examples I found in literature for constructing Stern Volmer plots to study interaction with HSA or BSA relies on quenching effect of the probe on the emission from tryptophan residue. The compound I am evaluating binds to site II in HSA and quenches the florescence of tyrosine in that binding site significantly compared to less pronounced effect on tryptophan based emission. However, I have not seen Stern Volmer plots based on tyrosine emission to study interaction with HSA, so would the results from this experiment be reliable ?
Thanks
Tyrosine fluorescence isn't often used because it is usually quite weak, but if you are getting a usable signal (and you are sure it is coming from the HSA rather than a contaminant), then I don't see any reason why you shouldn't use it. Since there is more tyrosine than tryptophan in most proteins, there is the possibility that the quenching is from more than one tyrosine residue.
Thanks for you reply. What do you think will be the best way to confirm that this signal is coming from HSA rather than a contaminant ?
Run a little of your HSA on SDS-PAGE with a heavy loading to check the protein purity. You should get a single band with very little else.
Run your HSA through a gel filtration column to remove any small molecules that might be bound to it or just in with it. Then see if there is any change in the Tyr fluorescence.
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