I am working on a protein which has a affinity for complex sugars and for the same I need to study its biophysical properties and interaction with heparin using fluorescence spectroscopy. Heparin is a Glycosaminoglycans with an avg. mol. wt. of 15kDa. Is it possible to do fluorescence quenching studies using heparin?
One experiment you could try is to test the effect of heparin on the intrinsic tryptophan fluorescence of the protein. Another experiment would be to attach an extrinsic fluorophore to the protein and test for quenching by heparin.
@Adam
Thanks for your suggestion, I think I will go for intrinsic tryptophan fluorescence. Do huge mol. like heparin interfere in the experiment?
Scattering of UV light by the heparin may be an issue. If there are any fluorescent impurities they could add background fluorescence. If there are any UV absorbing substances in the heparin they could cause an inner filter effect.
You can do controls with heparin alone and N-acetyltryptophanamide (NATA)+heparin to quantify any interference and, if it is not too serious, correct for it. The purpose of NATA is to serve as a surrogate for the protein tryptophan when testing for inner filter effect and scattering of the emitted light. The assumption is that NATA itself, which has no charge, has no interaction with heparin.
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