Nitrate Anion as a Probe for Electrostatic Interactions in Complexes Protein-Ligand

We are proposing a new technique for studying interaction of charged biomolecules. It is based on a difference between nitrate and chloride anion influence on electrostatic interactions in order to detect the role of positively charged guanidine groups of proteins. This comparison was demonstrated for human serum albumin (HSA) interaction with a fluo- rescent reporter, CAPIDAN. Nitrate anions lower their binding constant ( K). There are two causes that are responsible for that K decrease induced by nitrate. The first, rise of the ionic strength (like to chloride), and the second, a specific one: at the same concentration and ionic strength nitrate anion more significantly lowers K than chloride anion. The study of elec- tric conductivity of chloride and nitrate salts shows that nitrate anions better than chloride anions form complexes with gu- anidine cations. Therefore it can be assumed that the nitrate-chloride techniques detect a direct contact of negatively charged carboxyl of CAPIDAN with positively charged arginine residues of HSA. HSA site I includes Arg 218 and Arg 222. It is possible that the CAPIDAN carboxyl binds to one of these arginines.