Hydrophobic interactions between spin-label 5-SASL and humic acid as revealed by ESR spectroscopy

The spin-label probe 5-SASL (stearic acid spin-label with nitroxide free ra dical in position 5 of hydrocarbon chain), detectable by electron spin reso nance (ESR), was tested to evaluate pH and reaction time dependencies of hy drophobic interactions with humic acid (HA). Strong changes were observed i n 5-SASL ESR spectra in the presence of HA suspensions below pH 5, with dis appearance of the three isotropic narrow hyperfine lines of the nitroxide g roup (typical of free spin-label) and formation of "immobilized" 5-SASL spe ctra. These changes were interpreted as due to 5-SASL bonding with hydropho bic groups of HA, by van der Waals forces and/or hydrogen bonds, in very hy drophobic sites (probably water-protected) existent in HA below pH 5. Howev er, such sites are absent above pH 5, as demonstrated by a specific experim ent to check 5-SASL spectra reversibility. On the other hand, the HA suspen sion was more efficient in dissolving 5-SASL than water above pH 5. This fa ct also suggests the existence of "surface" hydrophobic sites, where the sp in-label binds to HA while maintaining the nitroxide group in contact with wafer, as evidenced by the typical free spin-label spectrum and hyperfine i nteraction splitting (a(0) = 1.574 mT). Also experiments checking 5-SASL re versibility bonding with HA were consistent with the supramolecular associa tion model to HA.