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 radical in position 5 of hydrocarbon chain), detectable by electron spin resonance (ESR), was tested to evaluate pH and reaction time dependencies of hydrophobic interactions with humic acid (HA). Strong changes were observed in 5-SASL ESR spectra in the presence of HA suspensions below pH 5, with disappearance of the three isotropic narrow hyperfine lines of the nitroxide group (typical of free spin-label) and formation of "immobilized" 5-SASL spectra. These changes were interpreted as due to 5-SASL bonding with hydrophobic groups of HA, by van der Waals forces and/or hydrogen bonds, in very hydrophobic sites (probably water-protected) existent in HA below pH 5. However, such sites are absent above pH 5, as demonstrated by a specific experiment to check 5-SASL spectra reversibility. On the other hand, the HA suspension was more efficient in dissolving 5-SASL than water above pH 5. This fact also suggests the existence of "surface" hydrophobic sites, where the spin-label binds to HA while maintaining the nitroxide group in contact with water, as evidenced by the typical free spin-label spectrum and hyperfine interaction splitting (a0 = 1.574 mT). Also experiments checking 5-SASL reversibility bonding with HA were consistent with the supramolecular association model to HA.
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Embrapa Instrumentação Agropecuária, R. XV de Novembro 1452, C.P. 741, 13560-970, São Carlos, SP, Brazil, IQSC-USP.
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Martin-Neto
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D004578:Electron Spin Resonance Spectroscopy; D006812:Humic Substances / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D013229:Stearic Acids / Q000737:chemistry*
D006812:Humic Substances; D013229:Stearic Acids; C031183:stearic acid