Interaction Kinetics of Sulfadiazine and N -Acetyl-sulfadiazine with Soil Humic Acid: ESR Investigations with Nitroxide Spin Label

The interaction of sulfadiazine (SDZ) and its main metabolite N-acetyl-SDZ (N-ac-SDZ) with model humic acid was investigated with stable paramagnetic nitroxide spin probes. Leonardite humic acid (LHA) was mixed with laccase to enhance the amount of reactive quinone groups of LHA and then incubated with nitroxide spin-labelled analogs of SDZ and N-ac-SDZ. The labeling at the pyrimidine moiety of SDZ leaves the aniline moiety susceptible to covalent binding to LHA, which is blocked by the N-acetylation. A broadened electron spin resonance (ESR) signal was observed for SDZ, which increased immediately after incubation and indicates strong restriction of the re-orientational motion of the spin probe, i.e., immobilization due covalent binding of the aniline moiety of SDZ to reactive quinone sites of LHA. A fast first-order reaction with a time constant of 17.6 ± 3.4 h of covalent binding was determined. The broadened ESR signal of N-ac-SDZ declined immediately after incubation with LHA and is caused by unspecific sorption to LHA, not by covalent binding. Short time constants of the bound and free SDZ were found for the reduction by the antioxidant sodium ascorbate demonstrating that SDZ and N-ac-SDZ are not physically entrapped by LHA.