Substance P (SP1-11) was exposed to a continuous flux of superoxide (O2-) or hydroxyl radicals (.OH) in a hypoxanthine (HX)/xanthine oxidase (86 mU) system in the presence of 1 mM deferoxamine and 40 mM D-mannitol or 50 μM FeCI3. 6H2O and 50 μM EDTA, respectively. O2- caused fragmentation between the Phe7 and Phe8, whereas .OH induced cleavage also between the Phe8 and Gly9. Reactive oxygen species H2O2 and HCIO did not cause fragmentation, but modification of the amino acid side chains and/or aggregation with altered hydrophobicity in reverse phase high performance liquid chromatography compared to native SP1-11. Furthermore, exposure of SP1-11 to phorbol myristate acetate preactivated neutrophils resuited in products similar to those observed upon exposure to superoxide or hydroxyl radicals in a cell-free HX/xanthine oxidase system. This study suggests that, in contrast to rigid proteins, fragmentation is relatively easily induced in a small peptide like SP1-11, perhaps due to strain on the peptide and t-carbon bonds caused by the movable, random coil configuration acquired by SP1-11 in an aqueous solution. Oxidative modification might modulate paracrine actions of SP1-11 at site of inflammation.
