Site-Specific Detection of Radicals on 伪-Lactalbumin after a Riboflavin-Sensitized Reaction, Detected by Immuno-spin Trapping, ESR, and MS
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- 作者：Trine K. Dalsgaard ; Mathilde Triquigneaux ; Leesa Deterding ; Fiona Summers ; Kalina Ranguelova ; Grith Mortensen ; Ronald P. Mason
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2013
- 出版时间：January 16, 2013
- 年：2013
- 卷：61
- 期：2
- 页码：418-426
- 全文大小：412K
- 年卷期：v.61,no.2(January 16, 2013)
- ISSN：1520-5118
文摘
Free radicals and other oxidation products were characterized on 伪-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of 伪-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.