We recently observed two 2,4-dinitrophenylhydrazine (DNPH)-reactive, proteins of 40 and 120 kDa in the bronchoalveolar lavage fluids of rats exposed to >95% O, for 48 h. The Ntemunal sequences of these proteins (16 amino acids) were both identical with -casein, which is a product of cytotoxic T-lymphocytes. To examine the contributions of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of -casein by HOC1. Following exposure to 200 mol equivalents of HOC1 at 4° C for 15 min, derivatization with DNPH, washing and trypsmization, the digests were analyzed by reverse phase HPLC. Whereas tryptic digests from HOCl-treated β-casein differed little from untreated protein with detection at 220 nm, the chromatograms at 365 nm, which is characteristic of DNPH-derived hydrazones, showed no peaks in unoxidized β-casein, but a few discrete peaks were observed in the HOCl-treated material. We have isolated and obtained initial structural characterization of selected prominent peaks from the 365 nm detection. One peptide, AVPYPQR, (amino acids 177-183), was isolated and identified. Gas phase sequence analysis confirmed all amino acid residues except me fourth cycle, which did not reveal a tyrosine. Mass spectrometric analysis of the peptide by both electrospray and MALDI identified the molecular ion MH* to be 1008.5 Da, which is an increase of 178 amu from the calculated monoisotopic MH' of the unmodified peptide of 830.45 Da. The data suggest oxidation of tyr to the corresponding quinone, which is subsequently derivatized with DNPH, and oxidized further with the loss of 2H. Electrospray data supported the site of modification at tyr. Our studies suggest a reasonably selective process of oxidation of β-casein and describe an approach that should be useful for the characterization of the oxidation of the other protein as well.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - Jan 1 1996|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)