Oxidation of bovine beta-casein by hypochlorite.
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(2) for 48 h. The N-terminal sequences of these proteins were both identical over 16 amino acids with rat beta-casein, which, in addition to its more common association with milk, is produced by cytotoxic T-lymphocytes, and has been found to have proinflammatory properties. Because of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of bovine beta-casein by HOCl. Following exposure to HOCl at 4 degrees C for 15 min, derivatization with DNPH, washing, and digestion with trypsin, the resultant peptides were separated by reverse-phase HPLC. One peptide isolated from a peak absorbing at 365 nm was identified as AVP(Y*)PQR, corresponding to amino acids 177-183 of bovine beta-casein. Analysis of the peptide by both electrospray and matrix assisted laser desorption ionization (MALDI) mass spectrometry identified a molecular ion MH+ of 1008.5 Da, which represents an increase of 178 Da from the calculated monoisotopic MH+ of the unmodified peptide of 830.45 Da. Daughter ion spectra of the doubly charged parent ion of the peptide further support the oxidation of the tyrosine to the quinone methide, with subsequent conversion to the corresponding hydrazone with DNPH. A second pair of products were identified as arising from oxidation of Y(193) within the tryptic peptide constituted by amino acids 184-202, and the corresponding chymotryptic cleavage side product, 191-202. Exposure of beta-casein to increasing amounts of HOCl revealed that M and Y residues were the most susceptible, although bovine beta-casein contains no C, and a single W, which would not be detected by our methods. The approach described in the present report can be used to evaluate the contributions of distinct mechanisms of oxidation in other experimental or pathological models.
Yang
C Y
CY
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Gu
Z W
ZW
Yang
H X
HX
Yang
M
M
Wiseman
W S
WS
Rogers
L K
LK
Welty
S E
SE
Katta
V
V
Rohde
M F
MF
Smith
C V
CV
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005609:Free Radicals; D006997:Hypochlorous Acid / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification; D010659:Phenylhydrazines / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D002364:Caseins; D005609:Free Radicals; D010446:Peptide Fragments; D010659:Phenylhydrazines; C004787:2,4-dinitrophenylhydrazine; D006997:Hypochlorous Acid; D014357:Trypsin