Photooxidation of specific residues in alpha-crystallin polypeptides.
Singlet oxygen is a biologically important, photochemically generated species that preferentially oxidizes His, Trp, and Met residues of protein molecules. Calf alpha-crystallin was photooxidized with use of meso-tetra(p-sulfonatophenyl)porphyrin (TPPS) and uroporphyrin (UP) as singlet oxygen generators. The effects of photooxidation were monitored by analyzing the changes in alpha-crystallin peptide maps obtained by reversed-phase HPLC using a photodiode array absorbance detector. The reaction led to the loss of six specific peptides, five of which contained photooxidizable residues. Peptides containing His-97 and His-154 from the A chain and Met-68 from the B chain are preferentially photooxidized, suggesting that those residues have access to singlet oxygen. Trp residues in the N-terminal region are converted to NFK, whereas Trp-60 in the B chain is not photooxidized strongly suggesting that the former are close to the surface of alpha-crystallin while the latter Trp residue is buried. Only one peptide that is lost from the peptide maps does not contain a photooxidizable group; however, this peptide does contain an apparently undigested Lys residue. It is suggested that it forms a cross-link with a photooxidized His residue.
McDermott
M
M
Department of Ophthalmology, Columbia University, New York.
Chiesa
R
R
Roberts
J E
JE
Dillon
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D010782:Photolysis; D011487:Protein Conformation; D014357:Trypsin
D003459:Crystallins; D010446:Peptide Fragments; D014357:Trypsin