MOLECULAR CHANGES DURING THE PHOTOOXIDATION OF α‐CRYSTALLIN IN THE PRESENCE OF UROPORPHYRIN

Abstract

Singlet oxygen reacts preferentially with three amino acids in proteins, His, Trp and Met. In order to study the specific molecular events that result from such oxidations, calf alpha-crystallin was photooxidized in the presence of uroporphyrin and the reactions were investigated by high performance liquid chromatography peptide mapping using a photodiode array detector followed by fast atom bombardment mass spectrometry (FAB-MS). From these studies, the following conclusions can be inferred: (1) Upon photooxidation residue Met-68 of the B chain is oxidized to Met sulfoxide, whereas residue Trp-60 remains intact. (2) Two of the 16 His residues in alpha-crystallin are photooxidized with an apparent pKa of ca 7.0 (3) FAB-MS analysis suggests that residue Lys-166 close to the C-terminal end of the A chain forms a cross-link with the His-7 residue close to the N-terminal end of the A chain. This may be either an inter- or intramolecular cross-link.