PROTOPORPHYRIN-SENSITIZED PHOTODAMAGE IN ISOLATED MEMBRANES OF HUMAN-ERYTHROCYTES

Protoporphyrin IX (proto) is a potent photo-sensitizer of membrane damage in human erythrocyte ghosts. Under aerobic conditions (pH 8, 10 °C), exposure of ghosts in the presence of proto to filtered blue light resulted in the formation of large (>250000 daltons) aggregates of membrane proteins, as visualized by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis. Correspondingly, there was a rapid disappearance of polypeptide bands 1, 2, 4.1, and 6 and a slower decay of the remaining bands. Postirradiation treatment of membranes with di-thiothreitol or pretreatment with N-ethylmaleimide had little effect on aggregation, indicating that intermolecular -S-S-bonds (exclusive of other bonds) are not produced to any appreciable extent in the photoreaction. Photoaggregates isolated by NaDodSO4-Sepharose chromatography were resistant to dissociation in boiling 1% NaDodSO4-8 M urea, which is strong supporting evidence that stable cross-links are produced. Amino acid analysis of total protein from exten-sively cross-linked membranes revealed significant losses of histidine, cysteine, tyrosine, and tryptophan, raising the possibility that some of these residues participate in cross-linking. The antioxidants butylated hydroxytoluene and N,N--diphenyl-p-phenylenediamine were effective inhibitors of lipid peroxidation (as measured by malonaldehyde formation) in irradiated membranes. However, protein cross-linking, photoinactivation of Na+,K+-ATPase and acetylcholinesterase, and loss of SH groups were unaffected by these agents, suggesting that lipid peroxidation does not mediate the observed protein damage. It follows that malonaldehyde, originating as a byproduct of peroxidation, probably plays no role in protein cross-linking. As further support for this conclusion, membranes in which cross-linking was induced by exogenous malonaldehyde were shown to be strongly fluorescent, with an emission maximum at 460 nm (excitation at 390 nm), whereas photooxidized membranes had no fluorescence in the 400-600-nm region. © 1979, American Chemical Society. All rights reserved.