Hemin-promoted peroxidation of red cell cytoskeletal proteins

I. Solar, J. Dulitzky, N. Shaklai*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Hemin-induced crosslinking of the erythrocyte membrane proteins was analyzed at three levels: (i) whole membranes, (ii) integrated or dissociated cytoskeletons, and (iii) isolated forms of the three main cytoskeletal proteins, spectrin, actin, and protein 4.1. Addition of H2O2 and hemoglobin to resealed membranes from without did not affect any of the membrane proteins. Hemin that can transport across the membrane induced, in the presence of H2O2, crosslinking of protein 4.1 and spectrin. Both free hemin and hemoglobin added with H2O2 induced crosslinking of integer cytoskeletons and mixtures of isolated cytoskeletal proteins, but hemin was always more active. Of the three major cytoskeletal proteins, spectrin and protein 4.1 were most active while the participation of actin was only minor. The yield of crosslinked products was increased in all reaction mixtures with pH, with an apparent pK above 9.0. Replacement of H2O2 by phenylhydrazine and tert-butyl hydroperoxide resulted in crosslinking of the same proteins, but with lower activity than H2O2. Bityrosines, which were identified by their specific fluorescence emission characteristics, were formed in reaction mixtures containing hemin and hydrogen peroxide and either spectrin or protein 4.1, but not actin. On the basis of fact that bityrosines were revealed only in reaction mixtures that produced protein adducts, formation of intermolecular bityrosines was analyzed to be involved in crosslinking of the cytoskeletal proteins. Since the levels of membrane-intercalated hemin are correlated with aggregation of membrane proteins, it is suggested that the peroxidative properties of hemin are responsible for its toxicity.

Original languageEnglish
Pages (from-to)81-89
Number of pages9
JournalArchives of Biochemistry and Biophysics
Issue number1
StatePublished - 15 Nov 1990


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