Identification of the disulfide-linked peptide in irreversibly sickled cell beta-actin

We have previously demonstrated that the membrane skeletons of irreversibly sickled cells (ISCs) dissociate more slowly at 37 degrees C, in high ionic strength Triton X-100 buffer, than do the membrane skeletons of reversibly sickled cells or control erythrocytes [Shartava et al. (1995) J. Cell. Biol. 128, 805-818], Furthermore, we demonstrated that the major cause of this' slow dissociation was a single posttranslational modification in ISC beta-actin. Two sulfhydryl groups (Cys(284) and Cys(373)) became inaccessible to thiol reagents because of this modification. We suggested the possibility that the modification was a disulfide bridge between Cys(284) and Cys(373) since the reducing agent dithiothreitol restored the sulfhydryl groups. In this article, we directly demonstrate the existence of the disulfide bridge between cysteine(284) and cysteine(373) in ISC beta-actin. We synthesized the associated ISC beta-actin tryptic cystine-peptide (KCF-CDVDIR), characterized it by HPLC, MS, and MSMS, and identified it in the tryptic digest of the ISC beta-actin. These results support our earlier suggestion that the oxidative change in ISC beta-actin is a major cause of the irreversible sickling phenomenon.