IDENTIFICATION OF THE DISULFIDE-LINKED PEPTIDE IN IRREVERSIBLY SICKLED CELL BETA-ACTIN

We have previously demonstrated that the membrane skeletons of irrever sibly sickled cells (ISCs) dissociate more slowly at 37 degrees C, in high ionic strength Triton X-100 buffer, than do the membrane skeleton s of reversibly sickled cells or control erythrocytes [Shartava et al. (1995) J. Cell. Biol. 128, 805-818], Furthermore, we demonstrated tha t the major cause of this' slow dissociation was a single posttranslat ional modification in ISC beta-actin. Two sulfhydryl groups (Cys(284) and Cys(373)) became inaccessible to thiol reagents because of this mo dification. We suggested the possibility that the modification was a d isulfide bridge between Cys(284) and Cys(373) since the reducing agent dithiothreitol restored the sulfhydryl groups. In this article, we di rectly demonstrate the existence of the disulfide bridge between cyste ine(284) and cysteine(373) in ISC beta-actin. We synthesized the assoc iated ISC beta-actin tryptic cystine-peptide (KCF-CDVDIR), characteriz ed it by HPLC, MS, and MSMS, and identified it in the tryptic digest o f the ISC beta-actin. These results support our earlier suggestion tha t the oxidative change in ISC beta-actin is a major cause of the irrev ersible sickling phenomenon.