High density sickle cell erythrocyte core membrane skeletons demonstrate slow temperature dependent dissociation

We have previously demonstrated that slow dissociation of HDSS membrane skeletons in high ionic strength Triton X-100 buffer was related to a posttranslational modification in beta-actin, in which a disulfide bridge was formed between cysteine 284 and cysteine 373[Shartava et al: J Cell Bio 128:805, 1995]. These previous dissociation assays were limited to two homozygous (SS) sickle cell patients and a single temperature (37 degrees C). In the current work, we have expanded the SS subjects to 9 and have carried out dissociation assays at 0, 24, 30, 34, and 37 degrees C. At 0 degrees C there was limited dissociation of spectrin and actin from normal(AA), low density sickle cell(LDSS), and high density sickle cell (HDSS) core skeleton up to 24 hr. The first order rate constants for dissociation of spectrin, at 0 degrees C, was 0.030-0.035 x 10-4 sec-1 for AA,LDSS, and HDSS core skeletons. However at 24, 30, 34, and 37 degrees C the rate of dissociation of spectrin from HDSS core skeletons was significantly slower than the rate of dissociation from AA core skeletons. Having determined the first order rate constants for spectrin dissociation at these specified temperatures, we then asked whether dithiothreitol (DTT) would hasten the dissociation of core skeletons. The presence of DTT caused the rate of dissociation of the HDSS membrane skeleton to become statistically indistinguishable from the rate of dissociation of AA membrane skeletons. This is consistent with the suggestion that reversible thiol oxidation is responsible for the slow dissociation of the HDSS membrane skeleton.