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Excess heme in sickle erythrocyte inside-out membranes: possible role in
thiol oxidation
SA Kuross, BH Rank and RP Hebbel
Department of Medicine, University of Minnesota Medical School,
Minneapolis.
It has been suggested that the development of sickle RBC membrane defects
might be related to abnormal amounts of membrane-associated heme (a term we
use in its generic sense to include hemoglobins, hemichromes, and free
heme). Techniques previously used to measure membrane heme, however, would
not distinguish between what is truly membrane-associated and what is
merely trapped in RBC ghost preparations. Consequently, we have examined
extensively washed inside- out membranes (IOM) prepared from normal and
sickle RBC. Approximately 25% of the sickle ghost heme is lost upon
conversion to IOM, but sickle IOM still have a significant excess (1.6 +/-
0.3 nmol heme/mg membrane protein compared with 0.7 +/- 0.2 nmol/mg for
normal IOM, P less than .001). Amounts of ghost heme are only poorly
predictive of amounts of IOM heme (r = .664). Preparation of IOM by using
isotonic lysis with saponin yields virtually identical amounts of IOM heme.
Small amounts of heme (less than 15%) can be displaced from IOM by using
manipulations that elute spectrin, displace electrostatically bound
proteins, or cleave the cytoplasmic portion of band 3. Treatment of IOM
with dithiothreitol (DTT), however, displaces the most heme (35%), and this
is almost reproduced (25% displacement) by the treatment of intact RBC with
DTT before IOM preparation. Sequential treatment with all manipulations
still leaves about 40% of the heme in sickle IOM, which indicates a
compartment more intimately associated with the membrane. At least part of
this is free heme without globin, as evidenced by abnormal binding of
radiochloroquine to sickle IOM. Conversely, some IOM-associated globin is
globin without heme because the measurement of globin per se markedly
overpredicts amount of IOM heme. There is a strong correlation between RBC
density and amounts of either ghost or IOM heme. Finally, the amount of
membrane thiol oxidation (as measured by thiol-disulfide-exchange
chromatography) does not correlate at all with ghost heme (r = .105), but
it correlates well with IOM heme (r = .877, P less than .001). These data
demonstrate that there are abnormal amounts of heme truly associated with
sickle RBC membranes, and they are consistent with the hypothesis that this
membrane-associated heme participates in the pathobiology of the sickle RBC
membrane, particularly those aspects perhaps related to thiol oxidation.
Volume 71,
Issue 4,
pp. 876-882,
04/01/1988
Copyright © 1988 by The American Society of Hematology
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