Inhibition of erythrocyte sickling by cystamine, a thiol reagent.

Abstract

Incubation of sickle cells with cystamine, a thiol reagent, resulted in the formation of an intracellular Sethylamine derivative. The rate of the reaction was dependent upon the cystamine concentration, the temperature, and the duration of the incubation. The cystamine-treated cells demonstrated a marked inhibition of sickling under hypoxic conditions, a decrease in their mean corpuscular hemoglobin concentration, and a significant increase in their oxygen affinity. The oxygen affinity of these cells was less dependent on their mean corpuscular hemoglobin concentration than that of untreated sickle cells. The minimum gelling concentration of Sethylamine deoxyhemoglobin S was slightly increased. Cystamine did not affect the intracellular pH nor the 2,3-di hosphoglycerate level. The exact contribution of the interrelated factors in cystamine inhibition of sickling (changes in oxygen affinity, mean corpuscular hemoglobin concentration, and minimum gelling concentration) has yet to be determined. Under hypoxic conditions, hemoglobin S (Hb S) molecules polymerize either in concentrated solutions (1, 2) or within erythrocytes (3, 4), giving rise to sickled cells. The polymerization of Hb S within the erythrocytes results in an abnormally low oxygen affinity (5, 6), which is dependent on Hb concentration within the cells containing Hb S (S cells) (7).Several agents have been reported to inhibit sickling, such as cyanate (8), nitrogen mustard (9), alkylurea (10), dimethyl adipimidate (11), and, more recently, bis(N-maleimidomethyl) ether (12). Cystamine, a thiol reagent (NH2-CH2-CH2-S-S-CH2-CH2-NH2), has been shown to bind to Hb (13). The Sethylamine Hb derivative migrated as a single electrophoretic band distinct from that of the unreacted Hb. This property provided a simple means of quantitation of the cystaminereacted fraction. The S-ethylamine Hb exhibited a high oxygen affinity and a reduced Bohr effect (14). In view of these properties, it was reasonable to suspect that cystamine could exert an antisickling effect.In the present report, the effects of cystamine on the sickling of S cells and on some related phenomena are presented.MATERIALS Fresh blood samples from individuals homozygous (SS), heterozygous (AS), and doubly heterozygous (SC) for sickle hemoglobinopathy and from normal adults were drawn into EDTA as anticoagulant. The composition of this anticoagulant was 1% EDTA, 0.6% NaCI, and 6% glucose (wt/vol); it maintained a constant level of 2,3-diphosphoglycerate (P2-G) in the erythrocytes during a minimum period of 7 days at 4°. Cystamine dichloride (2,2-dithio-bisethylamine dichloride) was provided was determined on packed frozen and thawed cells, at 370, with a Radiometer pH meter fitted with a microelectrode unit (type E 5021a). Erythrocyte P2-G level was determined according to Rose and Leibowitz (15). Hemolysates were prepared according to Drabkin (16). Hb electrophoresis was performed on cellulose acetate strips at pH 8.6 with Tris-EDTA-borate buffer (17); the proportions of the various frac...

Determination of deoxyhemoglobin S polymer in sickle erythrocytes upon deoxygenation.

“…These experimental and theoretical results on the oxygen dependence of intracellular polymer formation can be compared with published data on the relationship between cell sickling and oxygen saturation (34)(35)(36)(37). In theoretical curve are presented.…”

Therapeutic Applications of Cysteamine and Cystamine in Neurodegenerative and Neuropsychiatric Diseases

“…Cysteamine elicits both beneficial and harmful effects, some of which included ulcer formation and anti-angiogenic effects (23). Cystamine, the oxidized form of cysteamine, inhibits erythrocyte sickling in sickle cell anemia (24). Incubating sickle cells with cystamine leads to the formation of an S-ethylamine derivative and a decrease in sickling under hypoxic conditions.…”

Rational Approaches to Chemotherapy: Antisickling Agents

“…It seems to us that only amine-containing residues in a protein are reasonable candidates for modification with reagents mild enough to be pharmacologically acceptable. It is true that hemoglobin contains sulfhydryl residues, one of which, 193 Cys, reacts with mercaptan-blocking reagents (35,54,55) such as mercurials and disulfide compounds, but in vivo these would wreak havoc with a wide range of enzymes.…”