A Study of Membrane Protein Defects and a! Hemoglobin Chains of Red Blood Cells in Human ,8 Thalassemia*

The soluble pool of a hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, @* hemoglobin chain labeled with [SH]N-ethylmaleimide. This pool of soluble a chains was 0.067 f 0.017% of hemoglobin in blood of normal adult, 0.1 1 2 0.03% in heterozygous @ thalassemia and ranged from 0.26 to 1.30% in homozygous @ thalassemia intermedia. This elevated pool of soluble a chains observed in human @ thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble a chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In @ thalassemia intermedia the amount of insoluble a chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [SH]N-ethylmaleimide added to membrane ghosts prior to

/3 thalassemia intermedia, which usually does not necessitate transfusion, has been used to describe the many morphological, rheological, and biochemical defects of thalassemic red blood cells (see Refs. 1 and 2 for review). The cause of these abnormalities is the presence of free a chains, unpaired with /3 chains, which are unstable and oxidize. They bind to membrane and induce oxidation of lipids, proteins and lead to molecular cross-linking (3-5).
In the present study, we address the question of the relationship between the amount of soluble and insoluble a chain pool present in cell and membrane protein defects. For this purpose we have devised methods to evaluate the pools of a chains and the thiol groups of various membrane proteins.
* This work was supported by research grants from the Institut de la SantC et de la Recherche MCdicale and from D. R. E. T. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ll To whom correspondence should be addressed.
The soluble a chain pool present in all cells, from bone marrow cells to dense blood cells, was assessed by using a specific probe ( 3" hemoglobin chain labeled with [3H]NEM1 which combines with the soluble a chains present in cell lysates to form 3H Hb, separated and evaluated by electrophoresis. The insoluble a chains, remaining in the red cell ghosts after cell lysis and extensive washing of the ghosts, were evaluated by using polyacrylamide gel electrophoresis in the presence of urea and Triton X-100 (UT-PAGE) in order to separate simultaneously the various globin chains a, B, '7, and and the membrane proteins (6). The standard SDS-PAGE method was also used (7).
The thiol groups of the various membrane proteins were characterized by their assay with dithiobisnitrobenzoic acid and by the binding of [3H]N-ethylmaleimide to the red cell ghost prior to UT-PAGE and autoradiography.

EXPERIMENTAL PROCEDURES
Material-Parahydroxymercuribenzoate and phenylmethylsulfo-NEM was from Behring Diagnostics, [3H]NEM was from Du Pont-nyl fluoride were purchased from Sigma, soluene was from Packard, New England Nuclear, and antibodies were from Cappel and Jackson.
Patients-We investigated with their informed consent seven normal adult controls, two controls with high reticulocyte counts, one splenectomized control, 19 subjects with asymptomatic heterozygous @ thalassemia, and 10 patients with @ thalassemia intermedia who were not transfused during the 3 months preceding the study. Fractionation of RBCs by density on a discontinuous Stractan gradient (8) was performed for a splenectomized patient with @ thalassemia intermedia. Bone marrow cells were obtained from two normal bone marrow donors and one patient with @ thalassemia intermedia.
Preparation of Hemoglobin Subunits-Hemoglobin A was purified by DEAE-cellulose chromatography performed in 0.2 M glycine buffer, pH 7.8 (9). The subunits of Hb A were prepared by dissociation of Hb A in the presence of parahydroxymercuribenzoate followed by ion exchange chromatography, according to the method of Bucci et al. (10,11).
Preparation of Radioactive The ['HI@""" probe was able to combine to soluble cy chains in spite of the binding of two NEM molecules at the 0-93 and @-112 The abbreviations used are: NEM, N-ethylmaleimide; UT-PAGE, Triton X-100-polyacrylamide gel electrophoresis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RBCs, red blood cells. Measurement of the Free CY Chain Pool-Blood samples anticoagulated with heparin were washed immediately with sodium chloride 0.15 M by three centrifugations performed at 4 "C; the buffy coat was not removed. The packed cells were mixed with 3 volumes of water and subsequently frozen and stored in liquid nitrogen until analyzed. The membrane-free lysate of the thawed sample was obtained by centrifugation at 10,000 X g for 30 min at 4 "C. The hemoglobin concentration was adjusted to obtain 50 mg/ml in the final incubation mixture which was composed of 0.1 M sodium phosphate, pH 7.8, and also contained 13H]pNEM chains in a given Hb/p chain molar ratio.

Erythrocyte Defects in Human
The mixture was incubated at 0 "C usually for 1 h. A known volume of the incubation mixture, 3 pl, was submitted to cellulose acetate electrophoresis (11) performed with 0.03 M sodium phosphate buffer, pH 6.5, made 2 mM in KCN. Electrophoresis was performed for 90 min at 100 V and at room temperature. Subsequently, the strip was Gel Electrophoresis-SDS-PAGE of ghosts was performed according to Laemmli (7) using a 5-15% polyacrylamide gradient. A modification of the UT-PAGE described by Rovera et al. Zmmunodetection of Globin Chains-Blot transfer of proteins from gel to nitrocellulose was performed according to Waterborg and Harrington (16) and immunostained with the alkaline phosphatase to human Hb (17). reaction after having been incubated with rabbit polyclonal antibodies

RESULTS
The Soluble a Chain Pool-The electrophoresis radioactivity profiles of radioactive pNEM chains and of hemolysates incubated with pNEM chains are shown in Fig. 1. The free [3H] pNEM chains and those recombined with free a chains, i.e. present in reconstituted and radioactive Hb A are separated. We determined the best conditions to combine [3H]pNEM chains to all soluble and free a chains present in cell lysates or in artificial mixtures of purified a chains and Hb A. The molar ratio Hb/p""" chain of 20 used for blood studies, allowed all a chains to be combined in 1 h of incubation when the a chain pool was lower than 2% of the total hemoglobin as demonstrated by the analysis of artificial mixtures. With a molar Hb/pNE" ratio of 4 used to investigate bone marrow cells or blood cells fractionated by density, the recovery of a chains found to pNEM chains and present in Hb A was 100% for a chain pools ranging from 0 to 20% of Hb A. The standard deviation of the measurement was 8% of the theoretical a chain pool value. Storage of red blood cells in liquid nitrogen prior to analysis did not modify the a chain pool. Freezing in the presence of water and thawing necessary to lyse all red cells of patients with p thalassemia intermedia did not diminish the recovery of a chains added to the cells. The presence of the cell ghosts did not modify the soluble a chain pool. No exchange was observed between the added pNEM chain and PSH chain of Hb A during up to 16 h of incubation at 0 "C.
Values of the soluble a chain pool are shown in Fig. 2. Normal individuals had an a chain pool value ranging from 0.05 to 0.09% (mean, 0.067 k 0.017%). Two non-thalassemic individuals with a high reticulocyte count had an a chain pool of 0.074 and 0.080%, respectively. One control adult with a splenectomy had an a chain value of 0.09%. Heterozygotes for /3 thalassemia had an a chain pool ranging from 0.068 to 0.15% (mean = 0.11% f 0.03). The fl thalassemia intermedia condition was associated with an a chain pool ranging from 0.26 to 1.30%.
The a chain pool was heterogeneously distributed within red blood cells, as shown by density fractionation of blood cells from a splenectomized patient with p thalassemia intermedia. An 8-fold decrease of the a chain pool was observed from the light (2.4%) to the dense red cell fraction (0.3%) (Fig. 3). A 10-fold increase in the proportion of Hb F from light (1.9%) to dense cells (20.1%) was related to cell selection.
Bone marrow cells obtained from the same patient with p thalassemia intermedia contained 10% of soluble free a chains. The contamination of red blood cells was reduced by the analysis of cells contained in small fragments of bone marrow present in the sample obtained by aspiration. Bone marrow cells obtained from two normal controls had a much lower a chain pool, 0.20 and 0.14%, respectively. Red cells accounted for 60% of all cells.
Membrane Protein Defects-In thalassemia intermedia, the membrane protein profile obtained upon SDS-PAGE, used as reference for comparison to UT-PAGE, showed a 23% decrease of spectrin compared with the control value, a slight decrease of band 3, a 23% increase of bands in the 4.2-5 area, and the appearance of several minor bands between band 6 and globin. The ratio spectrinband 3 was decreased by 10% (Table I).
The electrophoretic pattern obtained by UT-PAGE is shown in Fig. 4. The proportion of various membrane proteins and of globin chains are shown in Table I1 and 111, respectively. The proportions of various globin chains evaluated as the percentage of membrane proteins are shown in Table 111. The polypeptide profile of red cell ghost proteins was identical for controls with or without splenectomy. In heterozygous /3 thalassemia there was only an increase in the a and globin chains in equivalent amount suggesting the increased binding of hemoglobin A to membrane. In splenectomized patients with @ thalassemia intermedia, the membrane protein evaluation showed a decrease in the proportion of spectrin (26%) and of ankyrin (34%) and an apparent increase in the actin fraction (103%, but not changed in SDS-PAGE), a high proportion of globin chains, mainly a chains and the presence of additional bands called U1-U7, located between the band 3 region and globin chains. A correlation was observed between the decrease of spectrin and the proportion of insoluble chains associated with cell ghosts (n = 8, R = 0.89, p < 0.005).         in dense cells ( Table V). The immunoblotting of UT-PAGE showed the presence of globin in various ghost protein fractions (Fig. 5) in B thalassemia intermedia.

Proportion of membrane proteins analyzed by UT-PAGE expressed as the percentage of total membrane proteins (without globin) of RBC separated on Stractan density gradient in 4 fractions from a splenectomized patient with a t9 thalassemia intermedia (fraction 1 is the lightest)
Free Sulfhydryl Groups-The present study showed a 27% decrease of the reactive thiol groups of ghost proteins, from 74.7 nM/mg of proteins in normal ghosts to 54.3 nM/mg in / 3 thalassemia intermedia. In the conditions used, the binding of [3H]NEM was specific to thiol groups because it was completely prevented by preincubation of ghosts with iodoacetamide (5 mM), a highly specific thiol reagent. To discriminate the very reactive from the low reactive thiol groups, we used different amounts of [3H]NEM added before, after, or with a subsaturating amount of non-radioactive NEM. The autoradiography showed, in Fig. 6 the dramatic decrease of thiol groups of membrane proteins which predominated in spectrin, ankyrin, and actin but also in uncharacterized fractions numbered 1, 2, 5, and 6 ( Table VI) other minor bands and the partial labeling of globin, mainly a chains. Fig. 7 shows the percentages of thiol groups with high or low reactivity in spectrin and ankyrin of a normal control and of the patient who had a very high soluble a chain pool (1.3%) and a very high proportion of insoluble a chains (30%) present in red cell ghosts. The most reactive thiol groups affected in j3 thalassemic ghosts were that of ankyrin decreasing from 25.2% in normal membranes to 3.5% and of spectrin 6.8% instead of 13.7% in normal membranes. In addition spectrin had more low reactive thiol groups in normal cells (49.4%) than in thalassemic cells (17.5%) while ankyrin exhibited 12.2% of the low reactive thiol groups in normal cells and 5.0% in j3 thalassemic cells.
the a chain present in bone marrow cells because of the cell heterogeneity and of non-heme proteins synthesized in addition to globin chains. This method is not suitable for studying the soluble a chain pool present in red cells which have lost the capability of protein synthesis. For these reasons we devised an affinity method to evaluate the soluble a chain pool present in all types of erythroid cells. The soluble a chain pool evaluated by this affinity method was relevant to the amount of free and soluble a chains present in intact cells in vivo at the time of sampling because the method fulfilled the conditions required for the quantitative evaluation of soluble a chains. The method is specific, very sensitive, and suitable for evaluating a chains in the presence of proteins other than hemoglobin. The sample processing is fast, performed at 0 "C, and is not associated with a loss of soluble a chains during 3 h of blood storage or upon freezing in liquid nitrogen and thawing in the conditions used.
In normal individuals, free a chains are present in red blood cells but in very small amounts, 0.067% f 0.017 of Hb. In the j3 thalassemia trait, the a chain pool was only slightly increased in spite of an unequal globin chain synthesis (alp ratio = 2) (19, 21, 22), suggesting a very efficient removal of soluble a chains in heterozygous j3 thalassemia, more by proteolysis (22-24) than by precipitation because of the absence of significant amounts of insoluble a chains associated with ghosts in heterozygous j3 thalassemia. However, selective removal of abnormal cells containing precipitated a chains could also explain the disappearance of unpaired a chains during maturation of reticulocytes and red cell aging in heterozygous B thalassemia.
In j3 thalassemia major, traces of soluble a chains able to combine with j3 chains and to form Hb A have been detected (25-27). The pool of soluble a chains observed in the present study (0.26-1.30%) was highly different from one patient to another. The amount of soluble a chains was modest with respect to total hemoglobin and to the imbalance of globin chain synthesis because of the instability of a chains (24,27) proteolysis (23, 24, [28][29][30], and removal of damaged cells by the reticuloendothelial system. In this regard four splenectomized patients with a relatively severe j3 thalassemia as determined by blood hemoglobin level had a high a chain pool (0.80-1.30). Two splenectomized patients with the lowest a chain pool (0.38-0.48) had a very mild form of j3 thalassemia suggesting that the soluble a chain pool could be related to the clinical severity of the disease in splenectomized and untransfused patients.
In j3 thalassemia intermedia, the soluble a chains are not restricted to reticulocytes as suggested by the distribution of the non-radioactive a chain pool ranging from 2.4% in most light cells containing 32% of reticulocytes to 0.3 in dense cells containing virtually no reticulocyte (<0.5%).
In bone marrow cells, a very high proportion of a chains exists in the soluble form (10% of Hb, i.e. 20% of a chains present in Hb). This high a chain pool in the bone marrow cells of a patient with B thalassemia intermedia contrasted with the small a chain pool value in normal bone marrow cells which, however, is 2-3 folds of that observed in normal blood, which is most probably related to the removal of the free and soluble a chains during maturation of normal red blood cell precursors. The much lower soluble a chain pool in reticulocytes (2.4%) than in bone marrow cells (10%) of the patient with /3 thalassemia intermedia is related to the precipitation and proteolysis of a chains and differential cell death during erythroblast maturation. Insoluble Hemoglobin Chains-Previous studies (3,32,33) of red cell ghosts from /3 thalassemic patients revealed an increase in their globin content. Results obtained in the present study using UT-PAGE showed a peculiar globin chain pattern in p thalassemia syndromes. In heterozygous / 3 thalassemia, the amounts of /3 and a globin chains present in ghosts were increased in similar proportion, suggesting that insoluble hemoglobin is increased and that most of the free a chains resulting from the inequal globin chain synthesis are removed or degraded. In this condition, the hemoglobin content of ghosts was not a simple contamination of soluble Hb because extensive washing of ghosts did not remove further hemoglobin, and ghosts from normal cells contained barely detectable hemoglobin but may more probably reflect an abnormal binding of Hb to membrane (34).
In homozygous /3 thalassemia, insoluble a hemoglobin chains have been found to be present in cell ghosts (3,33). In the present study ultrasonification of cell ghosts and subsequent centrifugation did not lead to separation of globin or hemoglobin from membrane, suggesting that hemoglobin chains were bound to membrane and not present as free inclusion bodies trapped in cell ghosts after hemolysis (35).
In addition we showed that non-a globin chains (p or /3 + y) were also present in ghosts and that their amount increased with cell density from 5.5% of total membrane proteins in light cells to 10.8% in dense cells. In contrast, the insoluble a chains decreased from 44.2 to 24% with cell density and the a/non-a ratio of globin present in ghosts dropped from 8 to 2.2. The increase in p and y chains in membrane ghosts in cells with increasing density indicates a reduced solubility of hemoglobin tetramers which may be related to increased oxidation in the thalassemic cells.
Membrane Proteins-In / 3 thalassemia intermedia changes in membrane proteins were amplified in the lightest cells which had a low hemoglobin content and a high amount of insoluble a chains. Both SDS-PAGE and UT-PAGE showed a reduction of spectrin and an increase of uncharacterized fractions. Band 3 was also reduced by SDS-PAGE but to a lower extent than spectrin chains. Consequently, the ratio Spectrinhand 3 decreased slightly in p thalassemia as shown in Table I. A slight deficit of spectrin has been also shown by Shinar et al. (33). Part of spectrin binding to intrinsic membrane proteins is reduced as shown by Platt and Falcone (36) for cells containing unstable hemoglobin, due to an increased oxidation. It is well known that oxidation of spectrin will increase the proportion of spectrin high molecular weight complexes (37,38) and impair the assembly of cytoskeleton (39,40). Diamide at very low concentration (3 p~) prevents the formation of the spectrin-4.1 complex in the presence of actin. Erythrocytes with inclusion bodies due to unstable hemoglobins have a lower spectrin and ankyrin content of membrane, an increase of the proportion of spectrin monomers, a decreased binding of spectrin to protein 4.1 and actin, an alteration of ankyrin structure decreasing the binding of normal spectrin to inside out vesicles of cells containing Heinz bodies (41,42). Shinar et 41. have recently shown (33) that in /3 thalassemia intermedia spectrin degradation products were not detectable in cell ghosts by the Western immunoblot of SDS-PAGE after the use of comprehensive antiproteolytic agents. The decrease in membrane spectrin is not specific to p thalassemia or to anemia due to unstable hemoglobin since it is also observed in hereditary spherocytosis (43), acquired disorders (44, and during blood aging in vitro (45) but not during in vivo aging of red cells fractionated by centrifugation (46). The decreased amount of normal ankyrin (34%) in /3 thalassemic cell ghosts was shown by UT-PAGE which separated ankyrin from spectrin / 3 chain better than SDS-PAGE ( 6 ) . The loss of ankyrin is similar to that of spectrin and has to be compared with the disappearance of ankyrin from the skeleton of cells containing unstable hemoglobins in which the ankyrin-spectrin association is altered, probably by oxidation (36).
Some proteins are present in increased amounts in p thalassemic cell ghosts. As suggested by Shinar et al. (33), these polypeptides could result from attachment of cytoplasmic proteins, including globin chains or polymers of native or degraded hemoglobin chains (35) and other cytosolic proteins or cross-linked membrane proteins, giving rise to higher molecular weight proteins (5), some of which are cleaved by reducing agents. Binding of globin to the membrane skeleton has been shown during aging of normal red cells in vivo (47,48) and in oxidation of normal red cells in vitro (49,50).
Topology of Sulfhydryl Groups-We confirm that in nontransfused patients with /3 thalassemia intermedia the free thiol groups of membrane proteins are reduced by 27% (51,52). Under the conditions used, 80% of normal membrane thiol groups are reacted with NEM (53). This reduction of thiol groups is not evenly distributed among membrane proteins. It affects primarily the most reactive thiol groups of spectrin (-32%) and ankyrin (-65%), in comparison to normal cells in which ankyrin contains a high proportion of the membrane most reactive thiol groups (Fig. 7). Thiol groups exhibiting low reactivity are also decreased in spectrin and ankyrin. Other unidentified protein fractions have modified reactivity of thiol groups. Heterogeneity of Free a Chains-The present study shows that the free a hemoglobin chains are present in different forms in /3 thalassemic cells: one soluble and able to combine with p hemoglobin chains and various types of insoluble a chains. One form of insoluble cy chain can be removed from membrane by dissociation in detergent. They migrate like a chains of hemoglobin in UT-PAGE which is very sensitive to small changes in protein structure induced by mutations or acetylation of NH2 terminus. Consequently, these cy chains seem to be intact a hemoglobin chains with regard to their globin structure. This hemoglobin chain fraction may be similar to that removed by high ionic strength used to induced inside out vesicles (54). Early studies have shown that the fingerprint of the tryptic peptides of the inclusion bodies were similar to that of normal a chain (55). Another type of a chain is linked to other membrane proteins by stable bounds revealed by the immunoblotting. These globin chains have an apparent molecular weight higher than that observed for free a chains and can be homopolymers of a chains (35) or crosslinked polymers with other proteins including spectrin, by disulfides or other linkages.