THE JOURNAL OF BIOI.OGICAL. CHEMIWHY Vol. 250. No. 22, Issue of November 25, pp. 8630-8634, 19X

Printed in U.S.A.

Patterns of Hemoglobin Assembly in Reticulocytes of Sickle Cell Trait Individuals*

(Received for publication, June 6, 1975)

JOSEPH R. SHAEFFER, MARY ANN LONGLEY,$ JOSEPH DESIMONE,~ AND LOIS J. KLEVE?

From the Department of Biology, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025

Venous blood was obtained from five sickle cell trait donors with relatively high hemoglobin S concentrations (40% of total hemoglobin) and five donors with unusually low hemoglobin S concentra- tions (25 to 30%). A fraction of cells with 15 to 20% reticulocytes was isolated from the blood and incubated with [3H]leucine in a medium supporting protein synthesis for various times from 1.25 to 60 min. Previous studies showed an imbalance in globin chain synthesis in reticulocytes of “low hemoglobin S” donors which suggested the presence of an a-thalassemia gene; reticulocytes of “high hemoglobin S” donors had balanced globin chain synthesis (DeSimone, J., Kleve, L., Longley, M. A., and Shaeffer, J. (1974) Biochem. Biophys. Res. Commun. 59, 564-569). In the present study the soluble phase of the ‘H-labeled reticulocytes was examined by electrophoresis on strips of cellulose acetate. The tetramer hemoglobins A and S were separated from each other and from a small pool of free, newly synthesized cy and p chains. Kinetics of labeling studies showed that the free a and /3 chains were intermediates in tetramer hemoglobin assembly.

The distribution of radioactivity between the cy and /3 chains of each of the electrophoretically isolated components was determined by separation of their globin chains on CM-cellulose columns. After 5 min of 3H-labeling of the reticulocytes from donors with 40% hemoglobin S the ratio of newly synthesized cy chains to p chains in the tetramer hemoglobins A and S ranged from 0.37 to 0.58. This ratio increased with longer labeling times. Almost all of the radioactivity of the free chain intermediates was in the cy chain. These results confirmed the presence of a significant pool of newly synthesized 01 chains and a normal pattern of hemoglobin assembly in which initially unlabeled (Y chains combined with labeled 0 chains when the cells were exposed to [“Hlleucine.

Conversely, in the reticulocytes of donors with 25 to 30% hemoglobin S the ratio of newly synthesized (Y chains to /3 chains in the completed hemoglobins A and S ranged from 0.96 to 1.37 and remained unchanged throughout the 3H-labeling period. The radioactivity of the free CY chain pool was substantially less than the total radioactivity of the PA and p” chain pools. These results confirmed the existence of a decreased pool size of soluble (Y chain intermediates and a pattern of hemoglobin assembly consistent with the presence of an a-thalassemia gene.

Individuals with sickle cell trait have both hemoglobin A and hemoglobin S in their peripheral blood. In most of these people the concentration of hemoglobin S is 35 to 45% of the total blood hemoglobin, the remainder being largely hemoglobin A (l-5). In a few individuals the blood concentration of hemoglo- bin S is as low as 25 to 30%. Neel et al. (6) suggested that genetic rather than environmental causes were responsible for the wide variation in blood hemoglobin S/hemoglobin A ratios

* This work was supported by Medical Genetics Center Grant GM 19513 from the National Institute of General Medical Sciences.

*Present address, Department of Pathology, The University of Texas Health Science Center Medical School, Houston, Texas 77025.

9; Present address, Center for Genetics, School of Basic Medical Sciences, University of Illinois Medical Center, Chicago, Illinois 60612.

ll Present address, Department of Human Biological Chemistry and Genetics. The Universitv of Texas Medical Branch, Galveston, Texas

because (a) the hemoglobin S concentration in a given individ- ual remained nearly constant and (b) many, although not all, of the relatives of an individual with sickle cell trait had similar amounts of hemoglobin S. Subsequent genetic and hematologi- cal data (7-10) suggested that sickle cell trait individuals with unusually low concentrations of hemoglobin S (25 to 30%) might carry a gene for a-thalassemia, an inherited disorder characterized primarily by a decrease in the production of hemoglobin (Y chains (11, 12). Presumably the products of the a-thalassemia gene interacted with those of the sickling gene to produce an unusually low hemoglobin S concentration in the circulating blood.

Recent studies in this laboratory showed that there was a relative deficit in (Y chain compared to non-a chain synthesis in the reticulocytes of sickle cell trait individuals with 26 to 32% hemoglobin S (13). Conversely, reticulocytes of individuals

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with 40 to 42% hemoglobin S had a balanced globin chain synthesis. These findings supported the hypothesis that sickle cell trait people with unusually low hemoglobin S concentra- tions carry an u-thalassemia gene. In this report we show that the detailed intracellular patterns of hemoglobin assembly in these reticulocytes were consistent with this conclusion. Some of these results were presented previously in a preliminary manner (14, 15).

METHODS AND MATERIALS

Isolation and Labeling of Intact Reticulocytes-Venous blood (100 to 125 ml) was withdrawn from 10 healthy, nonanemic black adults into heparinized syringes and chilled in ice at 4”. A fraction of cells with 15 to 20% reticulocytes was isolated from the blood by centrifuga- tion on gradients of Ficoll and Renografin, as described previously (16). Packed reticulocyte-rich cells (0.13 ml) were added to 0.39 ml of a Ilh-fold concentration of an incubation medium prepared without plasma as described by Boyer et al. (17) except for the omission of L-leucine in the amino acid supply. After addition of 0.03 ml of water, the reaction mixture was incubated for 5 min at 37”, and 2.0 mCi (0.10 ml) of L-[4,5-SH]leucine (60,000 mCi/mmol) were added. Replicate assays of this composition were incubated at 37” for various times from 1.25 to 60 min. The assays were chilled in ice at 4’, and 8 ml of a modified saline solution (0.13 M NaCl, 0.005 M KCl, 0.0075 M MgCl,) were added to each to stop the JH-labeling procedure.

Analysis of Soluble Products-Centrifuged 3H-labeled reticulocytes were lysed with four volumes (0.52 ml) of 2.5 mM MgCl, and constant, gentle stirring for 2 min at 4’. One VOhme (0.13 ml) of 1.5 M sucrose containing 0.15 M KC1 and 20 ~1 of 0.1 M L-leucine were added. The ghost fraction was removed by centrifugation for 15 min at 24,000 x g, and the resulting lysate was recentrifuged for 90 min at 105,000 x g to isolate the soluble fraction of the cells.

The distribution of protein radioactivity in the soluble phase was analyzed by electrophoresis of a 5~1 sample on a strip of cellulose acetate. The electrophoresis was done for 3 hours at 400 volts and 5” in a Tris/EDTtVborate buffer, pH 8.7. The strip was stained with ponceau S in 5% trichloroacetic acid to detect the major hemoglobin bands, dried, and cut into sections 2 mm wide. After each section was placed in a glass vial, 10 ml of a toluene-based liquid scintillation fluor were added. The fluor was prepared by adding 84 ml of Liquifluor (New England Nuclear) and 232 ml of Soluene 100 (Packard Instruments) to 2000 ml of toluene. The vials were counted in a liquid scintillation spectrometer. The recovery of protein radioactivity from the strip sections was routinely higher than 85% of that applied.

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In some experiments electrophoresis of the $H-labeled soluble phase on cellulose acetate strips was done in replicate. Sections of the unstained strips containing hemoglobin A or hemoglobin S or newly synthesized globin chains (see “Results and Discussion”) were excised and pooled with carrier globin A (30 mg), globin S (30 mg), or a mixture of the two globins (15 mg each), respectively, in acidified acetone. The globin mixture was washed several times with acidified acetone, and the (Y and fl chains were separated on a column (0.9 x 12 cm) of CM-cellulose at pH 6.8 as described by Clegg et al. (18). The eluted fractions were precipitated with 7% trichloroacetic acid and washed onto cellulose nitrate membrane filters for assay of radioactiv- ity in a dioxane-based liquid scintillation system described previously (19).

Muterials-The [3H lleucine was obtained from Schwarz/Mann, CM-cellulose (Serva, 0.65 meq/g) was purchased from Gallard-Schles- inger Chemical Manufacturing Corp. Cellulose acetate strips (Sepra- phore III) and the hemoglobin electrophoresis buffer were from Gelman Instrument Co., and cellulose nitrate filters (Bactiflex B6) were from Carl Schleicher and Schuell Co.

RESULTS AND DISCUSSION

Samples of blood from each donor were used to identify the presence of sickle cell trait on the basis of (a) the pattern observed after hemoglobin electrophoresis and (b) a positive turbidity test (Sickledex, Ortho Diagnostics) result. The rela- tive amounts of hemoglobins A and S in the blood were determined from the absorbance of these hemoglobins, stained after electrophoresis on cellulose acetate strips, as described previously (20). Five donors had hemoglobin S concentrations (40 to 42% S, 60 to 58% A) above the mean value (38% S, 62% A) reported for various North American populations (1, 3); five had hemoglobin S values (26 to 32% S, 74 to 68% A) near the lower end of the range. Total blood hemoglobin and serum iron concentrations of all donors were within normal limits.

Reticulocytes from each donor were labeled with [3H]leucine in a medium supporting protein synthesis, and the soluble phase of the cells was subsequently isolated (see “Methods and Materials”). Fig. 1A shows the pattern of protein radioactivity after electrophoresis of the soluble phase from reticulocytes pulse-labeled for 5 min. About 65% of the total protein radioactivity migrated with the tetramer hemoglobins A and S.

B

6

3 0 x4

z 0

2

2

0.

0

A. PULSE

a 68 GLOBIN

,

B. CHASE I

? +

FIG. 1. Patterns of protein radioac- tivity after electrophoresis of 5 pl of the soluble phase of 3H-labeled reticulocytes on cellulose acetate strips. A, reticulo- cytes from donor B.L. were pulse- labeled with LdH]leucine for 5 min. B, an identical batch of reticulocytes was pulse-labeled in a similar manner and subsequently incubated in a nonradi- oactive medium for an additional 30 min (chase). The positions of the stained bands of hemoglobins A and S are indi- cated at the top of each panel, and the line of sample application is denoted by zero.

DISTANCE (cm)

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The remainder migrated immediately anodal to the line of sample application and was not associated with any proteins present in concentrations sufficient to be stained by ponceau S. Previous studies with reticulocytes of rabbits (21, 22) and human beings with various anemias (20, 23) showed that this latter component represented small pools of free, newly synthe- sized a-hemoglobin and (Y- and /3-globin chains.

Patterns of protein radioactivity in the soluble phase of reticulocytes labeled for various times were qualitatively similar to that shown in Fig. IA. However, the distribution of protein radioactivity among the various components changed with time of labeling. Fig. 2 shows that the globin chain fraction was labeled initially faster than the tetramer hemo- globins. As time progressed, an increasingly larger fraction of the total soluble protein radioactivity migrated with hemo- globins A and S. This pattern of labeling kinetics suggested that the pools of newly synthesized chains were precursors to the completed, tetramer hemoglobins. To confirm this observa- tion, we incubated a batch of pulse-labeled reticulocytes, identical with those used in the assay of Fig. lA, for an additional 30 min in nonradioactive medium. Fig. 1B shows that, after this chase incubation, little radioactivity was associated with the soluble chain pools while that migrating with hemoglobins A and S was substantially increased. This result suggested that the newly synthesized globin and hemo- globin chains were intermediates in the assembly of the tetramer hemoglobins. Similar results were obtained in the previous studies with animal and human reticulocytes (20-22).

The temporal patterns of ‘H-labeling of the soluble protein components were similar for reticulocytes from sickle cell trait individuals with either high or low concentrations of hemoglo- bin S. However, there were definite and characteristic differ- ences between the two classes of donors in the rates of labeling of the a# and ps chain types in these soluble components. The electrophoresis of a 3H-labeled soluble phase from reticu-

I I I 5

I 10 15 20 TIME (min)

FIG. 2. Distribution of protein radioactivity between tetramer he- moglobins (Hbs) A and S (O- - -0) and free, newly synthesized OI- and P-globin chains (A-A) after electrophoresis of soluble fractions isolated from labeled reticulocytes. Replicate batches of reticulocytes from donor A.C. were incubated with [3H]leucine for various times, and the soluble phases of the cells were analyzed as described in the legend to Fig. 1. The total soluble protein is represented by the total protein radioactivity applied to the electrophoresis strip (O---O).

locytes of a donor with a relatively high concentration of hemoglobin S was done in replicate on cellulose acetate strips. The bands of the tetramer hemoglobin A were excised from the strips, pooled, and converted to globin in the presence of carrier globin A. The distribution of radioactivity between the (Y- and PA-globin chains was determined by separation of the two chains on a column of CM-cellulose (see “Methods and Materials”). Fig. 3A shows that after 5 min of labeling about 70% of the hemoglobin A radioactivity was in the p chain and only 30% in the cy chain. Similar results were obtained when the (Y- and OS-globin chains of the ‘H-labeled hemoglobin S tetramer were analyzed by chain separation chromatography (data not shown).

The extent of labeling of the p chain of either hemoglobin A or S was substantially higher than that of the complimentary (Y chain throughout the 40-min period of labeling of reticulocytes from a high hemoglobin S donor (Table I). However, for both hemoglobins A and S the fraction of radioactivity in the LY chain increased from about 30% at 5 min to higher than 40% at 40 min. Since each chain type, (Y or /3, has 18 leucine residues, the relative rate of [3H]leucine incorporation represented the relative rate of appearance of newly synthesized chains in the hemoglobins. The data were therefore consistent with the presence of a cellular pool of soluble (Y chains, largely unlabeled in the initial stages of the incubation with [3H]leucine, which combined with much smaller pools of newly synthesized, labeled PA and ps chains to form hemoglobins A and S, respectively. As time progressed, the specific radioactivity of

0.6 T:

04

02

I O i E

8

4" 06

0.4

02

0 k 20

Fraction Number

FIG. 3. Elution patterns from the chromatographic separation of the (Y- and P-globin chains of 3H-labeled hemoglobin A (HbA) in reticulocytes of (A) donor M.C. with a high hemoglobin S (HbS) concentration and (B) donor J.P. with a low hemoglobin S concentra- tion. Reticulocytes were incubated with [3H lleucine for 5 min, and the labeled soluble phases were analyzed by electrophoresis on 11 replicate cellulose acetate strips as described in the legend to Fig. 1. The separated hemoglobin A bands were excised and pooled in acidified acetone with 30 mg of carrier globin. The 3H-labeled globin samples were chromatographed on CM-cellulose columns, and the absorbance at 280 nm (---) and protein radioactivity (0- - -0) of each fraction are shown. Recovery of “H-labeled protein radioactivity from each column was 99%. The relative amounts of hemoglobins A and S in the blood of the donors are given at the top of each panel.

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TABLE I

Distribution of radzoactzvity inglobin chums of hemoglobins A and S

Reticulocytes from donor J.B. (A) with a high hemoglobin S concentration and donor A.C. (B) with a low hemoglobin S concentra- tion were incubated with [3H]leucine for various times. The total radioactivity migrating with each tetramer hemoglobin was deter- mined by electrophoresis of the soluble phases of the cells as described in the legend to Fig. 2. The separated hemoglobin A (HbA) and hemoglobin S (HbS) bands from replicate electrophoresis strips were converted to globin, and the percentage of hemoglobin radioactivity in the (Y and p chain components was determined by column chromatog- raphy of the labeled globin samples, as described in the legend to Fig. 3.

0.6.

0.4.

0.2

0.

I

8 0.6 elm

0.4.

QZ

0. :

0

e

If

!O

I. n HbA, HbS: 58142

; \ ’ HbA/HbS:72/28

I 40 60 FRACTION NUMBER

0 -c - , 80

0

9

A 1200 I

P) z ”

0

FIG. 4. Elution patterns from the separation of the pool of free, newly synthesized 01, j?’ and ps globin chains. Reticulocytes of donor E.S. (A) with a high hemoglobin S (HbS) concentration and donor B.L. (B) with a low hemoglobin S concentration were incubated with [3H]leucine for 5 and 20 min, respectively. The labeled soluble phases were analyzed by electrophoresis on three replicate cellulose acetate strips, and the regions (peaks) of protein radioactivity immediately anodal to the line of application (see Fig. 1A) were excised and pooled in acidified acetone with 15 mg of globin A and 15 mg of globin S. The SH-labeled globin mixtures were chromatographed on CM-cellulose columns, and the recoveries of protein radioactivity were (A) 60% and (B) 69%. Absorbance at 280 nm, -; W-labeled protein radioactivity, 0- - -0. HbA, hemoglobin A.

the (Y chain pool increased, and hence, the fraction of tetramer hemoglobin radioactivity in the cr chain was enhanced.

To provide additional evidence for this model, we deter- mined the distribution of radioactivity among the (Y and non-o chain types in the putative soluble globin pool. As expected, Fig. 4A shows that most of the radioactivity eluted with the (Y chain. Other workers, using methods different from those of this laboratory, previously reported the presence of small pools of newly synthesized a-globin and a-hemoglobin chains in rabbit (24-27) and human (28-30) erythroid cells. The present results suggested that the patterns of hemoglobin assembly in the reticulocytes of sickle cell trait donors with relatively high hemoglobin S levels were similar to those observed previously by us (20-22, 31) and others (24-28, 32) for the normal assembly of mammalian hemoglobins.

Conversely, when the hemoglobin A tetramer in reticulo- cytes from a donor with a relatively low hemoglobin S concentration was examined, Fig. 3B shows that, after 5 min of VH-labeling, about 55% of the radioactivity was in the (Y chain and 45% in the fi chain. Moreover, the rate of appearance of newly synthesized LY chains into hemoglobins A or S was approximately equivalent to that of newly synthesized ,0 chains throughout the labeling period for “low hemoglobin S” reticu-

Sample Incuba- Total

tion radioac- % In a % in f3 Ratio tnne tivity dP

mill

A. HbA/HbS:58/42 HbA 5

10 20 40

HbS 5 10 20 40

B. HbA/HbS:68/32 HbA 1.25

2.5 5.0

20.0 HbS 1.25

2.5 5.0

20.0

dpm

3122 27.2 72.8 0.37 8562 35.5 64.5 0.55

13460 39.4 60.6 0.65 45693 39.8 60.2 0.66

1843 34.8 65.2 0.53 5250 40.0 60.0 0.67

11378 41.8 58.2 0.72 33297 45.0 55.0 0.82

4713 53.9 46.1 1.17 11834 50.5 49.5 1.02 25787 49.0 51.6 0.96

121380 53.3 46.7 1.14 2545 54.8 45.2 1.21 6253 47.9 52.1 0.92

14635 52.2 47.8 1.09 62283 49.7 50.3 0.99

locytes (Table I). These data suggested that the specific radioactivity of the soluble (Y chain intermediates was about equal to or slightly higher than that of the PA or p” chain intermediates in these reticulocytes. The results, when com- pared with those from reticulocytes of donors with the higher hemoglobin S levels, were consistent with a decreased pool size of free, newly synthesized (Y chains relative to that of newly synthesized non-a chains. Indeed, Fig. 4B shows that the radioactivity of the free LY chain pool‘was substantially less than the total radioactivity of the /3 chain pools in the soluble phase of ‘H-labeled reticulocytes from a representative low hemoglobin S donor.

Finally, data from the reticulocytes of all the donors in each class were similar to those described above. Table II shows that, in the reticulocytes of the five sickle cell trait individuals with about 40% hemoglobin S and a control person, homozy- gous for normal hemoglobin A, total soluble (Y chain synthesis was equal to total fi chain synthesis, as reported previously (13). After 5 min of incubation with [3H]leucine, the ratio of newly synthesized (Y chains to p chains in the completed hemoglobins A and S ranged from 0.37 to 0.58. These results confirmed the presence of a significant pool of soluble o( chain intermediates and a normal pattern of tetramer hemoglobin assembly. Conversely, in the reticulocytes of the five sickle cell trait donors with about 25 to 30% hemoglobin S, total soluble LY chain synthesis was 60 to 80% that of total p chain synthesis (Table II). The ratio of newly synthesized (Y chains to p chains in the completed hemoglobins ranged from 0.96 to 1.37. These results confirmed the existence of a decreased pool size of

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TABLE II

Globin chain synthesis and hemoglobin assembly patterns in high and low hemoglobin S reticulocytes

Reticulocytes from five donors with high hemoglobin S concentra- tions and five donors with low hemoglobin S concentrations were incubated with [3H]leucine for 5 min. The relative amounts of hemoglobins A and S in the blood are given in the third column. The ratio of total soluble (Y chain to p chain synthesis was determined as described previously (13). The ratio of a chain radioactivity to fl chain radioactivity in the tetramer hemoglobins A and S was determined as described in the legend to Table I

DOnOr Sex Ratio Total adpmlpdpm in HbAlHbS soluble

ndpml&xn HbA HbS

Control J.S.

High HbS E.S. I.B. M.C. P.A. J.B.

Low HbS B.L. A.C. J.P.” R.P.” E.P.”

M A/A 1.00 0.50

F 58142 1.02 0.46 0.43 F 60/40 1.01 0.55 0.41 F 59141 0.95 0.37 0.42 F 58142 1.01 0.58 0.56 F 58142 1.15 0.37 0.53

F 72128 0.80 1.25 1.12 M 68132 0.76 0.96 1.09 M 71129 0.61 1.37 1.26 F 74126 0.71 1.17 1.04 F 71129 0.76O 1.15O 1.216

“J.P. and R.P. were son and daughter, respectively, of E.P.; the other donors were unrelated to each other and to the P. family.

a Values for incubation time of 2.5 min.

soluble LY chain intermediates and a pattern of hemoglobin assembly consistent with the presence of an a-thalassemia gene.

Studies of one family, recently concluded in our laboratory, showed the inherited nature of the a-thalassemia characteris- tics in sickle cell trait members with low hemoglobin S concentrations (33). Other investigators reported, from the results of hemoglobin synthesis studies, the presence of an cu.thalassemia gene in sickle cell trait individuals with hemo- globin S concentrations below 35% (34). This population of individuals, like ours, lived in the United States. Conversely, globin chain synthesis studies of a Nigerian population of sickle cell trait people did not support the presence of an a-thalassemia gene (5). Moreover, the curve of the distribution of hemoglobin S concentration among this latter population, unlike those reported for North American populations (1, 3), did not show a marked skewing toward lower hemoglobin S values. The apparent discrepancy in the findings between the Nigerian and United States populations may result from a heterogeneity in the expression of various a-thalassemia genes.

Previous studies by our laboratory showed that the presence of the a-thalassemia gene was partially responsible for the disparity between the concentrations of hemoglobins A and S in those people with about 25 to 30% hemoglobin S (35). Work in progress, however, suggests that the a-thalassemia gene is not responsible for the total amount of this disparity.

Acknowledgments-We are grateful to those individuals who volunteered blood samples, to the Research Hematology Labo- ratory, Texas Children’s Hospital and the Sickle Cell Disease

Research Foundation of Texas, Inc. for contacting them, and to Dr. Raymond Alexanian and his laboratory at this institution for their participation in the hematological aspects of this study.

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REFERENCES

Wells, I. C., and Itano, H. A. (1951) J. Biol. Chem. 188, 65-74 Tondo, C. V., and Salzano, F. M. (1962) Am. J. Hum. Genet. 14,

401-409 Wrightstone, R. N., Huisman, T. H. J., and Van Der Sar, A. (1968)

Clin. Chim. Acta 22, 593-602 Nance, W. E., and Grove, J. (1972) Science 177, 716-718 Esan, G. J. F., and Adesina, T. A. 0. (1974) Stand. J. Haematol.

13, 370-376 Neel, J. V., Wells, I. C., and Itano, H. A. (1951) J. Clin. Inuest. 30,

1120-1124 Cohen, F., Zuelzer, W. W., Neel, J. V., and Robinson, A. R. (1959)

Blood 14, 816-827 Weatherall, D. J. (1964) Ann. N. Y. Acad. Sci. 119, 463473 Huisman, T. H. J. (1960) Clin. Chim. Acta 5, 709-718 Weatherall, D. J., Clegg, J. B., Blankson, J., and McNeil, J. R.

(1969) Brit. J. Haematol. 17, 517-526 Weatherall, D. J., and Clegg, J. B. (1972) The Thalassaemia

Syndromes, 2nd ed.. un. 223-235. Blackwell Scientific Publica- tions, Oxford

. .

Schwartz, E., and Atwater, J. (1972) J. Clin. Inuest. 51, 412-418 DeSimone, J., Kleve, L., Longley, M. A., and Shaeffer, J. (1974)

Biochem. Biophys. Res. Commun. 59, 564-569 Shaeffer, J., DeSimone, J., and Kleve. L. (1974) Fed. Proc. 33,

1549 Shaeffer, J., Longley, M. A., DeSimone, J., and Kleve, L. (1974) in

Proceedings of the First National Symposium on Sickle Cell Disease, pp. 179-181, United States Department of Health, Education, and Welfare Publication No. (NIH) 75-723, Be- thesda

DeSimone, J., Kleve, L., and Shaeffer, ,J. (1974) J. Lab. Clin. Med. 84, 517-524

Boyer, S. H., Crosby, E. F., and Noyes, A. N. (1968) Johns Hopkins Med. J. 123, 85-91

Clegg, J. B., Naughton, M. A., and Weatherall, D. J. (1966) J. Mol. Biol. 19, 91-108

Shaeffer, J., Favelukes, G., and Schweet, R. (1964) Biochim. Blophys. Acta 80, 247-255

Shaeffer, J. R. (1973) J. Blol. Chem. 248, 7473-7480 Shaeffer, J. R. (1967) Biochem. Blophys. Res. Commun. 28,

6477652 Shaeffer, J. R., and Altenburg, L. C. (1974) J. Biol. Chem. 249,

2243-2248 Shaeffer, J. R., Trostle, P. K., and Baker, B. L. (1970) in Tenth

International Cancer Congress Abstracts, p, 285, Medical Arts Publishing Co., Houston

Baglioni, C., and Campana, T. (1967) Eur. J. Biochem. 2,480-492 Kruh, J., and Blum, N. (1968) Biochim. Biophys. Acta 161,

215-222 Tavill, A. S., Grayzel, A. I., London, I. M., Williams, M. K., and

Vanderhoff, G. A. (1968) J. Biol. Chem. 243, 4987-4999 Wolf, J. L., Mason, R. G., and Honig, G. R. (1973) Proc. Natl.

Acad. Sci. U. S. A. 70, 3405-3409 Modell, C. B., Latter, A., Steadman, J. H., and Huehns, E. R.

(1969) Brit. J. Haematol. 17, 485-501 Kohne, E., and Kleihauer, E. (1973) Res. Enp. Med. 161, 243-250 Gill, F. M., and Schwartz, E. (1973) J. Clin. Inuest. 52,3057-3063 Shaeffer, J. R., Trostle, P. K., and Evans, R. F. (1969) J. Blol.

Chem. 244, 4284-4291 Heywood, D., Karon, M., and Weissman, S. (1965) J. Lab. Clin.

Med. 66, 476-482 Shaeffer, J., DeSimone, J., and Kleve, L. (1974) Am. J. Hum.

Genet. 26, 78A Steinberg, M. H., Adams, J. G., and Dreiling, B. J. (1975) Brit. J.

Haematol. 30, 31-37 DeSimone, J., Kleve, L., Longley, M. A., and Shaeffer, J. (1974)

Biochem. Biophys. Res. Commun. 57, 248-254

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J R Shaeffer, M A Longley, J DeSimone and L J KlevePatterns of hemoglobin assembly in reticulocytes of sickle cell trait individuals.

1975, 250:8630-8634.J. Biol. Chem. 

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