Tile elucidation of the exact nature of the inclusion bodies, which have been observed within the cytoplasm of normoblasts and red blood cells of patients with severe or intermediate] 3-thalassemia, is essential for the understanding of the underlying abnormality in hemoglobin synthesis. It has been concluded, on the basis of their staining and solubility properties, that the inclusions consist of precipitated hemoglobin 1. This was confirmed by microspectrophotometry of single inclusion bodies, which display a typical Soret absorption maximum at 41o-42o mtL and, usually, the expected heme-protein ratio; furthermore, a typical alkaline hemochromogen spectrum was obtained from bulk preparations of inclusion bodies dissolved in NaOH and reduced by Na2S20 a (ref. 2). For closer chemical characterization, the precipitated intracellular material was analyzed by peptide fingerprinting; the preliminary results of this study are reported here in brief.

Difficulties were encountered because of the limited amount of available inclusion bodies, which had to be obtained from severely anemic patients and had to be freed as perfectly as possible of the soluble hemoglobins and all other cellular components. Blood samples were collected from unrelated splenectomized/~-thalassemic patients with a relatively high proportion of inclusion-carrying cells; after the red cells had been washed with cold saline, they were pooled. In other instances, blood was drawn from patients with/~-thalassemia by an exchange transfusion system and was not pooled. The red cells were washed thrice with cold saline and then subjected to repeated hemolysis and centrifugation in large volumes of very dilute phosphate buffers of pH 6. o and 8. o, in order to eliminate all s. luble hemoglobin:~. Stromal proteins and stroma-bound hemoglobin were further removed by washing the sediment twice with oI o/. o Na,, CO3 (ref. 4); the nucleoproteins from the nuclei of the numerous erythroblasts contained in the sample were dissolved out with IM NaC1 (ref. 5). After this stage the original material had been reduced to a minute amount of a finely dispersed brownish precipitate. The isolation procedure was followed by examining microscopically samples of the material stained by appropriate methods; as a result, the precipitate was shown to consist almost solely of inc! usion bodies. These were solubilized in o. o25-o. 1 M NaOH and then purified by shaking with large volumes of toluol, which removed the remaining stromal material. The globin moieties of this alkaline globin hemochromogen solution were precipitated by dropping the solution in acid acetone at-2o; the precipitate was washed repeatedly with cold acetone and dried; the amount of globin finally obtained was estimated at about 25 mg/Ioo ml of blood. This was hydrolyzed with trypsin (Worthington, 2> crystallized) as indicated by DIN'rzIs 6, ie 1: 5o enzyme to protein ratio, at 37~ for 12 h in an ammonia-ammonium acetate buffer (pH 9.0). The peptides were subjected to electrophoresis at pH 4-7 (pyridine-acetic acid-butanol, 2.5: 2.5: 5. o% in distilled water) followed by chromatography using as solvent systems either (a) pyridine-isoamyl alcohol-HzO (35: 35: 3 o, by vol.), proposed by BAt; LIOXl v, or (b) pyridine-acetic acid--butanol-H20 (5o: 15: 75: 6o, by vol.), as suggested by HILl., SWENSON AND SCHWARTZ 8. The chromatograms were developed with ninhydrin and assayed with stains specific for tyrosine, tryptophan, arginine and histidine'). For the sake of comparison, fingerprints of normal adult hemoglobin A, of hemoglobin F from