We have found a sickling variant, Hb S Antilles, O2PI3 23 Vai-][e), that has the same electrophoretic mobility as Hb S but a distinct isoelectric focus and produces sickling in the carriers of the Hb A/S Antilles trait. The carriers' erythrocytes tend to sickle at 02 partial pressures similar to those that induce sickling in Hb S/C disease. Pure deoxy-Hb S Antilles is 50% as soluble as deoxy-Hb S (saturating concentration = 11 g dl-1 compared to 18.4 for Hb S). MATERIALS AND METHODS Routine hematological parameters were determined by using a Coulter S Counter and standard procedures were used for the preparation of erythrocyte hemolysates. Methods for electrophoresis of hemoglobin on cellulose acetate and in citrate agar and of globin chains at alkaline and acidic conditions have been described (5). IEF was performed as in refs. 6 and 7. Abnormal hemoglobin was isolated by chromatography on DEAE-cellulose (Whatman DE-52) (8). After removal of hemin by precipitation with acid/acetone the abnormal chains were prepared by CM-cellulose/8 M urea chromatography (9), aminoethylated, and hydrolyzed with trypsin treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone (Worthington) (10).Tryptic digests were separated by reverse-phase HPLC using a ,uBondapak C18 column (Waters Associates) operated at room temperature. The buffers used were buffer A, 20 mM ammonium acetate, and buffer B, 50% (vol/vol) acetonitrile in 10 mM ammonium acetate, pH 5.7. All buffers were degassed and filtered before use. The column was equilibrated in 3% buffer B. Elution was performed at room temperature at a flow-rate of 1.5 ml/min and the absorbance of the fractions was monitored at 214 nm. A three-step linear gradient was applied for elution: 3-25% buffer B for 30 min, 25-50% for 10 min, and 50-60% for 15 min. Prior to elution, 1 mg of tryptic digest dissolved in 200 ,ul of 1o (vol/vol) acetic acid was centrifuged before analysis and 100 Al of the supernatant was injected into the column. Fractions were collected, and the acetonitrile was evaporated in a nitrogen stream at 60°C in a fume hood. After hydrolysis in 6 M HCl, the samples were analyzed in a Biotronik 6001E amino acid analyzer. Abnormal peptide ,-T3 was hydrolyzed by Staphyloccocus aureus V8 protease according to ref. 11 at 37°C for 15 hr. The S. aureus V8 peptides were characterized as described for the tryptic peptides. DNA of the propositus' leukocytes was digested with the restriction enzyme Mst II according to ref. 12 and the fragments were analyzed by Southern blotting.Sickling tests on fresh erythrocyte suspensions were done in vitro as previously described (13). Phthalate ester density profiles were determined with erythrocytes from several patients according to ref. 14. For preparative purposes, the propositus' erythrocytes were fractionated according to their density with a discontinuous albumin/Percoll gradient (15). Solubility of the abnormal hemoglobin was studied by measuring its Csat (saturating hemoglobin concentration) value. This was then compared...

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