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...

We have studied haplotype of ␤ S chromosome and ␣-globin gene status in 534 patients (255 adults and 279 children of whom 159 neonates) from Guadeloupe with various sickle cell-related conditions, namely SS (n = 298), SC (n = 170), S-␤-thal (n = 56), and other rare forms (n = 10). Haplotype data on ␤ S chromosomes confirm our previous observation that Benin type is the most prevalent (75%) ␤ S chromosome in Guadeloupe, in disagreement with the historical records. Comparison of the frequency of distribution of various ␤ S haplotypes between neonates and adults on the one hand and between SS and SC cases on the other shows that the current ␤ S haplotype distribution in this island is not distorted by haplotype-related differential survival. We also show that the frequency of ␣-thalassemia (−3.7 kb) in Guadeloupe is one of the highest recorded in this region involved in Atlantic slave trade and also failed to reveal any age-dependent increase in frequency. We conclude that the African component of Guadeloupe is distinct from that of Brazil and Cuba but is close to that of Jamaica. Am.

The Michaelis constants and the maximum velocities in the aminoacylation reaction of tRNATrp from beef liver, yeast and E. coli by pure beef pancreas tryptophan-tRNA ligase show that this mammalian enzyme recognizes and charges the two eucaryotic tRNAs with the same efficiency. The rate of aminoacylation of the procaryotic tRNATrp by the enzyme is three orders of magnitude lower. The pH optimum of aminoacylation is 8 for both eucaryotic tRNAs. The optimum magnesium concentration is different. The rate is maximum when magnesium concentration is stoichiometric to ATP concentration for tRNATrp from beef liver and 10 mM above ATP concentration for tRNATrp from yeast. The number of binding sites on the enzyme for the two eucaryotic tRNAs has been measured by equilibrium filtration on Sephadex G-100 and found equal to two.

The order of the partial reactions in the aminoacylation of tRNAE:k by tryptophan : tRNA ligase from beef pancreas has been explored by measurements of initial velocity and product inhibition according to the analytical framework described by Cleland [Biochim. Biophys. Acta, 67, 104-137, 173-187, 188-196 (1963)l. The kinetic patterns are suggestive of a ping-pong mechanism (bi-uni uni-bi). The order of addition of tryptophan and ATP that one can derive is crucially dependent on the conditions of magnesium concentration used. Random fixation of tryptophan and ATP is observed in studies performed at catalytically optimal magnesium concentrations. Linear-ordered fixation of the two substrates, ATP binding first, is observed under conditions where magnesium concentration is in excess, which are not catalytically optimal. The analysis of the inhibition patterns taking into account the calculated concentrations of the main active species Mg . ATP2-and Mg . PzO$-favors the random substrate-binding mechanism derived under optimal magnesium conditions. Under both conditions, adjusted and fixed magnesium, the release of products, AMP and tryptophanyl-tRNATrp, is ordered, AMP released first.The change in the product inhibition patterns of aminoacylation of tRNA& by beef pancreas tryptophan : tRNA ligase under different magnesium concentration conditions casts some doubt on the significance of the comparison of mechanisms derived for amino-acid: tRNA ligases of different sources.Many investigators have tried to compare the physical or species properties of amino-acid : tRNA ligases (molecular weight, quaternary structure, etc.) with their recognition and binding sequences of substrates in the aminoacylation reaction. For this pur-D