Trasko C S, Franzblau C, Troxler R F
Biochim Biophys Acta. 1976 Nov 1;447(4):425-35. doi: 10.1016/0005-2787(76)90080-0.
L-Azetidine-2-carboxylic acid, the naturally occurring lower homologue of L-proline, is incorporated into hemoglobin S (sickle hemoglobin) in vitro. Sickle erythrocytes from patients with sickle cell anemia incubated with L-[3H] azetidine-2-carboxylate synthesized radiolabeled hemoglobin which when isolated from cell lysates, co-chromatographed with hemoglobin S on DEAE-cellulose columns. The alpha/beta ratio of azetidine carboxylate incorporation into the globin chains of sickle hemoglobin was 0.94, which is consistent with the presence of four proline residues in each polypeptide chain. Incorporation of azetidine carboxylate into hot trichloroacetic acid-insoluble material in sickle erythrocytes indicated that the homologue was present in the polypeptide backbone of the globin chains of sickle hemoglobin. Amino acid analysis of the hot trichloroacetic acid-insoluble material from sickle erythrocytes which had been incubated with radiolabeled azetidine carboxylate indicated that 75% of the radioactivity could be accounted for as intact homologue while 20% of the radioactivity co-chromatographed with alanine. These results suggest that azetidine carboxylate is incorporated unaltered into hemoglobin S in addition to being metabolized to alanine in sickle erythrocytes prior to incorporation into protein. The kinetics of thermal precipitation of hemoglobin S (oxygen ligand) into which radioactive azetidine carboxylate or radioactive proline had been incorporated in vitro is identical. This observation, together with the behavior of hemoglobin S and the globin chains from hemoglobin S containing azetidine carboxylate during ion-exchange chromatography, indicates that homologue replacement of prolyl residues does not significantly alter the overall charge or stability of the hemoglobin S tetramer. Azetidine carboxylate did not inhibit uptake of radiolabeled proline by sickle erythrocytes suggesting that the homologue does not adversely affect amino acid transport in these cells.
L-氮杂环丁烷-2-羧酸是L-脯氨酸天然存在的低级同系物，可在体外掺入血红蛋白S（镰状血红蛋白）中。镰状细胞贫血患者的镰状红细胞与L-[3H]氮杂环丁烷-2-羧酸盐一起孵育，合成了放射性标记的血红蛋白，当从细胞裂解物中分离出来时，它与血红蛋白S在DEAE-纤维素柱上共同色谱分离。氮杂环丁烷羧酸盐掺入镰状血红蛋白珠蛋白链中的α/β比率为0.94，这与每条多肽链中存在四个脯氨酸残基一致。氮杂环丁烷羧酸盐掺入镰状红细胞中热三氯乙酸不溶性物质表明该同系物存在于镰状血红蛋白珠蛋白链的多肽主链中。对与放射性标记的氮杂环丁烷羧酸盐孵育的镰状红细胞中热三氯乙酸不溶性物质的氨基酸分析表明，75%的放射性可归因于完整的同系物，而20%的放射性与丙氨酸共同色谱分离。这些结果表明，氮杂环丁烷羧酸盐除了在掺入蛋白质之前在镰状红细胞中代谢为丙氨酸外，还未改变地掺入血红蛋白S中。体外掺入放射性氮杂环丁烷羧酸盐或放射性脯氨酸的血红蛋白S（氧配体）的热沉淀动力学是相同的。这一观察结果，连同血红蛋白S和含有氮杂环丁烷羧酸盐的血红蛋白S的珠蛋白链在离子交换色谱过程中的行为，表明脯氨酰残基的同系物替代不会显著改变血红蛋白S四聚体的整体电荷或稳定性。氮杂环丁烷羧酸盐不抑制镰状红细胞对放射性标记脯氨酸的摄取，这表明该同系物不会对这些细胞中的氨基酸转运产生不利影响。
