Molecular basis of the inhibition of β^s-chain-dependent polymerization by mouse α-chain: Semisynthesis of chimeras of human and mouse α-chains

The transgenic mouse models expressing β^s-globin genes do not fully exhibit the sickling phenotype, primarily as a result of the inhibition of β^s-chain-dependent polymerization by the mouse α-chains. The mouse α-chain differs from the human α-chain at 19 sequence locations. Of these, only α⁷⁸ and α¹¹⁶ are the known hemoglobin (Hb) S polymer contact sites. To define whether the inhibition of polymerization by the mouse α-chain is solely a consequence of the differences at these two sites or additional sites of sequence differences are also involved, we have constructed chimeric α-chains by employing the α-globin semisynthetic reaction (Sahni, G., Cho, Y. J., Iyer, K. S., Khan, S. A., Seetharam, R., and Acharya, A. S. (1989) Biochemistry 28, 5456-5461). Mouse α_1-30 was spliced with human α_31-141 using endoproteinase Glu-C to generate a chimeric α-globin (α^MH) containing eight of the 19 sequence differences of mouse α-globin. Similarly, human α_1-30 was spliced with mouse α_31-141 to generate another chimeric α-globin (α^HM) containing 11 sequence differences. The respective chimeric globins were purified, reconstituted with heme and β^s-chain into tetrameric hemoglobin, and the tetramers were purified by ion-exchange chromatography. The inhibitory potential of the chimeric α^MH-chain on the polymerization is 10-fold lower than that of the mouse α-chain. The absence of the α^31-141 region of the mouse α-chain relieves only a portion of the inhibition. The inhibitory potential of α^MH contributed by the mouse α_1-30 segment is significant although none of the sequence differences in this segment are located at any of the implicated polymer contact sites. The chimeric α^HM-chain also inhibits the polymerization, but the extent of inhibition is again lower (4-fold) than that of the full-length mouse α-chain. The results demonstrate that the inhibitory potential of mouse α-chains involves the sequence differences from both the α_1-30 and α_31-141 regions. Besides, since the sum of the inhibitory potential of either of these chimeric α-chains is lower than that of the intact mouse α-chains, we speculate that conformational changes that require the copresence of sequence differences in both portions of the mouse α-chain also contribute to the inhibitory propensity of the mouse α-chain.