MOLECULAR-BASIS OF THE INHIBITION OF BETA-S-CHAIN-DEPENDENT POLYMERIZATION BY MOUSE ALPHA-CHAIN - SEMISYNTHESIS OF CHIMERAS OF HUMAN AND MOUSE ALPHA-CHAINS

The transgenic mouse models expressing beta(S)-globin genes do not ful ly exhibit the sickling phenotype, primarily as a result of the inhibi tion of beta(S)-chain-dependent polymerization by the mouse alpha-chai ns. The mouse alpha-chain differs from the human alpha-chain at 19 seq uence locations. Of these, only alpha78 and alpha116 are the known hem oglobin (Hb) S polymer contact sites. To define whether the inhibition of polymerization by the mouse alpha-chain is solely a consequence of the differences at these two sites or additional sites of sequence di fferences are also involved, we have constructed chimeric alpha-chains by employing the alpha-globin semisynthetic reaction (Sahni, G., Cho, Y. J., Iyer, K. S., Khan, S. A., Seetharam, R., and Acharya, A. S. (1 989) Biochemistry 28, 5456-5461). Mouse alpha1-30 Was spliced with hum an alpha31-141 using endoproteinase Glu-C to generate a chimeric alpha -globin (alpha(MH)) containing eight of the 19 sequence differences of mouse alpha-globin. Similarly, human alpha1-30 was spliced with mouse alpha31-141 to generate another chimeric alpha-globin (alpha(HM)) con taining 11 sequence differences. The respective chimeric globins were purified, reconstituted with heme and beta(S)-chain into tetrameric he moglobin, and the tetramers were purified by ion-exchange chromatograp hy. The inhibitory potential of the chimeric alpha(MH)-chain on the po lymerization is 10-fold lower than that of the mouse alpha-chain. The absence of the alpha31-141 region of the mouse alpha-chain relieves on ly a portion of the inhibition. The inhibitory potential of alpha(MH) contributed by the mouse alpha1-30 segment is significant although non e of the sequence differences in this segment are located at any of th e implicated polymer contact sites. The chimeric alpha(HM)-chain also inhibits the polymerization, but the extent of inhibition is again low er (4-fold) than that of the full-length mouse alpha-chain. The result s demonstrate that the inhibitory potential of mouse alpha-chains invo lves the sequence differences from both the alpha1-30 and alpha31-141 regions. Besides, since the sum of the inhibitory potential of either of these chimeric alpha-chains is lower than that of the intact mouse alpha-chains, we speculate that conformational changes that require th e copresence of sequence differences in both portions of the mouse alp ha-chain also contribute to the inhibitory propensity of the mouse alp ha-chain.