CHIMERIC HEMOGLOBINS - HYBRIDS OF HUMAN AND SWINE HEMOGLOBIN - ASSEMBLY AND STABILITY OF INTERSPECIES HYBRIDS

Transgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely (H) alpha(2)(P) beta(2 ) (H = human, P = swine). In an attempt to establish whether the absen ce of the swine alpha and human beta((P) alpha(2)(H) beta(2)) hybrid i n vivo is a reflection of the lack of complementarity between the inte rspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine alpha and human beta chimeric tetramer . In contrast to the in vivo transgenic swine system, in vitro the hyb rid of swine alpha human beta chain is assembled readily and the hybri d exhibits normal cooperative oxygen binding. Both the swine alpha hum an beta and the human alpha swine beta interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even whe n mixed together. On the other hand, nearly complete exchange of (P) a lpha chain of (P) alpha(2)(H) beta(2) hybrid occurs in the presence of (H) alpha chain at pH 6.0 and room temperature, resulting in the form ation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest, that the thermodynamic stabili ty of (P) alpha(2)(H) beta(2) hybrid is lower compared to that of HbA. In contrast, (P) beta chain of (H) alpha(2)(P) beta(2) hybrid is refr actory to exchange with (H) beta chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of (H) alpha(2)(P) beta(2) is higher co mpared to that of HbA ((H) alpha(2)(H) beta(2)). The swine alpha human beta chimeric Hb undergoes subunit exchange reaction with human alpha -chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates th e lower thermodynamic stability of the intradimeric interactions of th e heterodimer even at neutral pH. A synergistic coupling of the intra- and interdimeric interactions of the swine alpha and human beta chain heterodimer is essential for the thermodynamic stability of the chime ric Hb under the physiological conditions. Accordingly, we speculate t hat the lower thermodynamic stability of (P) alpha(H) beta heterodimer (compared to the homodimers (H) alpha(H) beta and (P) alpha(P) beta) facilitates its segregation into the homodimers by subunit exchange re action involving either (H) alpha or (P) beta. This molecular aspect b y itself or possibly along with other cellular aspects of the swine sy stem results in the absence of (P) alpha(2)(H) beta(2) hybrid in trans genic swine expressing HbA.