Human apohemoglobin was found to induce fusion of various vesicles containing phosphatidylserine at low pH. The fusion of vesicles by apoHb was monitored by intermixing of internal aqueous contents, the resonance energy transfer assay which follows the mixing of membrane components and size increase from the electron microscopy and light scattering measurement. The fusion-pH profiles were similar to the binding profiles of apoHb to the same vesicles. Proteolytic digestion after hydrophobic labeling of PS and PS/PE vesicle-protein complex with dansyl chloride showed that a segment with a molecular weight of approximately 2500 penetrates the bilayer.
It was also found that PS/PC(1:1) vesicles not only induces fusion of PS/PC(1:1) vesicles at low protein concentration but also frament the same vesicles to form micellar complex at high protein concentration. The micellization of PS/PC(1:1) vesicles was confirmed by light scattering, gel filtration and electron microscopy.
The $[^{125}I]$ TID-labeling of the apohemoglobin in the vesicle-protein complex followed by CNBr cleavage of apohemoglobin showed that an N-terminal segment of β subunit with a molecular weight of approximately 6000 seems to be mainly involved in the fusion process but the whole sequences of both a and β chains participate in the micellization process. These results suggest that apoHb assumes different topologies in the vesicle-protein complex depending on the L/P ratio causing either fusion or fragmentation of vesicles.
사람의 apohemoglobin은 PS를 포함한 vesicle들을 낮은 pH에서 fusion을 일으켰으며, 특히 PS/PC(1:1)을 포함한 vesicle들에서는 단백질 농도에 따라 선택적으로 fusion도 일으키고 micellization도 일으켰다. Fusion은 internal aqueous content와 external lipid content간의 상호 mixing에 의해 증명되었으며, 실제로 전자현미경 사진으로 확인되었다. Micellization은 light scattering과 gel filtration chromatography로 실제로 크기가 작아진 것이 확인되었으며, 전자현미경 사진으로 특징적인 micellization의 모습이 확인되었다. Hydrophobic labeling 실험을 통해서 얻어진 결과, fusion에서는 apohemoglobin의 N-terminal 부분만이 bilayer에 파고들어가서 fusion을 야기시키는 것으로 보이며, micellization에서는 모든 segement가 관여하여 이 현상을 나타내는 것으로 관찰되었다. 따라서, fusion과 micellization 과정에서 apohemoglobin의 topology는 서로 다른 것으로 보여진다.
