Konishi Y, Feng R
National Research Council, Canada, Biotechnology Research Institute, Montreal, Quebec.
Biochemistry. 1994 Aug 16;33(32):9706-11. doi: 10.1021/bi00198a041.
The function and conformational stability of myoglobin were studied in vacuo by using an electrospray mass spectrometer. The electrospray technique gently transfers protein molecules from the solution phase to the gas phase, and solvent-free protein ions are produced in the mass spectrometer. Horse myoglobin was dissolved at neutral pH, Fe3+ in the heme was reduced to Fe2+ to produce the biologically active oxymyoglobin in solution, and then the protein was isolated in vacuo. A molecular ion (17,601 Da) corresponding to the molecular mass of oxymyoglobin (17,600.0 Da) was observed in the mass spectrum. This demonstrates that the protein retains a heme and an oxygen molecule in the gas phase. Since the biological function of myoglobin is to carry an oxygen molecule, this is the first observation that a protein is functional in the absence of solvent. Gas-phase "unfolding" of myoglobin was also studied. Collisions of accelerated protein ions with nitrogen curtain gas at a quadrupole guidance lens or argon gas introduced at a second quadrupole increase the "molecular temperature" of myoglobin, resulting in release of the heme from myoglobin. Apomyoglobin produced at the quadrupole guidance lens showed a larger collisional cross section than that of myoglobin, revealing conformational disordering of the protein. The gas-phase unfolding of horse and whale myoglobins and the alpha-chain of human hemoglobin induced at the second quadrupole were studied as a function of the argon gas thickness. Horse and whale myoglobins showed the same gas-phase stability, whereas the alpha-chain of human hemoglobin was less stable.(ABSTRACT TRUNCATED AT 250 WORDS)
利用电喷雾质谱仪在真空条件下研究了肌红蛋白的功能和构象稳定性。电喷雾技术能将蛋白质分子从溶液相温和地转移至气相，在质谱仪中产生无溶剂的蛋白质离子。马肌红蛋白在中性pH条件下溶解，将血红素中的Fe3+还原为Fe2+以在溶液中产生具有生物活性的氧合肌红蛋白，然后在真空中分离该蛋白质。在质谱图中观察到了与氧合肌红蛋白分子量（17,600.0 Da）相对应的分子离子（17,601 Da）。这表明该蛋白质在气相中保留了一个血红素和一个氧分子。由于肌红蛋白的生物学功能是携带一个氧分子，这是首次观察到蛋白质在无溶剂情况下仍具有功能。还研究了肌红蛋白在气相中的“展开”情况。加速的蛋白质离子与四极导向透镜处的氮气帘气或第二个四极处引入的氩气发生碰撞，会提高肌红蛋白的“分子温度”，导致血红素从肌红蛋白中释放出来。在四极导向透镜处产生的脱辅基肌红蛋白的碰撞截面比肌红蛋白的大，这表明该蛋白质存在构象紊乱。研究了在第二个四极处诱导的马和鲸肌红蛋白以及人血红蛋白α链的气相展开情况与氩气厚度的关系。马和鲸肌红蛋白表现出相同的气相稳定性，但人血红蛋白α链的稳定性较差。（摘要截短至250字）
