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Studies of non-covalent myoglobin interactions by electrospray ionization mass spectrometry Mark, Kevin J.

Abstract

Acid and base-induced unfolding of myoglobin in solution is monitored by electrospray ionization mass spectrometry. It is shown that acid-induced Mb unfolding causes the appearance of different charge state distributions in positive and negative ion modes compared to base-induced unfolding, suggesting different protein conformations in solution. Collision cross sections of both apomyoglobin and holomyoglobin ions are measured at various orifice-skimmer voltage differences (AVOS). The results show that at low AVOS, apomyglobin ions have greater collision cross sections than holomyoglobin ions, indicating that heme, when attached to the globin, helps maintain a more compact myoglobin structure. Coulomb effects in binding of heme in gas phase holomyoglobin ions are studied. Positive and negative ions are formed from solutions of Fe⁺² (ferromyoglobin) and Fe⁺³ (ferrimyoglobin). The energy that must be added to the resulting holomyoglobin ions to cause heme loss is measured with a triple quadrupole MS/MS system. With negative ions, neutral heme is lost, regardless of the charge state of Fe in solution. It is likely that the Fe⁺³ is reduced to Fe⁺² in the negative electrospray process. With positive ions, predominantly neutral heme loss is observed for ions formed from ferromyoglobin in solution, and positive heme loss for ions formed from ferrimyoglobin in solution. The energies required to induce neutral heme loss are similar for positive and negative ions. The energies required to induce charged heme loss from positive holomyoglobin ions are significantly less. Coulomb repulsion between the charged heme and charged protein appears to lower the barrier for heme loss. These results are consistent with a simple model of a long range Coulomb repulsion and a short range attraction between the heme and protein.

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