Interactions of Hemoglobin and Myoglobin with Their Ligands CN-, CO, and O^sub 2^ Monitored by Electrospray Ionization-Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 87; no. 19; p. 9538
• Main Authors: Sowole, Modupeola A, Vuong, Stephanie, Konermann, Lars
• Format: Journal Article
• Language: English
• Published: Washington American Chemical Society 06.10.2015
• Subjects: Experiments; Hemoglobin; Ligands; Mass spectrometry; Oxidation; Proteins
• ISSN: 0003-2700; 1520-6882

Electrospray ionization (ESI)-mass spectrometry (MS) can provide information on protein-ligand interactions via detection of the corresponding complexes as gaseous ions. Unfortunately, some systems are prone to dissociation upon transfer into the gas phase. The reversible oxygen binding to hemoglobin (Hb) has been extensively studied in solution using a wide range of biophysical techniques. In addition to O2, ferrous (FeII) Hb can bind CO. High affinity interactions with CN- are limited to the ferric (FeIII) state. In analogous fashion, CN-, CO, and O2 bind to myoglobin (Mb). It remains unclear whether any of these ligand-bound forms can be observed by ESI-MS. In this work we demonstrate the successful detection of MbCN, while MbCO and MbO2 do not survive under ESI-MS conditions. Control experiments suggest that an older report of "MbO2" detection by ESI-MS may involve the misassignment of oxidation artifacts formed under corona discharge conditions. The situation is more favorable for ESI-MS studies on Hb. The most intense signal in the HbCN mass distribution corresponds to the expected complex with four cyanide moieties bound. Ligand loss during ESI-MS is around 20%. HbCO is detectable as well, albeit with a more noticeable level of ligand dissociation (~50%) which produces the 2CO-bound state as the highest intensity ion in the spectrum. In addition, our data suggest that low levels of HbO2 can survive the transition into the gas phase, evident from +64 Da and +128 Da signals that can be assigned to Hb carrying two and four oxygen molecules, respectively. The application of collisional activation induces neutral ligand loss for all three Hb derivatives. It appears that this is the first report on the detection of MbCN, HbCO, and HbO2 in the gas phase. We hope that this work will pave the way towards future spectroscopic investigations of desolvated Mb and Hb, complementing the extensive literature on CN-, CO, and O2 bound globins in the condensed phase.