Characterization of conformational changes and noncovalent complexes ofmyoglobin by electrospray ionizationmass spectrometry, circular dichroism and fluorescence spectroscopy

摘要

Electrospray ionization mass spectrometry (ESI-MS) was employed tomonitor the hemerelease andthe conformational changes of myoglobin (Mb) under different solvent conditions, and to observe ligand bindings of Mb. ESI-MS, complemented by circular dichroism and fluorescence spectroscopy, was used to study themechanism of acid- and organic solvent-induced denaturation by probing the changes in the secondary and the tertiary structure of Mb. The results obtained show that complete disruption of the heme-protein interactions occurs when Mb is subjected to one of the following solution conditions: pH 3.2-3.6, or solution containing 20-30% acetonitrile or 40-50% methanol. Outside these ranges, Mb is present entirely in its native state (binding with a heme group) or as apomyoglobin (i.e. without the heme). Spectroscopic data demonstrate that the denaturation mechanism of Mb induced by acid may be significantly different from that by the organic solvent. Low pH reduces helices in Mb, whereas certain organic content level in solution results in the loss of the tertiary structure. ESI-MS conditionswere established to observe the H_2O- and CO-bound Mb complexes, respectively. H_2O binding to metmyoglobin (17 585 Da), where the heme iron is in the ferric oxidation state, is observed in ESI-MS. CO binding to Mb (17 595 Da), on the other hand, can be only observed after the heme iron is reduced to the ferrous form. Therefore, ESI-MS combinedwith spectroscopic techniques provides a useful means for probing the formation of ligand-binding complexes and characterizing protein conformational changes.