Observation of symmetric denaturation of hemoglobin subunits by electrospray ionization mass spectrometry.
Electrospray ionization mass spectrometry (ESI-MS) has been used to characterize the denaturation of porcine hemoglobin (Hb) induced by solvent changes. This work provides evidence for the symmetric nature of Hb denaturation and demonstrates that heme losses from α- and β-monomers occur in parallel, in response to the addition of acid and organic co-solvents in solution. When subject to one of the following solution conditions (pH 3.2-4.0 or 15-30% acetonitrile-water or 30-45% methanol-water solution), α- and β-globins undergo symmetric dissociation to release the heme groups, which is detected by ESI-MS. Circular dichroism (CD) and fluorescence spectroscopy (FS) data show that the acid-induced and organic solvent-induced heme release, as observed in the mass spectra, can probably be ascribed to different aspects of the conformational changes taking place in the protein. The acidity of the solvent has a significant effect on the secondary structure, whereas organic content level in solution (15-30% acetonitrile or 30-45% methanol) tends to destroy the tertiary structure of Hb globins, both leading to release of the heme from each subunit.
Key Laboratory of Analytical Chemistry of the State Ethnic Affairs Commission, College of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan, Hubei 430074, People's Republic of China. xwang@mail.scuec.edu.cn
Xian
X
Wang
Wenjie
W
Zhao
Xin
X
Lin
Bo
B
Su
Jianfeng
J
Liu
D000097:Acetonitriles; D000818:Animals; D002942:Circular Dichroism; D055554:Hemoglobin Subunits / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D011487:Protein Conformation; D011489:Protein Denaturation; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013552:Swine
D000097:Acetonitriles; D055554:Hemoglobin Subunits; D021122:Protein Subunits; D000432:Methanol; C032159:acetonitrile