Symmetric Behavior of Hemoglobin Alpha- and Beta- Subunits During Acid-induced Denaturation Observed by Electrospray Mass Spectrometry.
From: Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada.
Biochemistry
- Publish Date: Sep 2007
- ISSN: 0006-2960
- Volume: 46
- Issue: 37
- Pages: 10675-84
- Medium: Print
- Language: English
- Citation (JAMA): Boys Brian L, Kuprowski Mark C, Konermann Lars, et al. Symmetric Behavior of Hemoglobin Alpha- and Beta- Subunits During Acid-induced Denaturation Observed by Electrospray Mass Spectrometry.. Biochemistry Sep 2007;46:10675-84
Abstract
This work employs electrospray mass spectrometry (ESI-MS) and UV-vis spectroscopy for monitoring the mechanism of acid-induced hemoglobin (Hb) denaturation. The protein for these experiments has been freshly prepared from bovine blood. All three Hb derivatives studied (oxyHb, metHb, and cyanometHb) respond to gradual changes from pH 6.8 to 2.1 in a manner that can be described by a stepwise sequential unfolding mechanism: (alphahbetah)2 — > 2 alphahbetah — > 2 alphahfolded + 2 betahfolded — > 2 alphaaunfolded + 2 betaaunfolded + 4 heme (superscripts “h” and “a” refer to holo- and apo-forms, respectively). The results obtained on these freshly prepared samples are significantly different from those of similar experiments previously conducted on metHb obtained commercially as lyophilized powder. Those earlier experiments suggested a highly asymmetric behavior of the two globin chains, involving a heme-deficient dimer (alphahbetaa) as a mechanistically important intermediate on the (dis)assembly pathway. Importantly, heme-deficient dimers are virtually undetectable for the freshly prepared Hb derivatives studied herein at any pH. This apparent discrepancy is attributed to the occurrence of oxidative modifications in the commercial protein. Liquid chromatography and tandem mass spectrometry reveal significant levels of sulfoxide formation for all four methionine residues in commercially obtained metHb. The extent of these modifications for freshly prepared protein is lower by at least a factor of 10. It is concluded that the acid-induced denaturation of Hb follows a highly symmetric mechanism. The occurrence of other mechanisms (possibly involving asymmetric elements) under different solvent conditions cannot be ruled out.
Mesh Headings (Keywords): Animals, Cattle, Hemoglobins, Hydrogen-Ion Concentration, Oxidation-Reduction, Oxyhemoglobins, Protein Denaturation, Protein Subunits, Solvents, Spectrometry, Mass, Electrospray Ionization, Spectrophotometry, Ultraviolet
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