Symmetric Behavior of Hemoglobin - and - Subunits during Acid-Induced Denaturation Observed by Electrospray Mass Spectrometry
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- 作者：Brian L. Boys ; Mark C. Kuprowski ; Lars Konermann
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：September 18, 2007
- 年：2007
- 卷：46
- 期：37
- 页码：10675 - 10684
- 全文大小：266K
- 年卷期：v.46,no.37(September 18, 2007)
- ISSN：1520-4995
文摘
This work employs electrospray mass spectrometry (ESI-MS) and UV-vis spectroscopy formonitoring the mechanism of acid-induced hemoglobin (Hb) denaturation. The protein for these experimentshas been freshly prepared from bovine blood. All three Hb derivatives studied (oxyHb, metHb, andcyanometHb) respond to gradual changes from pH 6.8 to 2.1 in a manner that can be described by a stepwisesequential unfolding mechanism: (hh)2 2 hh 2 hfolded + 2 hfolded 2 aunfolded + 2 aunfolded+ 4 heme (superscripts "h" and "a" refer to holo- and apo-forms, respectively). The results obtained onthese freshly prepared samples are significantly different from those of similar experiments previouslyconducted on metHb obtained commercially as lyophilized powder. Those earlier experiments suggesteda highly asymmetric behavior of the two globin chains, involving a heme-deficient dimer (ha) as amechanistically important intermediate on the (dis)assembly pathway. Importantly, heme-deficient dimersare virtually undetectable for the freshly prepared Hb derivatives studied herein at any pH. This apparentdiscrepancy is attributed to the occurrence of oxidative modifications in the commercial protein. Liquidchromatography and tandem mass spectrometry reveal significant levels of sulfoxide formation for allfour methionine residues in commercially obtained metHb. The extent of these modifications for freshlyprepared protein is lower by at least a factor of 10. It is concluded that the acid-induced denaturation ofHb follows a highly symmetric mechanism. The occurrence of other mechanisms (possibly involvingasymmetric elements) under different solvent conditions cannot be ruled out.