Symmetric behavior of hemoglobin α- and β-subunits during acid-induced denaturation observed by electrospray mass spectrometry

被引:62
作者
Boys, Brian L. [1 ]
Kuprowski, Mark C. [1 ]
Konermann, Lars [1 ]
机构
[1] Univ Western Ontario, Dept Chem, London, ON N6A 5B7, Canada
关键词
NONCOVALENT PROTEIN COMPLEXES; HEME-BINDING; CONFORMATIONAL HETEROGENEITY; ELECTROSTATIC INTERACTIONS; OXYGEN-BINDING; GLOBIN CHAINS; GAS-PHASE; LOW PH; MECHANISM; KINETICS;
D O I
10.1021/bi701076q
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 ((Oxy)Hb, (met)Hb, and (cyanomet)Hb) respond to gradual changes from pH 6.8 to 2.1 in a manner that can be described by a stepwise sequential unfolding mechanism: ((alpha(h)beta(h))2 -> 2 alpha(h)beta(h) -> 2 (alpha(h)(folded) + 2 beta(h)(folded) -> 2 alpha(a) unfolded + 2 beta(a)(unfolded) + 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 (met)Hb obtained commercially as lyophilized powder. Those earlier experiments suggested a highly asymmetric behavior of the two globin chains, involving a heme-deficient dimer (alpha(h)beta(a)) 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 (met)Hb. 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.
引用
收藏
页码:10675 / 10684
页数:10
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