Symmetric Behavior of Hemoglobin α- and β- Subunits during Acid-Induced Denaturation Observed by Electrospray Mass Spectrometry

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.

Gas-phase H/D exchange and collision cross sections of hemoglobin monomers, dimers, and tetramers

“…Because the relative abundances of these species depend strongly on the pressure in the ion guide, it is difficult to draw conclusions about the relative abundances of these species in solution from the mass spectra, but it seems the levels of dimers and monomers are greater than expected from the equilibrium solution dissociation of hemoglobin. Recent studies using freshly prepared hemoglobin have shown that the presence of ␤-monomers in the ESI ␤ spectra (Figure 1a-c) are likely artifacts caused by the lyophilization process used to produce commercial hemoglobin [35,51]. Possibly in these experiments we see higher levels of dimers and monomers than expected because the hemoglobin is oxidized [35], the solutions contain 10% methanol, and multimers can dissociate in the electrospray and ion sampling processes.…”

“…Thus the levels of dimers and monomers in solution are expected to be very low. Nevertheless, all ESI MS studies of Hb show substantial levels of dimers and monomers in the spectrum [32,35,[42][43][44].…”

“…The spectra depend strongly on instrument operating conditions. At near-neutral pH, the ESI mass spectrum of hemoglobin can be dominated by ions of the intact tetrameric quaternary structure, and this has been attributed to the solution composition [32,35,[42][43][44]. Figure 1 shows the mass spectra of 100 M hemoglobin in 10 mM ammonium acetate and 10% methanol recorded here with the LIT-TOF apparatus operated at different quadrupole pressures with ⌬V OS ϭ 200 V and with no trapping.…”

“…For the ϩ14 ␤ 0 ␤ ␤ monomer (m 2 ϭ 15,608, ϭ 2348 Å 2 the ratio E/E 0 at 2.0 mTorr is 0.21 and at 12.5 mTorr, 7.5 ϫ 10 Ϫ5 . It has been argued that the observation of ions of dimers, tetramers, and higher order multimers such as hexamers and octamers with ESI is most likely a reflection of the presence of these species in solution [32,35,[42][43][44]. However hydrogen deuterium exchange experiments provide evidence that the monomers, dimers, and tetramers are present in solution, but that hexamers and octamers are artifacts of ESI [44].…”

“…Although commercial lyophilizate of bovine hemoglobin should not be used to infer any significant biological relevance [35], it is used here as a well established model for protein-protein interactions in the gas phase. We have measured the H/D exchange levels and collision cross sections of ions of the mono- 2 .…”