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.

The native structure of hemoglobin (Hb) comprises two ␣-and two ␤-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded ␣-and ␤-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric ␣-and ␤-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded ␣-and ␤-globin. However, some unexpected features are observed, e.g., a heme-deficient ␤-globin dimer that represents an off-pathway intermediate. Monomeric ␤-globin is capable of binding heme before forming a complex with an ␣-subunit. This observation suggests that holo-␣-apo-␤ globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes. (J Am Soc Mass Spectrom 2007, 18, 8 -16)

The exposure of solution-phase proteins to reactive oxygen species (ROS) causes oxidative modifications, giving rise to the formation of covalent +16 Da adducts. Electrospray ionization (ESI) mass spectrometry (MS) is the most widely used method for monitoring the extent of these modifications. Unfortunately, protein oxidation can also take place as an experimental artifact during ESI, such that it may be difficult to assess the actual level of oxidation in bulk solution. Previous work has demonstrated that ESI-induced oxidation is highly prevalent when operating at strongly elevated capillary voltage V(0) (e.g., +8 kV) and with oxygen nebulizer gas in the presence of a clearly visible corona discharge. Protein oxidation under these conditions is commonly attributed to OH radicals generated in the plasma of the discharge. On the other hand, charge balancing oxidation reactions are known to take place at the metal/liquid interface of the emitter. Previous studies have not systematically explored whether such electrochemical processes could be responsible for the formation of oxidative +16 Da adducts instead of (or in combination with) plasma-generated ROS. Using hemoglobin as a model system, this work illustrates the occurrence of extensive protein oxidation even under typical operating conditions (e.g., V(0) = 3.5 kV, N(2) nebulizer gas). Surprisingly, measurements of the current flowing in the ESI circuit demonstrate that a weak corona discharge persists for these relatively gentle settings. On the basis of comparative experiments with nebulizer gases of different dielectric strength, it is concluded that ROS generated under discharge conditions are solely responsible for ESI-induced protein oxidation. This result is corroborated through off-line electrolysis experiments designed to mimic the electrochemical processes taking place during ESI. Our findings highlight the necessity of using easily oxidizable internal standards in biophysical or biomedical ESI-MS studies where knowledge of protein oxidation in bulk solution is desired. Strategies for eliminating ESI-induced oxidation artifacts are discussed.

Prothymosin alpha (ProTalpha) is a small acidic protein that is highly conserved among mammals. The human form has 110 amino acid residues (M.W. 12.1 kDa; pI approximately 3.5) and is found to be expressed in a wide variety of tissues. ProTalpha plays an essential role in cell proliferation and apoptosis, and it is involved in transcriptional regulation of oxidative stress-protecting genes. Despite the multiple biological functions ProTalpha has, the protein does not adopt a well-defined three-dimensional structure under physiological conditions. Previous studies have shown that the interaction between ProTalpha and some of its protein targets is significantly enhanced in the presence of zinc ions, suggesting that zinc binding plays a crucial role in the protein's function. In this work, we use nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry to characterize the structure and dynamics of ProTalpha and its complexation with Zn2+. We found that zinc binding causes partial folding of the C-terminal half of ProTalpha, especially the Glu-rich region, while the N-terminal portion of the protein remains largely unstructured. The metalated protein also exhibits a significantly reduced flexibility. ProTalpha shows a high specificity for Zn2+, and the interactions with other divalent cations (Ca2+, Mg2+) are much weaker. On the basis of the site-specific information obtained here, as well as the results from previous studies, we propose that the conformational and dynamic changes upon zinc binding may act as an entropic switch that greatly facilitates the binding to other proteins.

The determination of solution-phase protein concentration ratios based on ESI-MS intensity ratios is not always straightforward. For example, equimolar mixtures of hemoglobin ␣-and ␤-subunits consistently result in much higher peak intensities for the ␣-chain. The current work explores the origin of this effect. Under mildly acidic conditions (pH 3.4) ␣-globin is extensively unfolded, whereas ␤-globin retains residual structure. Because of its greater nonpolar character, the more unfolded ␣-subunit can more effectively compete for charge. This leads to suppression of ␤-globin signals under conditions where the protein ion yield is limited by the charge concentration on the initially formed ESI droplets. More balanced intensities are observed when operating under charge excess conditions and/or in a solvent environment where both proteins are unfolded to a similar degree (pH 2.2). However, even in these cases the overall ␣-globin peak intensity is still twice as high as that of the ␤-subunit. The persistent imbalance under these conditions originates from the different declustering behaviors of the two proteins. A considerable fraction of ␤-globin undergoes incomplete desolvation during ESI, thereby reducing the intensity of bare [␤ ϩ zH] zϩ ions. When including the contributions of incompletely desolvated species, the overall ␣:␤ ion intensity ratio is close to unity. The ␣:␤ intensity imbalance can also be eliminated by a strongly elevated declustering potential in the ion sampling interface. In conclusion, important factors that have to be considered for the ESI-MS analysis of protein mixtures are (1) conformational effects, resulting in differential surface activities, and (2) properties [4][5][6][7], and their charge state distributions [8 -11]. Unfolded conformations generally result in higher ESI charge states than tightly folded structures, an effect that mirrors the lower compactness and the larger solventaccessible surface area of the unfolded state [12,13]. The combination of these features results in an unsurpassed selectivity that greatly facilitates the detection of coexisting species. One problem, however, that can complicate the analysis of ESI-MS data is that the measured ion intensities do not necessarily reflect the relative concentrations of the corresponding proteins in solution [14]. The apparent ionization efficiencies of different biomacromolecules can vary by several orders of magnitude [15]. The situation is further complicated by ion-suppression effects that may occur in protein mixtures and in the presence of other solutes [16 -19]. An improved understanding of the relationship between ESI-MS signal response and solution-phase concentration would be beneficial for a wide range of applications.The upper limit of the ionization efficiency in ESI-MS is determined by the molar concentration of excess charge, C q , on the initially formed electrospray droplets [19,20]. C q can be estimated based on the relationshipwhere K is the conductivity of the solution, ␥ is the surface tension of t...