We examine Hofmeister specific ion effects of electrolytes added to protein solution under conditions minimizing electrostatic attraction between cations and positively charged protein. Hemoglobin (Hb) in aqueous solution at the denaturing pH = 2.7 is investigated in the presence of several metal chlorides, along with sodium and potassium bromides, iodides and thiocyanates, using electrospray ionization mass spectrometry (ESI-MS). Salt concentration was varied to maximize peak intensity and bell-shaped profile in the ESI-MS spectrum. The α-chain of myoglobin is identified as the main pattern of the ESI-MS spectra in all Hb-salt systems. Both peak intensity and quality of the bell-shaped profile of the protein spectrum decrease in the cation order: K+ > > Mg2+ > Li+ > > Na+ > Ca2+ ≈ Cs+ > Rb+ for Hb-Metal Chloride systems, and decrease in the anion order: Cl- > Br- > I- > SCN- for systems of both Hb-NaX and Hb-KX salts. To quantify salt addition effects two Hofmeister specific electrolyte parameters HS, and PS are proposed. HS is the mean (Hb-salt)/Hb peak intensity ratio, measured for the nine peaks used for ESI-MS spectra deconvolution, taken at the same m/z values of the Hb profile. PS is the ratio between HS standard deviation and HS, and provides a specific perturbation parameter measuring the loss of protein structure. These two Hofmeister parameters give clear evidence of the effects induced either by KCl, MgCl2 and LiCl that enhance protein peak intensity, or by NaBr, NaI, NaSCN and KSCN that induce the protein fragmentation, due to electrolyte-mediated dissociation.
Keywords: ESI-MS; Hemoglobin; Myoglobin; Specific electrolyte effects.
Copyright © 2022 Elsevier Inc. All rights reserved.