Electrospray Ionization Mass Spectrometric Determination of the Molecular Mass of the ∼200-kDa Globin Dodecamer Subassemblies in Hexagonal Bilayer Hemoglobins

Abstract

The giant extracellular HBL 1 Hbs found in most terrestrial, aquatic, and marine annelids and in deep sea annelids and vestimentiferans are ϳ3.6-MDa complexes of globin subunits and nonglobin, linker chains and represent a summit of complexity for oxygen-binding heme proteins (1-4). The most extensively studied complex is the Hb from the common earthworm Lumbricus terrestris. Based on the finding of a ϳ200-kDa globin subassembly upon mild, partial dissociation of the Hb at neutral pH, a "bracelet" model of its quaternary structure was proposed to consist of twelve ϳ200-kDa globin subassemblies attached to a central scaffolding of 36 -42 linker chains (24 -32 kDa) (5). Scanning transmission electron microscopy mass mapping of the isolated globin subassembly showed it to have a mass of 202 Ϯ 26 kDa, consonant with it being a dodecamer of globin chains (ϳ17 ϫ 12 ϭ 204 kDa), [d] [22][23][24]. Based on these results, a Hb model was proposed consisting of 12 hexadecamer subassemblies and 24 linker chains. A key point in resolving the differences is a reliable mass for the globin subassembly. We present the results of an ESI-MS study that provides accurate masses for the globin subassemblies isolated from the HBL Hbs of L. terrestris and the marine polychaete Arenicola marina.

An electrospray ionization mass spectrometric study of the subunit structure of the giant hemoglobin from the leech Nephelopsis oscura

“…In general, the variation was small, the maximum decrease in mass being ϳ1.3% over the range 60 -160 V. At a given cone voltage, the observed masses of the subassembly were within experimental error of one another irrespective of the pH. Table 4 shows how the mean mass of the subassembly (n ϭ 3-5) varied with the declustering potential over the range 60 -160 V. The overall mass of the subassembly is dependent on the declustering potential, comparable to the effect observed earlier for similar subassemblies of Lumbricus and Arenicola HBL Hbs [19]. Declustering occurs when the positively charged protein ions are accelerated through a region that is intermediate in pressure between their generation at atmospheric pressure and analysis under high vacuum.…”

Mass Spectral Analysis of Asymmetric Hemoglobin Hybrids: Demonstration of Hb FS (α2γβS) in Sickle Cell Disease

“…Otherwise, the masses of the tetramers would be higher than expected which is not the case. The adducts are possibly addition of water molecules, ammonium, or alkali metal ions and appear to be fairly typical of noncovalent assemblies analyzed by ESI-MS (27). Also apparent in Fig.…”

Cofactor-dependent Assembly of the Flavoenzyme Vanillyl-alcohol Oxidase

“…With the development of new ionization techniques such as matrix-assisted laser desorption ionization (28) and electrospray ionization (29), and the coupling of these to time-of-flight analyzers, the detectable mass range of macromolecular systems by mass spectrometry has been enhanced extensively (30,31). Moreover, the relatively gentle phase transfer from solution to the gas phase, realized especially by electrospray ionization, has allowed the intact detection by mass spectrometry of larger multiprotein assemblies (32)(33)(34)(35). Several recent examples (36 -41) have revealed that biologically relevant parameters concerning multiprotein assemblies may now be investigated by mass spectrometry, and examples include protein complex topology, protein-ligand and protein-protein binding constants, and protein complex stability.…”