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

Abstract

The subunit structure of the giant, extracellular hexagonal bilayer (HBL) hemoglobin (Hb) from the leech Nephelopsis oscura was investigated by electrospray ionization mass spectrometry (ESI-MS) employing a maximum entropy deconvolution of its complex, multiply charged ESI spectra. The denatured unreduced Hb consisted of three monomer globin chains These giant complexes were found to have masses of ϳ3500 kDa and to consist of two types of polypeptide chains: heme-containing 16 -17 kDa globin chains and non-globin, linker chains of 24 -32 kDa containing 9 to 12 cysteines [4]. The amino acid sequences of the globin chains are clearly related to vertebrate and invertebrate globins [5] and they form disulfide-bonded dimer, trimer and tetramer subunits as well as noncovalent subassemblies, ranging from dimer to dodecamer [4].Over the last decade, ESI-MS has been shown to be the method of choice for the characterization of proteins [6,7] by providing highly accurate masses. In particular, in the case of heteromultimeric protein complexes, as exemplified by the HBL Hbs, ESI-MS provides an exquisitely accurate enumeration of the constituent chains and covalently bonded subunits, as well as a quantitative determination of free and disulfidebonded Cys residues [8]. Furthermore, the combination of nanoflow electrospray and time-of-flight (TOF) mass analyzers has recently allowed the investigation of the quaternary structure of large noncovalent protein complexes [9].We describe here the results of ESI-MS and ESI-TOF-MS studies of the polypeptide chain and subunit composition and noncovalent subassemblies of the HBL Hb from the leech Nephelopsis oscura.

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

“…This is commonly seen with ESI-MS of protein-protein complexes and has been attributed to clustering with solvent molecules or other species [52][53][54][55]. In an attempt to reduce clustering of the tetramer peaks, 100 M hemoglobin was run in 25% methanol, as described by Simmons et al [32].…”

Hemoglobina extracelular gigante de Glossoscolex paulistus: um extraordinário sistema supramolecular hemoproteico

“…4 113 Essa configuração espacial final, que consiste na estrutura quaternária da proteína, é chamada "modelo do bracelete", sendo que tal arquitetura é suportada por microscopia crioeletrônica. 107,110,114 Por outro lado, apesar de ser pertencente à mesma classe do anelídeo Lumbricus terrestris, que vem sendo intensamente estudado por diversas técnicas, 115-120 a hemoglobina de Glossoscolex paulistus permanece intrigante sob vários aspectos da sua relação estrutura/atividade.…”

Structural study of Carcinus maenas hemocyanin by native ESI‐MS: Interaction with L‐lactate and divalent cations

“…The 119 possibility of preserving the fragile non-covalent interactions through the electrospray 120 ionization (ESI) process has allowed probing quaternary structures of various assemblies and 121 following association and dissociation of non-covalent complexes and incorporation of small 122 organic molecules or inorganic ions (Potier et al, 1997;Rogniaux et al, 2001;Tahallah et al, 123 2002;van Duijn et al, 2006). The ESI source was also successfully used to study the 124 quaternary structure of high-molecular mass invertebrate respiratory pigments (Bruneaux et 125 al, 2008;Green et al, 2001;Green et al, 1998;Green & Vinogradov, 2004;Sanglier et al, 126 2003;Zal et al, 2002). 127 ESI-MS ability to characterize compounds by their mass with a high precision and to 128 maintain non-covalent interactions such as exist between hemocyanin and L-lactate and 129 divalent cations makes this technique a useful tool to probe the specificity of these 130 interactions and their effect on the quaternary structure of the complex.…”