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118 CHAPITRE 3. STRUCTURE DE L’HC DE C. MAENAS PAR ESI-MS FIG. 3.4 – Alkaline dissociation of Carcinus maenas hemocyanin by TEA. Left part, mass spectra of a whole hemolymph sample desalted in 10 mM AcNH4 with increasing quantities of TEA (0.005, 0.03 and 0.05 % TEA for pH 7.55, 8.55 and 9, respectively). m, monomers peaks, h, hexamer peaks (450 kDa), d, dodecamer peaks (900 kDa), dl, light dodecamer peaks (885 kDa). Right part, successive focuses on the monomers peak distributions. Six overlapping distributions can be observed. Masses obtained in non-covalent and denaturing conditions for each subunit are compared in table 1.
3.2. MANUSCRIT : STRUCTURAL STUDY OF C. MAENAS HC BY ESI-MS 119 Alkaline dissociation of complexes by TEA A native hemolymph sample was progressively dissociated in its constituting subunits by adding increasing quantities of TEA (figure 3.4). Before addition of TEA, one main species is observed and corresponds to a 900 kDa mass (dodecamer). A less intense signal is visible corresponding to a hexamer mass around 450 kDa and a slight monomer signal is also visible. It can be noted that the particular native sample shown here contains almost only dodecamers and only very few hexamers. Experiments on SEC-purified dodecamers and hexamers are detailed later. Upon TEA addition, the intensity of the dodecamer distribution decreases while the monomers peaks get more intense. A second distribution with higher m/z values is observed ; precise charge-state assignment is difficult for this due to the high mass of the complex. This distribution can correspond either to an 885 kDa or to an 864 kDa mass. Given that no subunit of 864/12 = 72 kDa is observed in denaturing conditions, this distribution can be identified unambiguously with the 885 kDa mass. This mass fits well with a dodecamer reassociated from the lightest subunits (below 74 kDa) produced by alkaline dissociation. The occurrence of this species before acidic reassociation suggests that a dynamic equilibrium between it and the free subunits exists as soon as dissociation of the dodecamers begins. Relative abundance of residual dodecamer after alkaline dissociation could vary slightly from one experiment to the other, but a massive dissociation was always observed. The monomer charge-state distributions can be examined to determine the different subunits which are present during the course of the dissociation (figure 3.4). Six main charge-state distributions are visible, corresponding to six subunits. The estimated masses are depicted in table 3.1. They could vary slightly from one experiment to another, but were almost always superior to the masses observed in denaturing conditions. Acidic reassociation of subunits by formic acid After alkaline dissociation of the complexes into their subunits, the preparation is acidified by progressive addition of formic acid (figure 3.5). The presence of dissociated subunits at various stages of the acidification process can be monitored by examining the distribution around m/z 4400, corresponding to the monomers with a 17 H+ charge. When pH decreases from 8.55 to 7.5, a progressive and partial reassociation into complexes is observed, mainly into light dodecamer (885 kDa) and dodecamer (900 kDa) complexes. In the meantime, a progressive disappearance of the Cm1 and Cm2 peaks corresponding to the lightest monomers suggests that these subunits are preferentially recruited for reassociation into complexes. When pH becomes strongly acidic (below the isoelectric point), complexes are dissociated anew and all subunits peaks are visible again.
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THESE DE DOCTORAT DE L’UNIVERSITE
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i Résumé / Abstract Réponse adap
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iv SOMMAIRE 4.2 Objectifs de l’é
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2 CHAPITRE 1. INTRODUCTION Les éco
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4 CHAPITRE 1. INTRODUCTION 1.1 Mili
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6 CHAPITRE 1. INTRODUCTION Les prin
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8 CHAPITRE 1. INTRODUCTION TAB. 1.1
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10 CHAPITRE 1. INTRODUCTION et cycl
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FIG. 1.5 - Localisation des princip
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(a) FIG. 1.6 - Fonctionnement d’u
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16 CHAPITRE 1. INTRODUCTION TAB. 1.
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18 CHAPITRE 1. INTRODUCTION FIG. 1.
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20 CHAPITRE 1. INTRODUCTION Différ
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24 CHAPITRE 1. INTRODUCTION le mili
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26 CHAPITRE 1. INTRODUCTION Capacit
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28 CHAPITRE 1. INTRODUCTION TAB. 1.
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30 CHAPITRE 1. INTRODUCTION 700 600
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32 CHAPITRE 1. INTRODUCTION férent
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34 CHAPITRE 1. INTRODUCTION (a) (b)
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40 CHAPITRE 1. INTRODUCTION 1 P 50
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42 CHAPITRE 1. INTRODUCTION presque
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44 CHAPITRE 1. INTRODUCTION affecte
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50 CHAPITRE 1. INTRODUCTION fure co
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52 CHAPITRE 1. INTRODUCTION amélio
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54 CHAPITRE 1. INTRODUCTION 1.5 Que
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56 CHAPITRE 2. ESI-MS ET MALLS APPL
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Page 78 and 79: The Structural Analysis of Large No
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168 CHAPITRE 4. PLASTICITÉ PHÉNOT
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170 CHAPITRE 4. PLASTICITÉ PHÉNOT
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172 CHAPITRE 5. ADAPTATIONS RESPIRA
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216 CHAPITRE 6. CONCLUSION ET PERSP
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Annexe A Détermination des masses
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229 10000 h280 .M h) h280−h337 80
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Annexe B Détermination des distrib
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233 Modèle 2 : 2 sous-unités lég
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235 1 0.9 0.8 6 s-u aléatoires 2 s
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Bibliographie Adair, G. S. The hemo
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BIBLIOGRAPHIE 239 Catalina, M. I.,
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BIBLIOGRAPHIE 241 Farmer, C. S., J.
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BIBLIOGRAPHIE 243 Hourdez, S. Adapt
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BIBLIOGRAPHIE 245 Mangum, C. P. Sub
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BIBLIOGRAPHIE 247 Rainer, J., C. P.
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BIBLIOGRAPHIE 249 Svedberg, T. et I
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BIBLIOGRAPHIE 251 Vanin, S., E. Neg
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Liste des figures 1.1 Cycle des mar
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LISTE DES FIGURES 255 4.17 Profils
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Table des matières Résumé Sommai
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TABLE DES MATIÈRES 261 4.3.5 Spect
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TABLE DES MATIÈRES 263 Table des m
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