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124 CHAPITRE 3. STRUCTURE DE L’HC DE C. MAENAS PAR ESI-MS FIG. 3.8 – Alkaline dissociation and acidic reassociation of Carcinus maenas hemocyanin coupled with chelation of divalent cations by EDTA. Whole hemolymph sample was washed by an EDTA mix (pH 9) as explained in the materials and methods section and salts were then removed by washing with 10 mM AcNH4 containing TEA at various concentrations. Left part, whole mass spectra, right part, focus in the monomer peaks m/z range. Spectra were acquired as follow : first spectrum, washing with 0.05 % TEA (pH 9.02) ; second spectrum, washing with 0.005 % TEA (pH 7.52) ; third spectrum, washing with 0.05 % TEA and addition of 2 mM lactic acid (pH 8.09) ; fourth spectrum, washing with 0.005 % TEA and addition of 2 mM lactic acid (pH 7.35). m, monomers peaks, h, hexamer peaks (450 kDa), d, dodecamer peaks (900 kDa), dl, light dodecamer peaks (885 kDa). On the third spectrum (pH 8.09, 2mM lactate), the hexamer peaks could correspond to a classical hexamer and to a "light" hexamer.
3.2. MANUSCRIT : STRUCTURAL STUDY OF C. MAENAS HC BY ESI-MS 125 3.2.4 Discussion Subunits masses in denaturing and non-covalent conditions The masses observed in denaturing conditions are in good agreement with those previously published by Sanglier and collaborators, also in denaturing conditions (Sanglier et al., 2003). The five most abundant subunits observed in Sanglier’s study are part of the six subunits observed here. In our study, a slight and variable difference is observed between masses estimated from denaturing and from non-covalent conditions. The higher masses estimated in non-covalent conditions can be accounted for by the occurrence of various adducts to the dissociated subunits in these conditions, such as alkaline ions, divalent cations or unremoved copper ions in the active site. These adducts are permitted by the unusual conditions used here, namely an alkaline dissociation observed in ESI-MS conditions preserving the remaining non-covalent interactions during the analysis. Desolvation conditions also differ between ESI-MS in denaturing and in non-covalent conditions, hence the amount of the remaining solvent is likely to be different. Complexes masses and abundances in non-covalent conditions The use of ESI-MS in non-covalent conditions enables to acquire signal for high-molecular mass non-covalent complexes in their native state (dodecamers and hexamers). The good agreement between dodecamer to hexamer ratios from ESI-MS and SEC confirms that our supramolecular ESI-MS data are relevant for studying complexes occurring in solution. For occurrence of 18-mer and 24-mer, aggregation is known to be possible during the ESI process but a slight fraction of 18-mer and 24- mer is also visible by SEC coupled with a light-scattering system (personal observation for Carcinus maenas hemocyanin ; Beltramini et al. (2005) for Penaeus monodon hemocyanin). However, 24-mers can be detected from purified 12-mers in figure 5. This is unlikely to come from a contamination during the purification step since no 18-mer is visible, but must be due to aggregation of the sample during preparation or during the ESI-MS process. Whatever the extent of aggregation process is for these high-mass complexes (18-mer and 24-mer), their concentration is always very low compared to dodecamer and hexamer. Even if they may really exist in the hemolymph of the animal and not be formed during the sampling and storage, they are probably of very little physiological significance due to this low concentration. In Sanglier and collaborators study, the hexamer peak intensity was higher than the dodecamer peak intensity, which is in contradiction with our results (figure 3.3). We found a lower signal for hexamers ; this is in accordance with the relative proportions found by SEC, with a typical hexamer abundance of 5 to 20 % of whole hemocyanin in native hemolymph. The difference with Sanglier’s study must be due to storage in liquid nitrogen whereas our samples were kept at -20°C, since partial
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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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The Structural Analysis of Large No
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174 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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