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116 CHAPITRE 3. STRUCTURE DE L’HC DE C. MAENAS PAR ESI-MS TAB. 3.1 – Subunit masses for Carcinus maenas hemocyanin obtained by ESI-MS Subunit name m/z value for the (17 H + ) peak Mass obtained by in denaturing Average mass Sanglier et al. ESI-MS (Da (2003) (Da) a ±s.d.) b Mass difference Examples of ESI-MS (Da) c denaturing ESI-MS masses estimated between noncovalent by non-covalent and (Da) d n°1 n°2 n°1 n°2 Cm1 73931 73922 ±1.3 4360 74116 74090 194 168 Cm2 74049 74043 ±1.1 4368 74218 74207 175 164 Cm3 75088 75073 ±3.2 4418 75106 75089 33 16 Cm4 75161 75187 ±5.8 4426 75253 75214 66 27 Cm5 75234 75224 ±1.8 4435 75344 75355 120 131 Cm6* 75459 75449 ±0.9 4450 75684 75629 235 180 *Dodecamer specific subunit a Nine masses were determined by Sanglier and collaborators ; the masses that were the closest to those we determined are reported here b Average masses and standard deviations are calculated from data for 14 different individuals ; the number of values for each subunit varies from 9 to 35 depending on the occurrence of each subunit in different individuals and in purified fractions for each individual c These masses were calculated from the monomers charge-state distributions from two different experiments d These differences were calculated using the average masses determined in denaturing conditions in this study ; molecular masses of potential adducts in non-covalent conditions are for example 127.1Da (the two Cu of the active site), 23Da (Na), 24.3Da (Mg), 40.1Da (Ca) and combinations thereof 3.2.3 Results Preliminary analysis of Carcinus maenas hemocyanin 28 different hemolymph samples from 14 different male individuals issued from control or acclimation experiments were analysed by ESI-MS in denaturing conditions. Six main subunits were most frequently observed, of which one was only observed in the dodecameric fraction when analyses were performed on SEC-separated, purified complexes. No covalently bound dimer was ever observed. Table 3.1 presents the masses obtained in our study for these subunits and the nearest masses obtained in a previous study by Sanglier and collaborators (Sanglier et al., 2003). In this previous study, a total of nine subunits were identified by the authors with four of them exhibiting low intensity peaks, the other five being identical to the subunits identified in our study. Hemocyanin analysis by ESI-MS in non-covalent conditions produced two main charge-state distributions corresponding to dodecameric and hexameric species (figure 3.3). The observed dodecamer to hexamer ratio is similar to the one determined by SEC ; this confirms that the desalting steps do not markedly alter the oligomerization equilibrium provided that the analysis is made just after desalting (storage shorter than 2 days at 4°C). Some high-mass distributions can also be observed with a lower intensity, corresponding to 18-meric and 24-meric complexes (up to 13 and 7 %, respectively).
3.2. MANUSCRIT : STRUCTURAL STUDY OF C. MAENAS HC BY ESI-MS 117 FIG. 3.3 – Mass spectrum of Carcinus maenas native hemolymph analysed in non-covalent conditions. Spectra were acquired using individual whole hemolymph samples. Aggregation state, estimated mass and m/z value for the main peak of each distribution are indicated. Insert : focus on the 10000 m/z area of a spectrum showing two overlapping distributions for the hexameric state. The higher mass hexamer was observed only in one individual and is likely to be made of six 81 kDa non-hemocyanin subunits (observed in denaturing conditions) (see the text for details). SEC experiments coupled with light-scattering mass determination (not presented here - personal observation) also revealed the presence of a slight amount of these high molecular-mass complexes. It is worth noting that for one crab, two different distributions were observed for the hexamer (figure 3.3, insert) ; this crab also harbored an original subunit with a molecular mass of 80962 Da (data not shown). The mass of the second hexameric species for this crab fits well with a complex made of six such subunits. This original hexamer could be a non-hemocyanin protein such as cryptocyanin which has a similar hexameric quaternary structure and is closely related to hemocyanin but cannot bind dioxygen (Terwilliger et al., 2005). Further analysis is required to unambiguously identify this protein.
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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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Dénaturant Natif
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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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