maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

168 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al.
weight; retention times do not vary between different hemolymph
samples (b) Masses obtained for LtHb. Values for MALLS is taken
from [32]. Partially native value for ESI-MS is based on the native
mass obtained by [63] for the dodecamer; it is calculated for 12
dodecamers and 36 linkers. The linker average mass is calculated
from [23] data, with abundances from [167] (27 659.94 Da). Values
for other methods are taken from [116] and include LS, STEM, EM
and SE determination. The model mass was taken from [32]. (c)
Masses obtained for Arenicola marina HBL-Hb. MALLS values are
from [89]. Partially native ESI-MS values were calculated from
native dodecamer mass from [64] and from the average linker dimer
mass calculated from [83] (50 945 Da). It is calculated for 12
dodecamers and 18 linker dimers. Values for other methods are
taken from [32], considering only data posterior to 1950. They includes
SE, SD and SAXS. The model mass was calculated from the
dodecamer mass calculated by [71] from denaturing ESI-MS data
and from the average linker dimer mass calculated from [83]. It
should be noted that the partially native ESI-MS determination for
HBL-Hb masses shares the linker data with the calculated model
mass.
Fig. (4). Comparison between masses estimated by MALLS,
ESI-MS and other methods for Hc and HBL-Hb.
Bars are mean values with s.d.; the dashed line corresponds to a
model calculated from masses obtained in denaturing ESI-MS (a)
Masses obtained for Carcinus maenas Hc. The same samples
(n=16) were analyzed by MALLS and ESI-MS except for one
which could not analyzed by ESI-MS. The model mass was calculated
for a dodecamer using an intensity-weighted mean subunit
mass with two copper atoms of 74 826.91 Da, obtained from all
samples in denaturing conditions. The SEC mass was obtained after
calibration of a Superose 6 column by markers of known molecular
HBL-Hb has been reported to involve independent folding of
individual domains followed by domain interaction for the
oligochaete Lumbricus terrestris Hb [110]. The stability of
the quaternary structure of annelid HBL-Hbs has been studied
by changing the chemical composition of the medium: by
varying pH, incubation in the presence of chaotropic salts, or
denaturing agents. There is an increasing interest in understanding
the dissociation and association process of this Hb
because it provides useful information about subunit interactions
necessary to maintain the quaternary structure. Moreover,
AmHb has been proposed as useful model system for
developing therapeutic extracellular blood substitute [7, 8]
and required a full study of subunit interactions in order to
find the correct storage and transfusion buffer composition.
In order to analyze the interactions between globin and linker
subunits constituting HBL-Hb, dissociation and reassociation
experiments were carried out under several conditions: exposure
to urea and alkaline pH and the effect was followed by
SEC-MALLS and ESI-MS. Thanks to the complementarity
of information obtained with ESI-MS and MALLS analysis,
Rousselot and collaborators have bring to light a novel and
complex mechanism of dissociation for AmHb compared to
other annelid extracellular Hbs studied to date [6]. Even
though the chemically-induced dissociation triggers partial
degradation of some subunits, spontaneous reassociation was
observed to some extent. Parallel dissociation of LtHb under
similar conditions shows striking differences that allow us to
propose a hypothesis on the nature of the intersubunit contacts
that are essential to form and to hold such a complex
quaternary structure.
6.2. An Application Example: The Case of Arenicola marina
HBL-Hb
MALLS analysis of partially dissociated AmHb at
slightly alkaline pH yielded profiles shown in Fig. (5), with
molecular masses Mw (Fig. 5a) and gyration radius Rg
(Fig. 5b) estimated during the elution profile at 3 different
incubation times. The estimated Mw (Fig. 5a) decreases during
incubation time and the polydispersity estimated by the
molar mass slopes assumes a downward curvature shape,
89