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The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 169
Fig. (5). Evaluation of the molecular weight and gyration radius during the dissociation process of AmHb.
Dissociation of AmHb followed by MALLS during the elution from a gel exclusion column (Superose 6-C). The solid curve represents the
refractive index (RI) profile overlaid with the dotted curve which represents the light scattering profil at 90° (LS), versus the retention time.
The RI and LS data have been scaled to make the comparison easier. (a) Distribution of the molecular weight values at different incubation
times: Mw profiles of AmHb immediately after exposure at pH 7.8 (red crosses), after 2 hours dissociation (black squares) and 24 hours dissociation
(blue triangles). The RI and LS profile correspond to a dissociation time of 2 hours. (b) Distribution of the gyration radius values at
different incubation times: Rw profiles of AmHb immediately after exposure at pH 7.8 (red crosses), after 2 hours dissociation (black
squares) and 24 hours dissociation (blue triangles).
characteristic of a less homogenous population. The polydispersity
of the peak I HBL indicates that it includes intermediates
of dissociation which are truncated HBL AmHb (Fig.
5a). Truncated HBL-Hb (partially dissociated HBL-Hb particles
lacking 1/6 to 1/2 of the HBL structure) are also observed
on the TEM images of I HBL fraction purified by gel
filtration (Fig. 6b). Even if the estimated gyration radius
average values (Fig. 5b) are close to the angular variation
detection limit of 10 nm, Rg decreases after 2 hours with an
important scattering and increase after 24 hours of incubation
for I 1 and I 2 . The same samples were analyzed by ESI-
MS under non-denaturing conditions and provided the results
shown in Fig. (7a,c). ESI-MS spectrum of partially dissociated
AmHb under non-denaturing conditions (Fig. 7a)
reveals the presence of a subunit of ~224 kDa which should
correspond to the 1/12 th subunits (D+L). However, ESI-MS
spectrum of entirely dissociated AmHb under denaturing and
non-denaturing conditions (Fig. 7c) revealed the presence of
a subunit of ~204 kDa which correspond to the dodecamers
D. Several simultaneous dissociations of a HBL-Hb structure
can be envisioned as proposed for Lumbricus Hb dissociation
[111]. However, the dissociation process of AmHb is
more complex to interpret. The dissociation leads to the
rapid formation of the 1/12 th subunits (D+L) through truncated
HBL. Indeed, SEC-MALLS (Fig. 6a), TEM (Fig. 7b)
and ESI-MS results (Fig. 7a,b) reveal the presence of a small
amount of truncated HBL at the early stage of the dissociation
process and the formation of one major peak I D (Fig. 5)
interpreted as the 1/12 th subunits (D+L) because of the
higher Mw of peak I D (MALLS and ESI-MS results,
Fig. 7a,b). All indicate that the dodecamer is still associated
with linkers at the beginning of the dissociation. Then, the
linkers dissociate from the dodecamer resulting in a decrease
of Mw (peak I D , Fig. 7c). The dodecamer does not dissociate
into stable trimers and monomers as observed for Lumbricus
Hb [111] but into higher Mw units (peaks I 1 and I 2 , Fig.
5a,7a), in low abundance and transitory. The denaturation of
these subunits is evident from the variation of the gyration
radius Rg (Fig. 5b). Rg increases while the molecular mass
decreases after 24 hours dissociation. These variations
90