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170 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al.
Fig. (6). Dissociation/reassociation properties of AmHb followed by SEC-MALLS and electron micrographs.
(a) After dissociation of AmHb (red), some rearrangements are observed when the sample is returned to neutral pH (blue). This rearrangment
is only observed in the presence of partially dissociated HBL-Hbs and the 1/12th subunits (D+L) and led to a recovery of the structure of the
1/12th subunits at different degree of polymerisation, and up to a completely reassociated HBL-Hb. (b) Electron micrographs of AmHb, negatively
stained showing self-association properties of Arenicola Hb. (Native) View of native AmHb. (Dissociated) View of truncated HBL and
dodecamers of AmHb isolated by gel filtration after dissociation. (Reassociated) View of reassociated AmHb isolated by gel filtration. The
yellow and pink arrows indicate the top and side view of the AmHb HBL-Hb, respectively.
of Rg are characteristic of an extended unfolded conformation
during the dissociation process. The decrease of Rg after
2 hours of dissociation is explained by the formation of
smaller subunits with smaller radius. The important scatter is
due to the presence of a mix of small structured subunits and
small destructed subunits which have a higher gyration radius.
After 24 hours of dissociation most of these dissociated
subunits are denaturated so the scattering is less important.
The extent of reassociation of AmHb was investigated by
MALLS after dissociation at alkaline pH. Since scattering
intensity is strongly dependent on particle radius, a small
amount of large particles in the sample would give a large
response with the light scattering detector, although their
amount, as measured by the RI response, is low. These interesting
properties allowed us to observe a reassembly of
AmHb which was not so easily observed using gel filtration
only (Fig. 6). The reassociation is limited, as revealed by the
RI profiles of peak I HBL after reassociation and the proportion
of reassociated HBL-Hb (Fig. 8). The observation of the
reassociation is characterized by the differences of the LS
signals for the I HBL peak before and after the reassociation
(Fig. 8). The reassociation is not observed after 1 hour of
dissociation at alkaline pH (Fig. 8c) and is less important as
pH increases (Fig. 8b) and coincides with the absence of
truncated HBL-Hbs (retention time between 20 and 25 min)
as revealed by MALLS profile (Fig. 8c). The reassociation is
confirmed by the TEM images of I HBL isolated by gel filtration
after reassociation process (Fig. 6b). We can distinguish
truncated HBL-Hbs in a more structured conformation than
before reassociation (Fig. 6a) and structured HBL-Hb similar
to native Arenicola HBL-Hb (Fig. 6b).
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