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

158 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al.
(Legend Fig. 1) contd….
on the basis of the measured dn/dc value of 0.185 ml/g. (d) ESI m/z spectrum of the ~200 kDa subassembly from AmHb (left) and LtHb
(right). Inset, result of deconvoluting the ESI spectrum over the m/z range 5700-7500 by MaxEnt. M, T and D set for the monomer chain, the
trimer and dodecamers subassembly respectively. The number after the colon indicates the number of charges on the ion. (reprinted from
[71], with permission from ASBMB). (e) MaxEnt-processed spectra of native AmHb (left) and LtHb (right) analyzed under denaturing conditions.
a1-2, b, c, d1-3 design the monomeric chains, T1-4; the trimer subunits and L1-4; the linkers monomer and D1-2; the dimeric linkers
(reprinted from [83] and [23], with permission from Elsevier for [23]). (f) Structural model proposed for AmHb (left) and LtHb (right). Drawing
of typical HBL-Hb on top view displaying the subunits for half a Hb molecule. Left: Each AmHb one-twelfth is constituted of one trimer
(T3, T4 or T5), with nine monomeric chains; M 9 T. The one-twelfth is detailed showing the possible arrangement of polypeptide chains [83].
Right: Each LtHb one-twelfth is constituted of three trimers T, with three monomeric chains; M 3 T 3 . (g) 3D reconstruction from crystallographic
data. Left: The 6.2 Å electron density of AmHb. Density corresponding to the globin chains is depicted dark violet, the linker chains
associated with the top of the molecule are shown in orange, while those associated with the bottom linker subunits are shown in blue (reprinted
from [81], with permission from Elsevier). Right: The 5.5 Å electron density of LtHb. Density corresponding to the globin chains is
depicted magenta, the linker chains associated with the top of the molecule are shown in yellow, while those associated with the bottom linker
subunits are shown in blue (reprinted from [80], copyright PNAS).
ied HBL-Hbs of the oligochaete Lumbricus terrestris (LtHb)
and the polychaete Arenicola marina (AmHb).
Prior to the use of ESI-MS and MALLS, the bracelet
model was the first model proposed for HBL-Hb based on
observation of dissociation products of LtHb. It consists of
12 dodecamers decorating a bracelet or framework of 30-40
linkers [93, 94] (Fig. 1a). On the basis of the mass provided
by STEM analysis (3.55 x 10 6 Da) and subunit masses estimated
by SDS-PAGE, the authors were able to propose a
structural model where the 12 dodecamers (2.13 x 10 5 x 12 =
2.56 x 10 6 Da) account for 72 % of the total mass, in excellent
agreement with the iron and heme content data [37].
Another proposed model suggested that LtHb was comprised
of 192 globin subunits and 24 linkers. This model was based
on native mass from light scattering measurement (4.1 x 10 6
Da), subunit masses from mass spectrometry and linker proportion
from capillary electrophoresis and HPLC experiments
[22, 95, 96].
Concurrent ESI-MS investigations of LtHb [23] and
AmHb [83] were led to obtain the molecular masses of their
constituent chains and subunits as well as their relative proportions.
Fig. (1d) shows the raw ESI-MS spectra of AmHb
and LtHb respectively obtained under non-denaturing conditions
and Fig. (1e) shows the resulting deconvolution on a
zero charge mass scale, using the MaxEnt analysis of the
ESI-MS spectra under denaturing conditions. The mass spectra
under denaturing conditions of the native HBL-Hb in
combination with those of reduced, reduced and carbamidomethylated
and unreduced carbamidomethylated forms
permit the determination of the total number of Cys residues
as well as the number of free and disulfide bound Cys residues.
ESI-MS has also provided unambiguous determination
of the nature of glycosylation, either of globin or of linker
subunits [69]. This detailed mass information coupled with
the MALLS determination of the molecular mass of the
whole HBL-Hb complexes has allowed to propose suitable
models for the quaternary structures with much greater confidence
than was possible heretofore [23, 82-85] (Fig. 1f).
Table 2 sums up representative masses and their SD of
polypeptide chains and the globin and linker subassemblies
observed in the native HBL-Hb of A. marina and L. terrestris
and the closest matching masses calculated for a dodecamers
globin subassembly and other possible combinations
of globin subunits. Intensity-weighted mean masses for
the globin subunits were derived from these data and used to
generate the calculated subassembly masses in Table 2.
When allowance was made for variations in the relative intensity
measurements, the estimated errors in the calculated
subassembly masses shown in Table 2 were < 0.05 %. It
should be noted that among the oligochaete and polychaete
Hb that have been investigated, there are two different patterns
of globin subunits [97, 98] as well as linker subunits
(see Table 1 and below). They are composed of one or more
~17 kDa monomer globin chains (M) and one or more disulfide-bonded
~50 kDa trimer subunits (T) [83, 84, 88, 99] and
the linker chains are either present as monomers or disulfidebound
dimers. This diversity in the small globin subunits can
obviously lead to more than one combination of these
subunits to give a dodecamer subassembly.
LtHb contains four globin chains (chains a-d) ranging in
mass from 15962 Da to 19390 Da, with the monomer
subunit (chain d) existing as three isoforms (d1-d3), and
chain a occurring as four glycosylated isoforms (a1-a4); the
latter forms four different disulfide-bound trimer subunits
with chains b and c [23]. Four monomeric linkers (L1-L4)
are observed (Table 2). AmHb contains eight different globin
chains (a1,a2,b1,b2,b3,c,d1 and d2) ranging in mass from
15 922 to 17 032 Da, with the b, c and d chains forming five
of the six possible disulfide-bound trimers (T1-T5)
(c+b1+d1, c+b1+d2, c+b1+d2, c+b2+d1, c+b2+d2, and
c+b3+d2) [83]. The two linkers (L1-L2) are disulfide-bound
to form hetero- or homo-dimers as detailed in Table 2.
The complete ESI-MS determination of the masses of the
constituent globin and linker chains and the disulfide-bound
trimer subunit of LtHb provided a calculated mass of
213.434 kDa for the assembly [M] 3 [T] 3 [23] (Fig. 1d, Table
2). Furthermore, the calculated masses for the Hb comprised
of 12 subassemblies and 36 or 42 linker chains are 3.523 and
3.687 MDa, respectively, in good agreement with the masses
determined by scanning transmission electron microscopy
and sedimentation equilibrium (3.56 ± 0.13 and 3.41 ± 0.39
MDa, respectively) [23]. However, the structural model of
the AmHb dodecamers proposed by Zal and collaborators
[83] (Fig. 1f) is slightly different than the model proposed by
Green and collaborators: D=M 3 T 3 . A model of the quarternary
structure of Arenicola marina HBL-Hb has been proposed
by Zal and collaborators based on ESI-MS analysis
and MALLS measurements (Fig. 1f). Three and six copies of
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