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

164 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al.
(Table 2) contd….
Chain/
Subunit
Mass Mass +
(Da) a heme b
(Da)
References
Chain/
Subunit
Mass Mass +
(Da) a heme b
(Da)
References
D1 50323.1 L1+L1 [83]
L4a 24169.9
D2 51981.5 L1+L2
L4b 24102.3
L4c 24019.0
a-Estimated error of measurement ± 1.0Da for M and ± 4.0Da for T
b-Mass(heme)= 616.5 Da
c-Weighted mean based on relative intensities [23]
d-Estimated error of measurement ± 5.0 Da
e-Calculated mass using average, intensity weighted masses of the apo-monomer (M) and apo-trimer (T) subunits: 15 983.2 and 52 696.1 Da for LtHb and 15 966.7 and 49 680.7 Da
for AmHb respectively. At each calculated mass, 12 hemes are added.
f-Subassembly proposed by Riggs and collaborators for LtHb [95, 96]
g-Representative measured mass of the dodecamers obtained by ESI-MS under non-denaturing conditions
were determined by denaturing ESI-MS. Given these
masses, the structure the most likely to explain dodecamer
masses was found to be M 3 T 3 (3 monomers and 3 trimers).
From this structure and using relative proportions from ESI-
MS data, a random assembly model of the dodecamers can
be performed. For M and T, chains are selected from the
existing pool for each species with probabilities depending
on their proportion in the ESI-MS spectrum, and incorporated
independently of each other in the dodecamer. Calculated
expected masses are 213 436 ± 319 Da and 204 342 ±
80 Da for Lumbricus and Arenicola, respectively. These are
in good agreement with measured masses of 214 200 Da and
204 600 Da. The calculated expected range and the experimental
mass range are both larger for Lumbricus than for
Arenicola. This supports the M 3 T 3 model for these species
and suggests than the broader experimental mass range for
Lumbricus is due to a higher number of possible combinations
for dodecamer assemblies (200 versus 140 for Arenicola).
It is worthwhile to note that masses obtained by the
statistical model and by simple intensity-average mass calculation
are the same since the model incorporates probabilities
based on intensity from ESI-MS. However the statistical
approach also gives the expected range and the standard deviation
which are in good agreement with experimental data
and thus supports the model.
Another work investigated the theoretical mass distribution
of the whole assembly for Lumbricus terrestris and
Riftia pachyptila HBL-Hb [32]. Considering subunit masses
and proportions from ESI-MS data, mass distributions were
calculated for dodecamers, for 12 dodecamers, for linkers
and finally for the whole HBL-Hb structure. For the dodecamer
distribution, the M 3 T 3 model was used for Lumbricus
while Riftia dodecamers were built from a random number
of monomers and dimers totalizing 12 globin chains. At each
step additions of different subunits were considered independent
from each other. Expected masses for the whole
assemblies were 3 516 ± 16 kDa and 3 284 ± 10 kDa for
Lumbricus and Riftia HBL-Hb, respectively. These masses
were shown to be in very good agreement with the mean of
known masses for Lumbricus (3 524 ± 481 kDa) and the
mean of all the known polychaete HBL-Hb masses for Riftia
(3 209 ± 389 kDa). MALLS values of 3 755 ± 80 kDa and
3 503 ± 13 kDa were obtained for Lumbricus and Riftia, respectively
[32, 85]. The corresponding differences represent
6.8 % and 6.7 % variations. The higher heterogeneity of
Lumbricus linker subunit masses compared to Riftia results
in calculated variation values (ratio range/expected mass) of
30.2 % for Lumbricus and 15.9 % for Riftia, for the linker
subassembly.
Probabilistic approaches are thus a convenient way to
reproduce theoretical assembly from subunit masses and
proportions. Expected masses and variations can support a
model when compared to experimental measurements and
ought to be closer to biological reality since they deal with
mass distribution rather than with a unique determined mass.
5. REBUILDING RESPIRATORY PIGMENT STRUC-
TURE FROM EXPERIMENTAL DATA: THE
EXAMPLE OF CRUSTACEAN HC
In his thorough study of respiratory pigments by sedimentation
velocity and sedimentation equilibrium, Svedberg
examined sedimentation constant and molecular mass for
various decapod crustacean Hc [104]. Two values were determined
for sedimentation constants: 16.9 S and 23.4 S. The
forms present in the hemolymph depended on the species.
Remarkably, Svedberg had observed the two main Hc forms
found in crustacean hemolymph and had accurately determined
their sedimentation constant values. However, the
determined masses (360 kDa and 640 kDa instead of 450
kDa and 900 kDa, respectively) were underestimated, as was
the case for annelid hemoglobins.
Nowadays MALLS is a rapid and convenient way to determine
the mass of an unknown Hc complex and to assess
the polydispersity of the different fractions. Fig. (2) presents
a typical MALLS analysis displaying the calculated molecular
mass versus elution volume during a SEC experiment on
Rimicaris exoculata hemolymph [105]. Crustacean samples
analyzed by SEC-MALLS usually exhibit one or two Hc
peaks with molecular masses around 900 kDa and 450 kDa.
Since the known mass of a single subunit is about 75 kDa,
these masses fit unambiguously with dodecameric and hexameric
forms, respectively. This is confirmed by TEM observation
of hemolymph samples in which globular particles
are visible either isolated (hexamers) or associated by two
(dodecamers). When analyzing Hc from different decapod
species, dominant forms can be observed within each group
and permit to outline some evolutionary patterns [106] (see
85