(Received for publication, November 1, 1995; and in revised form, January 26, 1996)
From the
The deep-sea tube worm Riftia pachyptila Jones
possesses a well developed circulatory system and a large coelomic
compartment, both containing extracellular hemoglobins. Fresh vascular
blood is heterogeneous and contains two different hemoglobins (V1 and
V2), whereas the coelomic fluid is homogeneous and comprises only one
hemoglobin (C1). Their molecular weights have been determined by
scanning transmission electron microscopy mass mapping (STEM) and by
multi-angle laser light scattering (MALLS). Both methods yielded
approximately the same molecular weights with masses significantly
higher than the literature data for V1. V1, V2, and C1 had M of 3396 ± 540
10
, 393
± 71
10
, and 410 ± 51
10
by STEM, and 3503 ± 13
10
,
433 ± 8
10
, and 380 ± 4
10
by MALLS, respectively. Transmission electron
micrographs of V1 are typical of an hexagonal bilayer hemoglobin (HBL
Hb). When submitted to dilution or osmotic shock, V1 dissociates into
halves and one-twelfth subunits like annelid HBL Hbs. V1 is resistant
to urea treatment, indicating that hydrophobic interactions play a
small role in its quaternary structure. Conversely, V1 Hb is rather
unstable in solution without denaturant, a property which seems to be
characteristic of vestimentiferan HBL Hbs and could be explained by an
important number of hydrogen bonds.
Deep-sea hydrothermal vents are characterized by unusual
chemical and physical parameters, including high pressures, high
temperature gradients, and high concentrations of toxic elements such
as sulfides and heavy metals(1) . In this extreme environment,
flourishing animal communities live. Riftia pachyptila Jones (2, 3, 4) is the most spectacular and one of
the best studied species of these communities. This animal is strictly
endemic to this ecosystem and inhabits sulfide-rich environments at
depths of about 2600 m. This autotrophic organism is devoid of
digestive tube and derives the energy necessary for its growth and
metabolism from chemoautotrophic sulfur-oxidizing endosymbionts
harbored in a special, highly vascularized organ, the
trophosome(5, 6, 7) . Since the symbionts are
isolated from ambient water and inorganic nutrients, Riftia has to supply them with O, H
S and
CO
. Transportation of these inorganic nutrients is
facilitated by several different extracellular hemoglobins (Hbs) (
)present in the well developed circulatory system and the
large coelomic compartment of Riftia. These Hbs have a high
affinity for oxygen(8, 9) , an ability to reversibly
bind sulfide(10, 11, 12, 13) , and a
moderate ability to combine with carbon dioxide(14) . Although
the functional properties of Riftia Hbs have been well studied
(reviewed in (15) ), there are some uncertainties on their
number, distribution between the body compartments, and
structures(8, 11, 16) . Terwilliger et
al.(8) described two Hbs with different molecular weights
in the blood of R. pachyptila; a large one with M
of about 1700
10
(FI), and a
small one of M
400
10
(FII).
The M
of FI was unusual when compared to HBL Hbs
from annelids (17) or other
vestimentiferans(16, 18, 19) . Arp et al.(11) concluded that the FII fractions from vascular and
coelomic fluids were identical and that these compartments may
therefore be confluent at the molecular size of the lower M
Hb. However, this possibility was excluded on
the basis of statistical results of their distribution between the two
compartments(20) . To date, these conclusions have not been
confirmed with structural data.
Indeed, the number and the molecular weight of the different Hbs and their distribution between the different body compartments remain unclear in Riftia and other Vestimentifera. Hence, the aim of the present work was first to identify and purify R. pachyptila Hbs, and then to evaluate their relative molecular masses using the most accurate techniques to date: scanning transmission electron microscopy (STEM) mass mapping and multi-angle laser light scattering (MALLS). With the determination of the complete polypeptide chain composition of these Hbs, reported in a companion study(27) , these mass estimations will allow us to elaborate proper models of the quaternary structure of these multimeric proteins.
Figure 1: Elution profiles of R. pachyptila hemoglobins on Superose 6-C gel eluted with Riftia saline buffer. A, freshly frozen vascular blood contains two Hbs (V1 and V2). B, freshly frozen coelomic fluid contains only one Hb (C1). C, stored vascular blood contains four Hbs (V1, V2, V3, and V4). D, stored coelomic fluid contains two Hbs (C1 and C2). (See text for description of stored and freshly frozen conditions.)
Figure 2:
Elution profiles of purified Hbs of R.
pachyptila V1 (A) and A. marina (B),
after dilution at 1:600 in 0.05 M Tris-Cl buffer (pH 7.5). The arrows and numbers indicate the different peaks
obtained. (1, M
3000
10
; 2, M
1500
10
; 3, M
220
10
).
Purified V1 Hb was
also incubated with increasing concentrations of urea in saline buffer
for 30 min to 8 h (Table 1). After 8 h of incubation with 2 M urea, the native peak was unaltered; a small peak (400 kDa)
appeared after 2-h incubation with 4 M urea and a third one
(150 kDa) after 8 h. In the presence of 8 M urea after only
30-min incubation, V1 dissociated into three major components with
masses 400, 220, and 150 kDa, respectively.
Figure 3: Electron micrographs of purified R. pachyptila Hbs and native A. marina Hb, negatively stained with 2% uranyl acetate. A, HBL Hb V1, face view (fw) and edge view (ew); the inset shows a dissociating molecule. B, view of A. marina HBL Hb and one-twelfth subunits (ot). C, small ring-shaped Hb V2. D, small ring-shaped Hb C1, the aggregate next to the scale bar is a contamination by HBL Hb. Scale bar = 30 nm.
Figure 4: STEM mass mapping measurements of R. pachyptila Hb V1. Histogram of the masses of 331 particles.
Figure 5: MALLS analysis of R. pachyptila Hb V1. A, molecular mass (g/mol) versus elution volume (ml). B, gyration radius (nm) versus elution volume (ml).
Gel filtration performed on freshly frozen body fluids from R. pachyptila revealed two Hb fractions for vascular blood (V1
and V2) and one for coelomic fluid (C1). Therefore we assume that V3,
V4, and C2 found only in stored samples are dissociation products of
V1, V2, and C1. The elution profile of stored Riftia vascular
blood was similar to that obtained for Lamellibrachia sp.,
another vestimentiferan worm(18) . However, in this last case,
the body fluids were not collected separately before gel filtration,
and these results were obtained on mixed fluid. Anyway, Riftia Hbs M values determined by FPLC are in
agreement with those found in Lamellibrachia sp. (18) for V1, V2, or C1, but they are different from those
reported previously for R. pachyptila larger Hb(8) .
Indeed, Terwilliger and co-workers(8) , also working on mixed
fluid, obtained two majors fractions with masses around 1700 kDa (FI)
and 400 kDa (FII). They interpreted the unusual mass of FI by the
pigment instability as well as the broadness of the elution peak that
may contain some intact molecules whose molecular masses would
correspond more closely to 3000 kDa(8) , the usual mass of HBL
Hbs.
Our experiments on dilution and/or osmotic shock on V1 Hb and A. marina Hb could also explain this unusual mass. Gel
filtration performed after incubation of both hemoglobins in 0.05 M Tris-Cl buffer (pH 7.5) revealed in both cases three peaks which
could correspond to the dissociation of the whole molecule into halves,
and one-twelfth subunits. This scheme of dissociation was also found
for Lumbricus terrestris HBL Hb(28, 29) . V1
Hb is rather unstable and dissociates rapidly in agreement with first
observations (8, 30) , suggesting that the instability
of R. pachyptila vascular Hb might be due to changes in
pressure as the protein is brought to the surface or during work at
atmospheric pressure. This relative instability might well be an
inherent property of vestimentiferan Hbs, since the same phenomenon was
observed for Lamellibrachia sp. (19) and for Tevnia jerichonana. ()Furthermore, the quaternary
structure of HBL Hbs from alvinellids, which are vent annelids, is not
similarly affected by pressure changes(30) .
Surprisingly, V1 Hb was resistant to dissociation by urea since a small amount (about 30%, Table 1) of dissociation products were observed even at high urea concentrations. The same was true for Lamellibrachia sp.(19) . However, V1 Hb produced the same dissociation products as L. terrestris HBL Hb at alkaline pH with the appearance of three peaks corresponding to subunits approximately 350, 130, and 60-90 kDa(31) . As in L. terrestris, the 350-kDa component is not exactly one-twelfth of the native molecule since it contains some linker chains(31) . Since urea is known to destabilize hydrophobic interactions(32) , these should play a small role in the quaternary structure of V1 Hb. This is consistent with the fact that V1 is rather unstable in solution without denaturant, which could be explained by an important number of hydrogen bounds.
Molecular masses of R. pachyptila Hbs measured by MALLS and STEM mass mapping are statistically different (Student's t test, p < 0.001). This discrepancy may be due to the rather harsh preparative treatments necessary for STEM mass mapping as opposed to MALLS, which analyses molecules directly after the purification step. This is also reflected by the lower standard deviations obtained with MALLS. However, both methods yield a usual mass for V1, corresponding to a classical HBL Hb as found in annelids and vestimentifera, and in agreement with the TEM micrographs.
V2 and C1 Hbs had masses corresponding to the smaller ring-shaped structures revealed by electron micrographs. Similar structures are also found in pogonophoran(33) . Both MALLS and STEM mass mapping provide statistically different masses for V2 and C1 suggesting that these two Hbs are distinct molecules. However, as determined by MALLS V2 is larger than C1, while the opposite was found with STEM mass mapping. Even if MALLS estimations are considered more accurate, it is therefore hazardous to conclude that V2 and C1 are indeed distinct molecules. More accurate data on the complete polypeptide chain composition of these Hbs are necessary to resolve this crucial point.