(Received for publication, November 21, 1995; and in revised form, January 26, 1996)
From the
The dissociation of the 3500-kDa hexagonal bilayer (HBL)
hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm
salts, urea and the heteropolytungstates
[SiW
O
]
(SiW),
[NaSb
W
O
]
(SbW) and
[BaAs
W
O
]
(AsW) at neutral pH was followed by gel filtration, SDS-polyacrylamide
gel electrophoresis, and scanning transmission electron microscopy.
Elution curves were fitted to sums of exponentially modified gaussians
to represent the peaks due to undissociated oxyHb, D (
200 kDa),
T+L (
50 kDa), and M (
25 kDa) (T =
disulfide-bonded trimer of chains a-c, M = chain d, and L = linker chains). OxyHb dissociation
decreased in the order Gdm
SCN > Gdm
Cl > urea >
Gdm
OAc and AsW > SbW > SiW. Scanning transmission electron
microscopy mass mapping of D showed
10-nm particles with masses of
200 kDa, suggesting them to be dodecamers (a+b+c)
d
.
OxyHb dissociations in urea and Gdm
Cl and at alkaline pH could be
fitted only as sums of 3 exponentials. The time course of D was
bell-shaped, indicating it was an intermediate. Dissociations in SiW
and upon conversion to metHb showed only two phases. The kinetic
heterogeneity may be due to oxyHb structural heterogeneity. Formation
of D was spontaneous during HBL reassembly, which was minimal (
10%) without Group IIA cations. During reassembly, maximal (
60%)
at 10 mM cation, D occurs at constant levels (
15%),
implying the dodecamer to be an intermediate.
The giant, hexagonal bilayer (HBL) ()extracellular
Hbs and chlorocruorin of annelids and vestimentiferans are
60 S
proteins with an acidic isoelectric point, high cooperativity of oxygen
binding, and a characteristically low iron and heme content, about two
thirds of normal(1, 2, 3, 4) . They
represent in many ways a summit of complexity for structures containing
globins(5) . The most extensively studied Hb is that of the
common North American earthworm Lumbricus terrestris. Although
it has been the subject of numerous studies since Svedberg determined
its mass by centrifugation in 1933, the molecular architecture of this
complex of
180 polypeptide chains remains uncertain in the absence
of a crystal structure. An early SDS-PAGE study showed that it
consisted of at least six subunits(6) , four of which were
globins, comprising a monomer subunit M (7) and a
disulfide-bonded trimer T(8) , the remainder being linkers,
chains of 24-32 kDa. The amino acid sequences of the T and M
subunits have been determined(9, 10) . Although only
three linker chains were thought to exist(11) , only one of
which had been sequenced (12) , a recent ESI-mass spectroscopy
study provided a detailed inventory of all the constituent polypeptide
chains and indicated the existence of four linker chains(13) .
Here we report the results of a study of the dissociation and
reassembly of Lumbricus Hb, which support the role of the
dodecamer of globin chains [3T+3M] as a principal
intermediate in both processes.
where a is the amplitude, a
is the center, a
is the width of the
gaussian, and a
is the width of the exponential.
The EMG is asymmetric with an exponential tail on the right side; the
falloff rate of the tail is controlled by the parameter a
. The areas of the individual peaks were plotted
as percent of total versus time and fitted to sums of
exponentials,
using PSI-Plot software (Poly Software International, Salt Lake City, UT) employing the Marquardt-Levenburg method. The acceptability of fits was judged by the absence of systematic trends in the plot of residuals with time.
Figure 1:
FPLC elution profile at 280 nm of
partially dissociated Lumbricus oxyHb in 0.1 M TrisCl buffer, 1 mM EDTA, pH 7.0. A,
exposure to 1.5 m Gdm
Cl; B, exposure to 12.4
mM SiW. The insets show the unreduced SDS-PAGE of
native Hb (lane 1) and the indicated fractions. The
undissociated peak is labeled HBL, and the three dissociated
peaks are the dodecamer D, the trimer and linker subunits T+L, and
the monomer subunit M. The profiles were obtained with different
columns. Note that in B peak M is overlapped by the SiW
peak.
Figure 2:
EMG
fits to the elution profiles at 280 nm obtained by FPLC. A,
dissociation of oxyHb in 1.22 m GdmCl after 28 h; B, reassociation of completely dissociated oxyHb in 10 mM Ca
after 48 h. In A, the buffer also
contained 1 mM EDTA. The differences in the elution volumes
for the same subunits is due to the use of different
columns.
Figure 3:
Zero time dissociation of Lumbricus oxyHb. A, percent undissociated oxyHb versus concentration of GdmSCN, Gdm
Cl, urea, and
Gdm
OAc. B, percent undissociated oxyHb (HBL)
and peaks D, T+L, and M versus concentration of
Gdm
SCN and Gdm
OAc. Each species was determined by
resolution of FPLC profiles using the EMG function and is expressed as
percent of total area.
Fig. 3B shows the relative
percent of the four peaks as a function of increasing concentrations of
GdmSCN and Gdm
OAc. The relative proportion of the dodecamer
D is much less in Gdm
SCN than in Gdm
OAc, the weakest
dissociating agent.
Figure 4:
Time course of Lumbricus oxyHb
dissociation in 0.1 M TrisCl buffer, pH 7.0. A,
in 4 M urea and 1 mM EDTA; B, in 4 M urea and 2.5 mM Ca
. The dotted
lines show the two exponential functions fitted with the resulting
residual below. C, in 4 M urea after 144 h as a
function of
[Ca
].
Figure 5:
Time course of Lumbricus oxyHb
dissociation in 1.75 m urea in 0.1 M TrisCl
buffer, pH 7, 1 mM EDTA. A, peak HBL; B,
dodecamer; C, T+L subunits; D, M subunit,
expressed as percent of total area. The insets show the
dissociation over the first 200 h. The fits shown are to the sum of the
three exponentials together with the resulting residuals. Note that the
dodecamer reaches a maximum after
250 h (B) and then
decreases and the absence of any induction period in the formation of
peaks T+L and M (C and D,
respectively).
Figure 6:
STEM images of Lumbricus Hb
undissociated peak obtained by FPLC after exposure to 0.25 M GdmSCN (A and C) and 0.6 M Gdm
SCN (B and D) and of peak D obtained by
FPLC at neutral pH, subsequent to exposure to 4.12 mM SiW (E) and pH 8.3 (F). All samples were in 0.05 M PIPES, pH 7.0. C and D represent 2.5-fold
magnifications of selected areas from A and B,
respectively. The extent of dissociation of the Hb was 10% in A and C and 89% in B and D. The scale
bar in F represents 50 nm in C-F and 125
nm in A and B. Note the presence of deficient HBL
structures lacking and in A-D.
Figure 7:
Histograms of STEM masses of unstained,
cryolyophilized specimens of the undissociated (HBL) peak
obtained by FPLC of Lumbricus oxyHb dissociated in the
presence of GdmSCN to the extent of 89% of total (A) and
of peak D fractions obtained by FPLC subsequent to exposure to SiW at
neutral pH (B) and at pH 8.3 (C). They correspond to
the STEM images shown in Fig. 6, panels B, E,
and F, respectively.
Fig. 6(E and F) shows typical
views of unstained, cryolyophilized peak D obtained by dissociation in
SiW and at pH 8.3, respectively; the observed particles are 10 nm
in diameter and histograms of the STEM masses within the range
150-250 kDa (Fig. 7, B and C) had
corresponding mean masses of 200 ± 26 kDa and 195 ± 21
kDa, respectively.
Figure 8:
Time courses of dissociations in 0.1 M TrisCl buffer, 1 mM EDTA. A and B, OxyHb at pH 7.0 in 4.2 mM and 12.6 mM SiW. C and D, OxyHb at pH 8.0 and 8.2,
respectively. D and E, MetHb, following oxidation of
oxyHb with K
Fe(CN)
and NaNO
,
respectively, and their removal by gel filtration. The exponential fits
are shown as dotted lines together with the plots of residuals versus time below each panel.
It is well known that HBL Hbs dissociate at pH
8(7, 22) . Fig. 8(C and D)
shows the time courses of dissociation at pH 8.0 and 8.2. Again, it is
evident that a third, rapid phase occurs within the dead time of the
FPLC (
2 min). Thus, there appear to be three dissociation
processes with t
1 min, 2-22 h, and
50-1200 h.
An early observation by Ascoli et al.(23) suggested that oxidation of earthworm Hb led to the
dissociation of its quaternary structure. We reinvestigated this
phenomenon because Lumbricus oxyHb was slowly altered to the
met form during the dissociations in urea and GdmCl. Fig. 8(E and F) shows the time courses of
dissociation following the conversion of oxyHb to metHb and the removal
of oxidant by gel filtration.
The fitted parameters for all the dissociations are provided in Table 1.
Figure 9:
Reassembly of HBL structure following
complete dissociation of Lumbricus oxyHb (absence of HBL) in 8 M urea in 0.1 M TrisCl buffer, pH 7, in the
absence and presence of group IIA cations. A, time course of
reassembly in 1 mM EDTA; note that mainly subunit D is formed
with
1% HBL. B, time course of reassembly in 5 mM Mg
. C, reassembly at 240 h versus [Ca
]. D, time courses of
reassembly in 10 mM Ca
,
Mg
, and
Sr
.
Reassociation, starting with peaks T+L and M isolated by gel
filtration of oxyHb dissociated in 4 M urea, shows that a
spontaneous reassociation of T and M to about 20% D had occurred within
6 h prior to the first FPLC (Fig. 10), even though
reassembly to the HBL was almost nonexistent (
1%). Fig. 10also shows the reassembly time courses in 2.5 mM and 10 mM Ca
; although the relative
contents of T and M declined steadily, the level of peak D remained
fairly constant at 10-15%. STEM images of unstained
HBL[T+L+M] are indistinguishable from those of
native Hb, and the mass distributions are similar to those determined
for native Hb(13) . The time courses of HBL reassembly could be
fitted reasonably well with a single asymptotic exponential.
Figure 10:
Time course of reassembly of HBL
structure in 0.1 M TrisCl buffer, pH 7, in 2.5 mM Ca
(circles) and 10 mM Ca
(squares). The curves represent least squares fits to asymptotic exponentials Y = 24 - 23exp(-0.23t) and 46 -
38exp(-0.46t), respectively. The empty squares represent the time course of D as percent of total during
reassembly in 10 mM Ca
; the curve represents a least squares fit to a single asymptotic exponential Y = 11 +
7.3exp(-0.2t).
Fig. 3summarizes the
effect of urea and several Gdm salts on the dissociation of Lumbricus oxyHb determined by FPLC at zero time. The order of
decreasing effectiveness is GdmSCN > Gdm
Cl > urea
> Gdm
OAc, with the order of the anions in line with the well
known Hoffmeister series(25, 26) .
The order of
increasing effectiveness of the three heteropolytungstates, SiW <
SbW < AsW, appears to be correlated with their total charge and
mass, -8 (3239 Da), -18 (7178 Da), and -27 (11,732
Da), respectively, and not with the surface charge density. Although
SiW is spherical, SbW is a trigonal pyramid, and AsW is a
parallelliped, the charge per unit area is approximately the same:
-1.8, -2.0, and -2.1/100 Å,
respectively(27) .
The
time courses of oxyHb dissociation in 1.75 m urea (Fig. 5A) and 1.22 m GdmCl at neutral pH
can be satisfactorily represented as the sum of three first-order
processes with t
1-2 h, 30-50 h,
and 400-500 h (Table 1). Fig. 4also shows that
there are at least three processes occurring in the dissociation of
oxyHb in 4 M urea in the absence and presence of
Ca
.
Three first-order processes are also observed
in oxyHb dissociation at alkaline pH, t
1
min, 2-20 h, and 50-1200 h (Table 1). OxyHb
dissociation in the presence of SiW (Fig. 8, A and B) can be fitted with two first-order processes, t
10-40 h and 400-1300 h (Table 1). The
latter values correspond roughly to the t
for the
two slower dissociation processes in urea and Gdm
Cl and at
alkaline pH.
Two points must be considered before discussing
possible mechanisms for the dissociation of Lumbricus oxyHb.
1) Whether slow oxidation of oxyHb to metHb could be responsible for
one of the dissociation processes observed. MetHb dissociation (Fig. 8, E and F, and Table 1) consists
of two phases: a small (10%) initial dissociation (t
2 h), followed by a dissociation that is slower
by more than 1 order of magnitude than the slowest phase of the oxyHb
dissociations (t
13,000-35,000 h versus 50-1300 h). Hence, dissociation due to metHb
formation can be neglected. 2) Can the dissociation of the oxyHb be
accompanied by a partial disruption of the tertiary and secondary
structures of the globin subunits? It is known that myoglobin does not
evince any conformational alterations at urea concentrations less than
5 M(35, 36) . Hence, it is unlikely that 4 M urea affects either the M or the disulfide-bonded T subunit.
Figure 11: Schematic representation of possible processes in the dissociation of Lumbricus oxyHb HBL structure (A), the formation of deficient HBLs missing and (B), and a simple two-step reassembly to HBL structures (C).
The
dissociation of the dodecamer in the presence of urea and GdmCl
requires two exponentials for a satisfactory fit with t
100-200 h and 2700-5000 h. (
)The slower process has a t
close to
that determined for the dissociation of the metdodecamer, which is
about an order of magnitude faster than the dissociation of the metHb.
It is likely that oxydodecamer dissociation (t
100-200 h) occurs mostly in the later stages of oxyHb
dissociation, following the accretion of peak D observed in the first
50-300 h (Fig. 5B).
There seem to be two
simple explanations for the kinetic heterogeneity of oxyHb
dissociation. 1) Since peak HBL, whose area is a measure of
undissociated HBL structures, contains ``complete'' HBLs as
well as the deficient HBLs lacking and of the structure, one
explanation is that the observed three first-order processes reflect
the dissociation of the complete and deficient HBLs. However, the STEM
appearance and STEM mass distributions at a late stage of dissociation (Fig. 6, B and C, and 7A) indicate
the presence of limited numbers of deficient HBLs. 2) Another
possibility is that the native Hb consists of three unequal populations
of HBL structures differing in their stabilities toward dissociation,
each population of HBLs exhibiting its own rate of dissociation in the
presence of a given concentration of the dissociating agent. In this
view, the deficient HBLs are likely intermediates in the overall
dissociations. Our results suggest that the initial, rapid oxyHb
dissociation with tof
1 min at alkaline pH and
1-2 h in 1.75 m urea and 1.22 m Gdm
Cl,
which is not observed in the case of SiW, may be related to the ease of
penetration into the Hb interior. The penetration of OH
and its reaction, e.g. with salt bridges stabilizing
some intersubunit contacts, should be much more rapid than the
penetration by urea or Gdm
Cl and their binding to enough peptide
groups and/or side-chain groups of the different subunits to effect a
similar destabilization. This notion is consistent with the probable
inability of the heteropolytungstates to penetrate into the Hb interior
and the consequent occurrence of only two first-order processes (t
10-40 h and 400-1300 h), comparable to the
two slower processes observed in urea and Gdm
Cl and at pH 8.0 (t
22-53 h and 400-1200 h, Table 1).
In contrast to the kinetic heterogenity of HBL dissociation (Fig. 4, 5, and 8), the time course of HBL reassembly ( Fig. 9and Fig. 10) is readily fitted with a single asymptotic exponential. The first step of dodecamer formation (Fig. 11) appears to be relatively fast; hence, the observed process is likely to be the second step of dodecamer combination with linker subunits to form HBL structures (Fig. 11). Peaks intermediate between HBL and D occur in the elution profiles of reassociating mixtures (peaks I1 and I2 in Fig. 2B and Fig. 9B). At present, we do not know whether they are intermediates or reassembly-incompetent side-products.
Furthermore, the recent three-dimensional reconstructions from
cryoelectron microscopic images of Eudistylia chlorocruorin, Macrobdella Hb, Lumbricus Hb, and reassembled HBL
missing one of the linker subunits of Lumbricus Hb by Lamy and
collaborators (40, 41) ()demonstrate that
all the HBL structures are very similar. An obvious explanation is that
HBL structures may not require a fixed stoichiometry of globin and
linker subunits. Hence, structural heterogeneity of Lumbricus Hb may lie at the heart of the kinetic heterogeneity of its
dissociation.