| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, November 21, 1995; and in revised form, January 26,
1996) From the
The dissociation of the
The giant, hexagonal bilayer (HBL) (
where a
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 Tris
Figure 2:
EMG
fits to the elution profiles at 280 nm obtained by FPLC. A,
dissociation of oxyHb in 1.22 m Gdm
Figure 3:
Zero time dissociation of Lumbricus oxyHb. A, percent undissociated oxyHb versus concentration of Gdm
Fig. 3B shows the relative
percent of the four peaks as a function of increasing concentrations of
Gdm
Figure 4:
Time course of Lumbricus oxyHb
dissociation in 0.1 M Tris
Figure 5:
Time course of Lumbricus oxyHb
dissociation in 1.75 m urea in 0.1 M Tris
Figure 6:
STEM images of Lumbricus Hb
undissociated peak obtained by FPLC after exposure to 0.25 M Gdm
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 Gdm
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
Figure 8:
Time courses of dissociations in 0.1 M Tris
It is well known that HBL Hbs dissociate at 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 Gdm 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 Tris
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
Figure 10:
Time course of reassembly of HBL
structure in 0.1 M Tris
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 Gdm 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
The
time courses of oxyHb dissociation in 1.75 m urea (Fig. 5A) and 1.22 m Gdm Three first-order processes are also observed
in oxyHb dissociation at alkaline pH, t 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 (
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 Gdm 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 t
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) (
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 8754-8762
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 > GdmCl > urea >
GdmOAc 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 GdmCl 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.
)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.
Materials
L. terrestris Hb was prepared
as described previously in 0.1 M TrisCl buffer, pH 7.0,
1 mM EDTA, 2 mM phenylmethanesulfonyl fluoride, from
live worms collected around London, Ontario (Carolina Wholesale Bait
Co., Canton, NC)(4, 6) . The concentration of the Hb
was determined from the absorbance of the native form at 280 nm or of
the cyanmet form at 540 nm(4) , employing the respective
extinction coefficients, 2.063 ± 0.032
mlmg
cm and 0.442
± 0.013
mlmgcm(13) .
The Gdm salts were purum grade from Fluka AG (9470 Buchs, Switzerland)
and urea was from Sigma. The heteropolytungstate salts KSiW
(K
[SiWO].14H
O,
3239.2 Da), NaSbW
(Na
[NaSbWO].24HO,
7178.4 Da) and AsW
(Na
[BaAsWO].60HO,
11,731.7 Da) were prepared according to Klemperer(14) .Analytical Gel Filtration
Low pressure, isocratic
gel filtration was carried out at room temperature (20 ± 2
°C) employing an FPLC system (Pharmacia Biotech Inc.) and 1 
30-cm columns of Superose S12 or S6 (Pharmacia). Flow rate was 0.4
ml/min and the eluate was monitored at 280 nm. A constant amount of
protein in a constant sample volume, 800 µg/200 µl, was
loaded each time.Polyacrylamide Gel Electrophoresis
Polyacrylamide
gel electrophoresis in the presence of 0.1% SDS was carried out using
the buffer system of Laemmli (15) and slab gels (1.5 mm
10 cm 8 cm) of 8-20% acrylamide. The gels were
electrophoresed for 2-4 h, stained in 0.125% Coomassie Brilliant
Blue R-250 in 45% methanol, 7.5% acetic acid, and destained in 25%
methanol, 7.5% acetic acid.Optical Spectrophotometry
The absorption spectra
over the the 200-650 nm range were obtained using an OLIS
(Bogart, GA) spectrophotometer employing a Hewlett Packard diode array
detector or a Hitachi model 2000 spectrophotometer.Fitting of Elution Profiles
The elution curves
were either digitized on a Summagraphics Summasketch MM18 tablet using
a Sigma Scan version 3.0 (Jandel Scientific, Corte Madera, CA) or
acquired using the Easyest System 8 (Keithley Instruments, Inc.,
Rochester, NY) and an IBM PC/386 computer. The elution curve was then
fitted as a sum of four EMGs, each representing the undissociated Hb
(HBL) and peaks D, T+L, and M, employing least squares
minimization (Peak Fit version 2.0, Jandel Scientific). The EMG
function is a convolution of a gaussian and a decreasing exponential
and is known to represent well the shape of chromatographic elution
peaks(16, 17, 18) ,
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,
Dissociation of Lumbricus OxyHb
The dissociating
agent was dissolved in 0.1 M TrisHCl buffer, pH 7.0, 1
mM EDTA, and Hb stock solution added to obtain the desired
concentration, 3.6 mg/ml. The following dissociating agents were
employed: urea, GdmSCN, GdmCl, GdmOAc, SiW, SbW, and
AsW. The dissociations of oxyHb at neutral pH and at pH 8.0 and 8.2 in
0.1 M TrisCl buffer, 1 mM EDTA, were followed
by FPLC at neutral pH.Oxidation of Lumbricus Hb
The oxidation of Lumbricus oxyHb was effected by the addition of potassium
ferricyanide (Fisher) or of sodium nitrite (Aldrich) at molar ratios
relative to heme, ranging from 1 to 1000, in 0.1 M TrisCl buffer, pH 7.0, 1 mM EDTA. The conversion of
oxyHb to metHb was monitored using optical spectrophotometry over the
450-650 nm range and was complete in 5-20 min. The metHb
solution was then immediately passed over a 1.5 20-cm Sephadex
G-25 column to remove the oxidizing agent, and the progress of the
dissociation measured using FPLC at neutral pH.Reassembly of HBL Structure from Completely Dissociated
OxyHb
Lumbricus Hb (30-60 mg/ml) was dissociated
in the presence of 4-8 M urea in 0.1 M TrisCl buffer, pH 7.0, 1 mM EDTA at room
temperature. The completeness of the dissociation to T+L+M
was checked by FPLC, urea was removed by dialysis against 2 1.5
liters of TrisCl buffer for 2 h and the reassociation
followed by FPLC in the presence of Group IIA cations
Mg
, Ca, and Sr over the 0-50 mM range; the solutions were kept at
7 °C. Alternatively, the T+L+M fractions were obtained by
preparative gel filtration of oxyHb exposed to 4 M urea,
pooled, concentrated by pressure filtration using Centricon 10
concentrators (Amicon Division, W. R. Grace & Co., Danvers, MA),
and its reassociation followed in the absence and presence of
Ca.STEM Imaging and Mass Measurement of Unstained
Protein
The mass measurements were performed using the STEM at
the Brookhaven National Laboratory(19) . Preparation of the
unstained specimens with TMV fibers as internal mass standards was
carried out as described by Kapp et al.(20) . Negative
staining was with 0.5% (w/w) uranyl acetate. The STEM was operated at
40 kev, a dose level <10 e/0.1 nm and a
resolution of 0.25 nm. An interactive program (19) was
used to select the electron micrographs on the basis of clean
background and apparent quality of TMV fibers and protein particles; it
computes the background and permits the operator to select the TMV
segments for internal mass calibration and the particles for mass
measurement. At least 3-5 good TMV segments are generally chosen
to calculate the internal mass calibration. The individual particles
are selected based on clean background around the particles and absence
of visible flaws.
Dissociation of OxyHb by Urea and Guanidinium Salts
Elution Profile of the Dissociation
Products
Fig. 1A shows a typical FPLC elution
profile of oxyHb dissociated at neutral pH in the presence of 1.5m GdmCl. In addition to the undissociated Hb (HBL), three
peaks are observed, D, T+L, and M, at elution volumes
corresponding to approximately 200, 60, and 25 kDa. The unreduced
SDS-PAGE (inset) shows that the subunit content of
undissociated Hb is similar to the native Hb, peak D consists of
subunits T (54.5 kDa; chains a+b+c) and M (16.6 kDa,
chain d), peak T+L is the envelope of unresolved peaks
due to subunit T and the four linker subunits L (L1-L4: 24.1,
24.9, 27.6, and 32.1 kDa), and peak M is the monomer (the masses
provided here are from a recent mass spectrometric study of the Hb; (13) ).
Cl buffer, 1 mM EDTA, pH 7.0. A,
exposure to 1.5 m GdmCl; 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.
Fitting of Elution Profiles
Fig. 2A shows a representative fit of an FPLC elution curve with four EMG
functions. The elution volumes of the four peaks remained unchanged
throughout the course of the dissociation to ±3%, as did the
fitted variables a (peak position). Fig. 2B shows a similar fit of an elution curve
obtained following reassociation of T+L+M in 10 mM Ca.
Cl 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.
Dissociation of OxyHb at Zero Time
In these
experiments, the oxyHb was loaded on the column right after mixing with
the dissociating agent and subjected to FPLC. The time elapsed between
mixing and complete penetration of the sample into the column was
110-120 s. Plots of percent dissociation versus the
concentration of the dissociating agent are shown in Fig. 3A.
SCN, GdmCl, urea, and
GdmOAc. B, percent undissociated oxyHb (HBL)
and peaks D, T+L, and M versus concentration of
GdmSCN and GdmOAc. Each species was determined by
resolution of FPLC profiles using the EMG function and is expressed as
percent of total area.
SCN and GdmOAc. The relative proportion of the dodecamer
D is much less in GdmSCN than in GdmOAc, the weakest
dissociating agent.Time Course of OxyHb Dissociation in 4 M Urea
and the Effect of Ca
Fig. 4A shows the time course of oxyHb dissociation in 4 M urea;
although it is almost complete within 2 h, peak D remains constant
indicating its stability in 4 M urea. Fig. 4B shows the time course of oxyHb dissociation in 4 M urea
in 2.5 mM Ca; it can be fitted as the sum of
two exponentials. However, since substantial dissociation occurs within
the dead time of the FPLC method (2 min), there appear to be at
least three separate dissociation processes with apparent t
1 min, 1 h, and 50 h. Fig. 4C illustrates the effect of
[Ca] on the dissociation of oxyHb in 4 M urea after 144 h.
Cl 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].
Kinetics of OxyHb Dissociation
Fig. 5A shows the time course of oxyHb dissociation in 1.75 m urea and Fig. 5(B-D), show the
corresponding time courses of peaks D, T+L, and M, respectively.
The insets show the initial phases. The points shown represent
averages of values determined in three separate experiments. For all
the processes shown in Fig. 5and for oxyHb dissociation in 1.22 m GdmCl (results not shown), at least three exponentials
were necessary to obtain a good fit as judged by the absence of any
trends in the plots of residuals versus time provided at the
bottom of each panel. Table 1summarizes the amplitudes and
kinetic constants determined from the fits.
Cl
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).
STEM Imaging and Mass Mapping of Undissociated
OxyHb and Peak D
Fig. 6shows views of unstained, cryolyophilized
undissociated oxyHb obtained at 11% (A and C) and 89% (B and D) dissociation, respectively. Fig. 7A shows a histogram of the STEM masses of the
complete HBL structures observed at 89% dissociation. Although the mean
mass, 3540 ± 260 kDa (n = 120), is similar to
the value 3560 ± 130 kDa obtained previously for native
Hb(13) , the distribution of masses is more asymmetric at the
lower end.
SCN (A and C) and 0.6 M GdmSCN (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.
SCN 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.
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.Dissociation of OxyHb by Heteropolytungstates, at
Alkaline pH and upon Conversion to MetHb
The complex heteropolytungstate anions
SiW, SbW, and AsW are known to form 1:1 complexes with metMb at neutral pH with
association constants in the 10
to 10
M range and concomitant formation of
hemichrome type visible absorption spectra (21) . All three
dissociate oxyHb; a typical elution curve is shown in Fig. 1B. Fig. 8(A and B)
shows the time courses of dissociation in 4.12 mM and 12.4
mM SiW, together with the fits to sums of two exponentials.
Cl 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 KFe(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.
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.Cl. 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.Reassembly of HBL Structure
Fig. 9shows some representative results obtained with
the reassembly of HBL structures from completely dissociated oxyHb. In
the absence of Group IIA cations, reassembly of was limited, generally
much less than 10%. However, in the first 24 h, there is a spontaneous
formation of dodecamer as illustrated in Fig. 9A. The
same result is also observed in Fig. 9B, which depicts
the time course of reassembly to HBL in 5 mM Mg. Fig. 9C illustrates the
effect of cation concentration on the extent of HBL reassembly, and Fig. 9D shows that although there may be differences in
the extent of reassembly achieved initially, the final [HBL]
is remarkably similar for all three cations after 200 h.
Cl 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.
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.
Cl 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).
A Dodecamer [3T+3M] Is Observed in All
Dissociations of Lumbricus OxyHb
The dissociation of the HBL
structure at neutral pH by Gdm salts and heteropolytungstate anions and
at mildly alkaline pH ( Fig. 1and Fig. 3) provide
remarkably similar pictures; a 200-kDa dodecamer D
([3T+3M]), deficient in linker subunits, is always
formed in addition to the M, T, and L subunits. In particular,
dissociation in the weakest dissociating agent, namely GdmOAc (Fig. 3), shows that D accounts for about half of the initial
dissociation products. The time course of dissociation in 4 M urea (Fig. 4A) also shows that D accounts for
40-50% of the dissociation products. In addition, it appears that
D is fairly stable in the presence of 4 M urea, in agreement
with earlier findings(24) .SCN > GdmCl > urea
> GdmOAc, with the order of the anions in line with the well
known Hoffmeister series(25, 26) .,
respectively(27) .Effect of Ca
Ca on Urea Dissociation of
OxyHb exerts a markedly protective effect
on the quaternary structure of oxyHb in the presence of 4 M urea (Fig. 4). Although dissociation is almost complete
(95%) after 2 h in 4 M urea (Fig. 4A),
even 2.5 mM Ca reduces dissociation to
75% after 144 h in 4 M urea (Fig. 4B).
The maximum protective effect is reached at
[Ca] 10 mM (Fig. 4C). Alkaline earth (Group IIA) cations are
known to stabilize the HBL structure of annelid Hbs with respect to
dissociation at alkaline pH(22, 28, 29) , at
acid pH(30, 31) , as well as thermal unfolding and
autoxidation(32) . In some cases, such as Amphitrite Hb (33) and Myxicola chlorocruorin (34) ,
Ca is necessary for maintaining the HBL structure
even at neutral pH.The Kinetic Heterogeneity of Lumbricus OxyHb
Dissociation
Our results show that dissociation of oxyHb
followed by FPLC over several weeks is not accompanied by alteration in
the properties of either the starting material or the products. 1) The
elution volumes of the undissociated Hb (HBL) and of the products of
its dissociation (peaks D, T+L, and M) remain unaltered. 2) The
subunit compositions of all the peaks as judged by SDS-PAGE remain
unchanged. 3) The STEM images of the HBL peak at an early (10%) and a
late stage of dissociation (89%) indicate no major alterations in
dimensions (Fig. 6, A-D). Furthermore, the STEM
mass distribution at 89% dissociation (Fig. 7A)
compared to that of the native Hb (13) exhibits only a slight
asymmetry at the low end, probably due to the presence of a relatively
small number of ``deficient'' HBLs, missing and of the HBL
structure that can be observed in Fig. 6(A-D).Cl 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. 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 tfor the
two slower dissociation processes in urea and GdmCl and at
alkaline pH.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. Possible Mechanisms of OxyHb Dissociation
Several
simultaneous dissociations of a HBL structure can be envisaged (Fig. 11A): to D+L(1) ,
T+L+M(2) , D+T+L+M(3) , and (4) dissociation of D from (1) and (3) to
T+M. STEM images of Hb at 11% and 89% dissociation (Fig. 6, A-D) show the presence of deficient HBLs, partially
dissociated Hb particles lacking and of the HBL structure
(represented schematically in Fig. 11B). We do not know
whether these deficient HBLs are intermediates or not. The time course
of the appearance of D, which reaches a maximum in the the initial 10%
of the dissociation and decreases thereafter (Fig. 5B),
is consonant with the formation of deficient HBLs with concomitant
formation only of D (Fig. 11B), being the initial stage
of HBL dissociation. The latter is reminiscent of the time course of
formation of an intermediate B in a simple set of consecutive
first-order reactions A 
B C(37) . However, this
simple scheme does not fit our results, since: 1) oxyHb dissociation
can not be represented by a single exponential and 2) the time courses
of T+L and M appearances (Fig. 5, C and D) do not exhibit an induction period and consequently, an
inflection point in their curves, as does the appearance of the final
product C in the foregoing model. The latter result suggests that
processes(1) -(3) occur simultaneously.
Cl
requires two exponentials for a satisfactory fit with t 100-200 h and 2700-5000 h. ()The slower process has a tclose 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).of 1 min at alkaline pH and
1-2 h in 1.75 m urea and 1.22 m GdmCl,
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 GdmCl 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 GdmCl and at pH 8.0 (t 22-53 h and 400-1200 h, Table 1).Role of the Dodecamer in HBL Structure
Reassembly
Our results ( Fig. 9and Fig. 10)
demonstrate that dodecamers are formed both in the presence and absence
of the linker subunits L and in the absence and presence of Group IIA
cations. Likewise, in the presence of Mg and
Sr (Fig. 9B), but not
Ca, there is an increase in D which occurs in the
first 24 h, prior to the formation of any significant amount of HBL.
These facts suggests that formation of the dodecamer precedes that of
HBL and that the dodecamer is an obligatory intermediate in the
reassembly of the HBL structure (Fig. 11C). In the
presence of Ca (Fig. 10), the formation of HBL
is accompanied by only a small decrease in D, the latter remaining at a
fairly constant level between 10 and 15%. At optimum concentrations of
Mg, Ca, and Sr,
10 mM in all three cases (Fig. 9C), the
extent of HBL reassembly reaches 50-60%, again indicating a
dominant role for Group IIA cations in stabilizing the HBL structure. Is Subunit Stoichiometry Constant in HBL
Structures?
Native Lumbricus Hb examined by STEM mass
mapping and sedimentation equilibrium, exhibits a fairly broad range of
masses from 3200 to 3900 kDa(13) . HBL structures can be
reassembled from T+L subunits(38) , and we have recently
shown them to have STEM masses ranging from 2500 to 3600 kDa. ()Although the distribution is asymmetrical with a tail at
lower masses than 3000 kDa, the surprising observation is that a
considerable fraction of the masses are higher than 3000 kDa, the mass
of native Hb, 3560 kDa, minus the contribution of the M subunit, 575
kDa(13) . A possible explanation is that there may occur
extensive formation of ``pseudo-dodecamers'' consisting of 4T
subunits instead of [3T+3M], which preserves the local
3-fold symmetry found in the dodecamer crystal(39) . )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.Conclusion
The results presented here extend our
earlier findings (24, 42) and provide conclusive
evidence for the dodecamer [3T+3M] being the principal
structural intermediate in the dissociation and reassembly of Lumbricus Hb.
)[SiWO].14HO;
NaSbW, sodium heni-cosatungstononaantimonate (III),
Na[NaSbWO].24HO;
NaAsW, sodium tetracontatungstotetra-arsenate(III),
Na[BaAsWO].60HO;
PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid
chromatography; EMG, exponentially modified gaussian; Gdm, guanidinium;
SNC, thiocyanate; OAc, acetate; STEM, scanning transmission electron
microscopy; PIPES, piperazine-N,N`-bis[2-ethanesulfonic
acid]; TMV, tobacco mosaic virus.)))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. S. Santiago, F. Moura, L. M. Moreira, M. M. Domingues, N. C. Santos, and M. Tabak Dynamic Light Scattering and Optical Absorption Spectroscopy Study of pH and Temperature Stabilities of the Extracellular Hemoglobin of Glossoscolex paulistus Biophys. J., March 15, 2008; 94(6): 2228 - 2240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Weber and S. N. Vinogradov Nonvertebrate Hemoglobins: Functions and Molecular Adaptations Physiol Rev, April 1, 2001; 81(2): 569 - 628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. N. Green, R. S. Bordoli, L. G. Hanin, F. H. Lallier, A. Toulmond, and S. N. Vinogradov Electrospray Ionization Mass Spectrometric Determination of the Molecular Mass of the ~200-kDa Globin Dodecamer Subassemblies in Hexagonal Bilayer Hemoglobins J. Biol. Chem., October 1, 1999; 274(40): 28206 - 28212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhu, D. W. Ownby, C. K. Riggs, N. J. Nolasco, J. K. Stoops, and A. F. Riggs Assembly of the Gigantic Hemoglobin of the Earthworm Lumbricus terrestris. ROLES OF SUBUNIT EQUILIBRIA, NON-GLOBIN LINKER CHAINS, AND VALENCE OF THE HEME IRON J. Biol. Chem., November 22, 1996; 271(47): 30007 - 30021. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Krebs, A. R. Kuchumov, P. K. Sharma, E. H. Braswell, P. Zipper, R. E. Weber, G. Chottard, and S. N. Vinogradov Molecular Shape, Dissociation, and Oxygen Binding of the Dodecamer Subunit of Lumbricus terrestris Hemoglobin J. Biol. Chem., August 2, 1996; 271(31): 18695 - 18704. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||
ABSTRACT
