(Received for publication, May 22, 1995; and in revised form, September 25, 1995)
From the
The thermodynamics of assembly of the allophycocyanin hexamer
was examined employing hydrostatic pressures in the range of 1 bar to
2.4 kbar and temperatures of 20 to -12 °C, the latter made
possible by the decrease of the freezing point of water under pressure.
The existence of two processes, dissociation of the hexamer into
dimers, ()
3 (
), and
dissociation of the
dimers into monomers, (
)
+
have been recognized previously by changes in
the absorbance and fluorescence of the tetrapyrrolic chromophores owing
to added ligands. The same changes are observed in the absence of
ligands at pressures of under 2.4 kbar and temperatures down to
-12 °C. On decompression from 2.4 kbar at 0 °C,
appreciable hysteresis and a persistent loss of 50% in the absorbance
at 653 nm is observed. It results from the conformational drift of the
isolated subunits and is reduced to 10% when the highest pressure is
limited to 1.6 kbar. The thermodynamic parameters of the reaction
+
can be determined from pressure effects
on perchlorate solutions of allophycocyanin, which consist of dimers
alone. Their previous knowledge permits estimation, under suitable
hypotheses, of the thermodynamic parameters of the reaction
3(
)
(
)
from the overall pressure
effects on the hexamers. Both association reactions have positive
enthalpy changes, and the whole hexamer assembly is made possible by
the excess entropy.
Allophycocyanin is one of the phycobiliproteins of
phycobilisomes, the accessory light-harvesting complexes in
cyanobacteria and red algae. Allophycocyanin has been isolated as a
trimeric species ()
(Zilinskas et al.,
1978; MacColl et al., 1981) of 110 kDa to which we shall refer
here as a hexamer. Conversion of (
)
into
dimers is obtained by the addition of 1 M
perchlorate (MacColl et al., 1971; MacColl et al.,
1981; Huang et al., 1987) or by lowering the pH (MacColl et al., 1980). Dissociation of
dimers into
+
monomers has been achieved only in the presence of urea at
8 M concentration (Erokhima and Krasnoviskii, 1974).
Hydrostatic pressure has been used successfully to explore the
thermodynamics and mechanism of assembly of oligomeric proteins. (Silva
and Weber, 1993). In this report, we describe how the dissociation of
()
into
dimers and then into
+
monomers occurs when the solutions of the protein are
subjected to hydrostatic pressures in the range of atmospheric to 3
kbar and temperatures in the range of 10 to -11 °C. The
decrease in the freezing point of water under pressure makes it
possible to reach a temperature of -20 °C at 2.4 kbar while
maintaining the liquid state. In the absence of perchlorate or urea,
complete dissociation of allophycocyanin takes place only at these low
temperatures, and this has enabled us to show that the process of dimer
dissociation by the cold results in a state very similar to that
obtained by addition of a classical chemical denaturant, urea, but as a
freely displaceable equilibrium. Separation of the two sequential
processes of hexamer and dimer dissociation cannot be unequivocally
done from the spectral changes under pressure or temperature alone.
However the isolation of the pressure effects on the
dimers
by starting with allophycocyanin solutions in 1 M perchlorate,
in which the dissociation of the hexamer into dimers is complete, has
permitted us to obtain the volume and enthalpy changes of the
association of the monomers into dimers. Starting with these data, we
have derived the corresponding quantities for the reaction
(
)
3
from the overall spectral
changes with pressure and temperature, in absence of any added ligands.
We thus provide a complete thermodynamic description of the pressure
dissociation of a heterohexamer, (
)
, into its
ultimate subunits, the
and
monomers.
The generation and measurement of pressure and the use of
spectroscopic methods to determine the degree of dissociation, the
changes in volume (V) and the changes in the standard
free energy of association of the aggregate (
G) are
described elsewhere (Paladini and Weber, 1981a, 1981b; Silva et
al., 1986; Weber, 1986). The pressure bomb utilized in this study
has been described previously (Paladini and Weber, 1981a). The bomb
temperature, measured with a thermocouple, was decreased below room
temperature using a methanol circulator and thermostat. Condensation on
the bomb windows was avoided by passing a stream of dry nitrogen.
For absorbance measurements, the pressure bomb was placed in the
sample compartment of a photodiode array spectrophotometer (3000 Array,
of SLM-Aminco). Fluorescence spectra, excited at 600 nm, were
determined with a double monochromator spectrofluorometer controlled by
software from ISS (Champaign, IL). We recall that change in volume upon
reaction, V, may be obtained by two different procedures
(Ruan and Weber, 1989). One involves the change in degree of
dissociation with pressure at constant concentration
(
V
); the other involves the change
in pressure required to reach the same degree of dissociation at two
different protein concentrations
(
V
). Unless otherwise stated the
values designated here as
V are exclusively
V
.
Polarizations of fluorescence were measured and corrected for window birefringence as described by Paladini and Weber (1981a). The sample was excited at 600 nm, and the emission reached the photodetector through an R-59 cut-off filter.
Allophycocyanin from Spirulina platensis was purchased from
Sigma, dissolved in 100 mM sodium phosphate buffer (pH 7.5),
and stored at 4 °C. TrisHCl (100 mM, pH 7.5) was
employed in all the pressure experiments because of the minimal
variation of its pK with pressure (Neumann et al., 1973).
Decreasing temperature causes an increase in pH of 0.03 pH
units/°C. Thus, at -10 °C, an increase in pH of
approximately 1 unit with respect to the value at 20 °C is
expected. The
dimers were obtained by the addition of 1 M NaClO
to Tris
HCl buffer followed by
adjustment of the pH. The purity of the protein was checked by
SDS-polyacrylamide gel electrophoresis; only two bands were observed
corresponding to the
and
subunits.
The pressure was increased in steps of 200 bar, allowing 15 min for equilibration at each pressure and temperature prior to recording measurements. Control experiments showed that changes in absorbance and fluorescence spectra were completed within 5 min and remained stable for 90 min thereafter.
Figure 1: Effects of hydrostatic pressure at 0 °C on the absorption of allophycocyanin hexamer. From top to bottom: 1.0 bar, 0.6 kbar, 1.0 kbar, 1.2 kbar, 1.4 kbar, 1.6 kbar, 1.8 kbar, 2.2 kbar, and 2.4 kbar. Protein concentration was 30 µg/ml.
Figure 2: Continuous lines, absorption spectra of allophycocyanin in 1 M perchlorate at 0 °C. From top to bottom: 1.0 bar, 0.8 kbar, 1.0 kbar, 1.2 kbar, 1.4 kbar, 1.8 kbar, 2.0 kbar, 2.2 kbar, and 2.4 kbar. Dashed line, absorption spectrum of allophycocyanin after the addition of 8 M urea at room temperature and atmospheric pressure. Protein concentrations, 15 µg/ml.
In a previous
study (MacColl, 1983) it was shown that the hexameric species
()
of allophycocyanin on excitation of the
chromophores at 600 nm exhibits a fluorescence emission with a peak at
662 nm, whereas the emission from the
dimers obtained by the
addition of 1 M perchlorate has a maximum at 640 nm, with a
lower yield. The addition of 8 M urea, which dissociates
into
+
, decreases the fluorescence
intensity without further spectral shifts. Fig. 3shows the
fluorescence spectra of solutions in neutral buffer (a), 1 M perchlorate solutions (b), at apressure of 2.4 kbar
at 0 °C (c), and in 8 M urea (dashed
line). The fluorescence changes follow quantitatively the
corresponding absorbance changes.
Figure 3: Emission spectra of allophycocyanin. a, in the hexameric state; b, in 1 M perchlorate; c, in 8 M urea. The dashed line is the fluorescence in neutral buffer at 2.4 kbar and 0 °C. Excitation at 600 nm.
The limiting fluorescence emission spectrum is very similar to that seen in the presence of urea so that the data of both absorption or fluorescence can be used to monitor the dissociation of hexamer into monomers.
By comparison of Fig. 1and Fig. 2, it is possible to deduce that by the
application of pressures of up to 2.4 kbar at 0 °C, the two
sequential reactions, ()
3 (
)
and
+
occur with the absorbance, and
also fluorescence, changes belonging to them running smoothly into each
other as dissociation proceeds. Isolation of the corresponding
thermodynamic parameters becomes a matter of fitting the data to a
preassigned model. Success in this procedure critically depends on the
hypotheses made on the spectral characteristics assigned to the
reactions and on the precision of the measurements. To minimize the
uncertainties involved, we used a more direct procedure.
Allophycocyanin in 1 M perchlorate solution is in the form of
dimers, and the thermodynamic parameters for the dimer-monomer
equilibrium can be determined by the effect of pressure on these
solutions following experience with several other protein dimers (Silva
and Weber, 1993). Making use of this data, we can set up the equations
corresponding to the two-step dissociation of the hexamers into
and
monomers and derive the thermodynamic parameters of the
dissociation of the hexamer into dimers brought about by the
application of pressure.
Fig. 4shows the decrease in the maximal
absorbance of the allophycocyanin dimer in 1 M
perchlorate as pressure is increased at 0 °C. The decompression
pathway (lower curve) exhibits a slight hysteresis. After
decompression, the spectral properties of the dimer were fully
recovered, indicating complete reversibility of the dissociation
reaction at this protein concentration. The application of high
pressure (2.4 kbar) at 22 °C promoted a fast dissociation (to
= 0.47) that reached a plateau in less than 5 min and was
practically constant over longer times. Degrees of dimer dissociation
(
) were calculated on the assumption that the plateau values of
absorption or emission observed at pressures close to atmospheric
pressure correspond to zero dissociation and at highest pressures (or
lowest temperatures) correspond to complete dissociation (see as
example Fig. 1and Fig. 2). The relation of the degree of
dissociation to the dissociation equilibrium constant (K
) for a dimer is
On-line formulae not verified for accuracy
Figure 4:
Compression (filled circles) and
decompression (empty circles) effects on the absorbance at 615
nm, at 0 °C, of allophycocyanin in 1 M perchlorate. The
absorbance ratios were calculated by
(Abs/Abs
) where p is
the pressure applied. Protein concentration was 15 µg/ml. Inset, plot of ln(
/(1 -
)) against
pressure for the absorbance change with pressure of Fig. 2.
where C is the total concentration of
protein expressed as dimer. In terms of the characteristic dimer
concentration C
at which
=
, becomes
On-line formulae not verified for accuracy
Fig. 4, inset, shows the plot of
ln(/(1 -
)) against pressure for the data
of the figure. The volume change of association (
V) and
the dissociation constant at 1 bar calculated for the reaction
+
are summarized in Table 1.
The volume change found for association of the
and
monomers
(104 ml
mol
) is of the magnitude observed for
other dimer-monomer equilibria (Silva and Weber, 1993).
It is also
possible to monitor the dissociation and denaturation of
allophycocyanin hexamers by measuring the decrease in fluorescence in
neutral buffers solution at 0 °C (Fig. 5). The fluorescence
at the highest pressure resembles that obtained with 8 M urea,
a further indication that the combined effects of pressure and low
temperature results in a state of the tetrapyrrolic pigments that
mimics that of denaturation at room temperature by urea. Returning
pressure and temperature to initial conditions resulted in recovery of
the fluorescence properties of the protein parallel to the changes in
absorbance, and the values of V and K
from the fluorescence data are in close agreement with those of
absorbance (Table 1).
Figure 5: Pressure effects on the fluorescence spectrum of allophycocyanin at 0 °C in neutral buffer, excited at 600 nm. Protein concentration was 10 µg/ml. From top to bottom: 1.0 bar, 0.2 kbar, 0.6 kbar, 1.0 kbar, 1.4 kbar, 1.6 kbar, 1.8 kbar, 2.0 kbar, and 2.4 kbar.
To determine the protein concentration
dependence of the pressure effects on the dimer, compression at 0
°C was performed at two different protein concentrations. Fig. 6shows that the pressure curve was uniformly shifted to
higher pressures at the higher protein concentration. The pressure
difference at = 0.5,
p
is 330
bar, while the theoretical for
V = 100 ml
mol
is
p
= 400
bar. The 20% difference of
p
between
observed and computed values is within the range generally observed in
other dimers (Silva and Silveira, 1992; Silva and Weber, 1993).
Figure 6: Concentration dependence of the pressure-induced dissociation at 0 °C of the dimer (1 M perchlorate solutions) investigated by fluorescence. The protein concentrations were 5 µg/ml (black circles) and 40 µg/ml (empty circles). The isolated symbol represents the fluorescence level achieved after decompression. Relative changes are given as ratio of spectral areas (Area p/Area atm), where p is the applied pressure.
The dissociation reaction was also examined by varying the temperature at constant pressure (Fig. 7). Starting at atmospheric pressure at 22 °C (solid symbol in Fig. 7, upper left) an increase in pressure to 1.6 kbar produced a very small change in absorbance (filled circles), but a considerably larger one at 2.4 kbar (empty circles). At 2.4 kbar and -11 °C, the final absorption ratio reached was nearly the same as observed by increase in pressure at 0 °C (Fig. 4). At 1.6 kbar, the freezing point is -9 °C, and while the maximum dissociation reached is thus limited, the slope of the change in absorbance with temperature is such as to indicate that the lower limit of absorbance would be reached at a temperature not much different than that at 2.4 kbar.
Figure 7:
Effect of temperature in the range 24 to
-12 °C at 2.4 and 1.6 kbar on the absorbance at 615 nm. The
absorbance ratio was calculated by
(Abs/Abs
). The dashed line shows the change promoted by the shift in pressure at room
temperature. Protein concentration was 45
µg/ml.
On-line formulae not verified for accuracy
where C is the concentration of protein
expressed as hexamer. In terms of the characteristic concentration
C
at which
=
, reads
On-line formulae not verified for accuracy
If the constants C and
C
, as well as the protein concentration as
hexamer, C
, are given, and can be coupled by a simple iterative procedure to yield the
values of
and
at which, at any fixed conditions of
temperature and pressure,
On-line formulae not verified for accuracy
In this resolution, the characteristic values of
C and C
at each
pressure, C
(p) and
C
(p), are related to the
corresponding values at atmospheric pressure,
C
(0) and C
(0), by
the equations
On-line formulae not verified for accuracy
where V
and
V
are the standard volume changes of the respective association reactions 3(
)
(
)
and
+
. The parameters for the dimer monomer
equilibrium C
(0) and
V
are known from the observations on perchlorate solutions of
allophycocyanin so that the coupling of the sequential equilibria
depends upon the choice of the parameters
C
(0) and
V
.
Employment of the independently obtained data on dimer dissociation in
perchlorate solutions involves the assumption that the thermodynamic
parameters of the dissociation of the dimer in 1 M perchlorate
do not materially differ from those that correspond to the dimers
generated on dissociation of the hexamers in the absence of
perchlorate. We consider this uncertainty preferable to that involved
in the simultaneous determination of four unknown parameters instead of
two. If we thus employ the two parameters obtained from the
observations on 1 M perchlorate solution, we can compute, for
any pair of the parameters, C
(0) and
V
, the proportions of hexamers, dimers, and
monomers at each pressure. As we know ( Fig. 2and Fig. 3)
that the respective contributions to absorption at 636 nm (see below)
or to fluorescence are in the ratios 1:0.27:0.05, we can construct the
spectral change curve corresponding to the parameters employed,
C
(0) and
V
, and
compare it with the experimental observations. Fig. 1shows that
increasing pressure to 2.4 kbar at 0 °C results in a progressive
decline in both absorption peaks at 620 and 653 nm but with the
decrease in absorption at 620 nm occurring at higher pressures than at
653 nm. The 653 nm peak has been assigned to exciton splitting of the
absorbance owing to interaction of nearby dimer chromophores within the
hexamer, which can be affected by pressure somewhat differently than
the overall absorbance. The average absorption across the whole
spectrum should be unaffected by the exciton splitting because the
exciton splitting is expected to occur with conservation of the
oscillator strength. The monotonic decrease in absorption over the
whole band cannot be resolved into contributions from hexamers and
dimers individually represented by any specific region of the spectrum;
instead we must take the integrated absorption over the whole band as a
measure of the hexamer dissociation. The solid line in Fig. 8corresponds to the unsmoothed average absorption between
the limits of 620 and 653 nm. It virtually coincides with the
absorption change in the center of mass of the whole band, at 636 nm.
This latter set of values was used as characteristic of the changes in
hexamer concentration. At 0 °C and 2.4 kbar, the absorption
throughout the whole band reaches very low limiting values, indicating
that for this range of pressures dissociation at 0 °C of hexamers
into the
and
monomers is virtually complete. Best fitting
of the observed spectral changes obtains for K
/K
= 0.07, or
C
= 0.22 ± 0.06 nM and
V
= 100 ± 5 ml
mol
. The fractions of hexamers, dimers, and monomers
computed for these values of C
(0) and
V
, together with their fractional
contribution to the absorbance deduced from Fig. 1and Fig. 2permits calculation of a spectral curve quite close to the
experimental points of decrease in absorption at 636 nm at 0 °C, as
shown in Fig. 9. The difference in the spectral curves that
assume dimer contributions of 0.27 (full line) and 0 (broken line) is generally insignificant. The cause is clearly
the minimal dimer fraction present at any degree of dissociation, so
that we virtually observe a hexamer-monomer equilibrium, in which we
are able to separate the constants characteristic of the two stages
because of the previous observations on the perchlorate solutions that
independently define the parameters C
(0) and
V
. We note that the coincidence of the
observed and expected absorbance changes does not depend on the exact
value of C
so long as
C
/C
1. This
relation between the dissociation constants appears almost inevitable
from the smooth changes in absorption (Fig. 1) or fluorescence ( Fig. 3and Fig. 5) of solutions of allophycocyanin in
buffer compressed at 0 °C toward the corresponding spectra observed
in 8 M urea and the equally smooth changes in absorbance on
cooling the solutions below 0 °C (Fig. 7). However, we note
that the conclusions reached by the agreement of the data of Fig. 1and Fig. 5with the result of the computations (Fig. 9) substantially depend on specific hypotheses about the
molecular origin of the absorption spectrum and on the assumption that
the dissociation constant for the dimer-monomer equilibrium in buffer
is not significantly smaller than that determined for the dimer in 1 M perchlorate.
Figure 8: Dependence of the relative absorbance change on the pressure for the data of Fig. 1. Filled circles, 653 nm; half-filled circles, 636 nm; empty circles, 620 nm. The line is the unsmoothed integration of the absorbance for the whole band, which is seen to coincide with the center of mass of the absorbance, 636 nm, as demanded by the conservation of the oscillator strength in exciton splitting.
Figure 9:
Spectral curves constructed according to
the proportions of hexamers, dimers and monomers calculated with use of
the parameters C = 3.2
nM, C
= 0.22
nM,
V
= 104 ml
mol
,
V
=
100 ml mol
. Solid line, absorbance change
with pressure for a dimer contribution of 0.27; dashed line,
absorbance change for zero dimer contribution. Points,
experimental values of the relative absorbance change at 636
nm.
Fig. 10illustrates the hysteresis
that occurs on reversal of the changes in absorption with pressure.
Though there were considerable differences between compression and
decompression associations calculated from the absorbances at both 653
and 620 nm, reassociation on decompression appears to be virtually
complete for the 620 nm absorbance but only about 50% complete for the
653 nm absorbance, indicating an imperfect association that does not
restore completely the conditions necessary for the recovery of the
initial exciton splitting. This imperfect reassociation, which bespeaks
a loss of free energy after dissociation, evidently arises from changes
in the conformation (``conformational drift'') of the
separated subunits of oligomeric proteins. It is observed in the
dissociation of some dimers by dilution (Xu and Weber, 1980; Silva and
Silveira, 1991; Erijman et al., 1993) and in other dimers and
higher order aggregates dissociated by hydrostatic pressure (King and
Weber, 1986; Silva et al., 1986; Ruan and Weber, 1989, 1993).
The theory of these effects is discussed by Weber(1986, 1992). When the
free energy loss by conformational drift is not too large,
reassociation of subunits on decompression can be virtually complete,
but the recovered aggregate shows persistent changes in properties
characteristic of a material of ``fading memory'' (Gurtin,
1968; Truesdell, 1985). In the present case, the hysteresis and the
incomplete recovery of properties is sufficient to establish the
existence of new hexamer-dimer-monomer equilibria with modified
thermodynamic parameters. Fig. 10shows also that the absorbance
at 653 nm is almost fully reversible after compression to 1.6 kbar at 0
°C, conditions that bring about half-dissociation. The hysteresis
of the decompression curve is much less pronounced, and the
reassociation is nearly complete. Similar results have been previously
described for the pressure dissociation of the apoprotein of tryptophan
synthase subunit (Silva et al., 1986). They
require that the loss of free energy of association owing to
conformational drift increase with the degree of dissociation reached
by the protein aggregate and that the major part of the loss of free
energy occur at conditions of nearly complete dissociation.
Figure 10:
Effects
of compression up to 2.4 kbar followed by decompression to atmospheric
pressure at 0 °C on the relative absorbances at 653 nm (dark
circles) and 620 nm (empty circles) of allophycocyanin in
neutral buffer. Dark half-circles, absorbance at 653
nm on decompression from 1600 bar to atmospheric pressure. After
decompression, the temperature was raised to 22 °C (dashed
line). Absorbance ratios equal
Abs/Abs
where p is
the applied pressure. Protein concentration was 30
µg/ml.
Regardless of exact values, all calculations confirm that the association of the subunits is an entropy-driven phenomenon with substantial positive enthalpies that oppose association.
The pressure dissociation of the aggregates is determined by the preferential destabilization of the apolar contacts (Weber, 1993). This destabilization results in a sufficient increase in the enthalpy of association to shift the equilibrium toward dissociation, while the changes in entropy on compression are much less important.
Figure 11: Changes in fluorescence polarization in the temperature range of 22 to -11 °C at 2.4 kbar of allophycocyanin in aqueous buffer solution. Filled circles, decrease in temperature. Empty circles, subsequent increase in temperature. The hysteresis is consistent with a loss of 15-20% of the free energy of hexamer-dimer association.
Recent studies of the effects of low temperatures on protein folding have shown that supercooling of a single-chain protein in solution causes its denaturation (Sturtevant, 1977; Griko et al., 1988; Privalov, 1990). The studies of protein dissociation by hydrostatic pressure carried out thus far have been limited to temperatures above 0 °C, and the relatively small changes in protein conformation that have been detected in the separated subunits have been considered to be the result of a limited conformational drift, which never achieves the structural disorganization characteristic of protein denaturation (King and Weber, 1986; Silva and Weber, 1993). The decrease in the freezing point of water that occurs under pressure makes it possible to expand the accessible temperature range down to -20 °C at a hydrostatic pressure of 2.4 kbar. Thus one can attain, under equilibrium conditions, the temperatures of the supercooled solutions in which polypeptide denaturation has been observed.
The temperature effects observed at constant pressure show
that the association of allophycocyanin is maintained by a large
entropy change that opposes a considerably positive enthalpy change, a
situation that appears to obtain generally for the association of
subunits into oligomers (Lauffer, 1975; Weber, 1993; Silva and Weber,
1993). Allophycocyanin dissociation shows more clearly than other cases
the entropic contribution to the protein assembly process, since at
room temperature, the state of aggregation of the protein is barely
affected by pressure alone. Table 1establishes the
entropy-driven character of the reversible folding and association of
+
subunits of allophycocyanin into dimers; thus an
increase in entropy drives the entire process of association of the
unfolded monomers into the complete allophycocyanin hexamer.
Allophycocyanin compression and decompression curves exhibit hysteresis that is more pronounced the greater the extent of dissociation. Hysteresis in the compression-decompression cycle has been observed in most of the oligomers studied (Silva and Weber, 1993). It is attributed to occurrence of a process that follows the separation of the subunits and reduces their affinity for each other. It also results in delayed recovery of the original spectroscopic and enzymic properties of proteins after decompression (King and Weber, 1986; Silva et al. 1986; Ruan and Weber, 1989). As discussed elsewhere (Weber, 1986) this phenomenon appears to be the consequence of the substitution of intersubunit contacts by solvent-subunit contacts. The phenomenon of hysteresis requires that after a reduction in pressure, the degree of dissociation reaches a value that maintains itself for a time, which is at least of the order of the whole compression-decompression cycle (Everett and Smith, 1955; Cooper, 1988; Silva and Weber, 1993). This behavior is well known in the mechanics of solids as characteristic of ``materials of fading memory'' (Truesdell, 1985), and the observation of the effects of hydrostatic pressure on many oligomers shows that they fall naturally in this category. While materials of fading memory may be expected to share many features of their thermodynamics and dynamics (Gurtin, 1968), proteins are far more heterogeneous than most materials in this category and will inevitably require a theory peculiar to themselves.
The volume change of association found for hexamer- dimer
equilibrium (100 ml/mol) is intermediate between the one found for
dimers (60-150 ml/mol) and that for tetramers (around 220 ml/mol)
(Silva and Weber, 1993). Volume changes calculated from data at
different temperatures are very similar (Table 1), suggesting
that the conformational drift of the monomers separated by compression
at different temperatures does not influence appreciably the volume
change on association, at least in this case.
The observation that the dissociation constants for the hexamer into dimers is approximately 200 times smaller than the dissociation constant of the dimer into monmers in dilute neutral buffer solutions predicates that the former dissociation is inevitably followed by the latter and that the dimer is not an intermediate form that can exist in appreciable concentration in such media, although it is stabilized by 1 M perchlorate. The absence of dimer intermediates has been observed in the dissociation of the tetramers of glyceraldehide phosphate dehydrogenase (Ruan and Weber, 1989) and glycogen phosphorylase (Ruan and Weber, 1993). Its physiological significance may lie in the need for rapid hydrolytic removal of the protein following the initial stage of dissociation.
The reversibility observed in the reassembly of the
dimers after they are converted into
+
subunits by the
combined use of pressure and low temperature indicates that inside the
cell this step in the assembly process of phycobiliproteins can occur
spontaneously. On the other hand, the association into native hexamers
may require a particular set of conditions or the presence of cofactors
in order to proceed inside the cell, since it is not completely
reversible after decompression to atmospheric pressure or increase in
temperature, in the dilute buffered solutions studied here. However,
the extent of hexamer recovery is proportional to temperature and
protein concentration, and inside the cyanobacterial cell the
concentration of phycobiliproteins is extremely high. Thus it is also
possible that the hexamer assembly proceeds driven by the law of mass
action, with some aid from the temperature.
The dissociation-denaturation behavior of allophycocyanin observed in this study is consistent with previous results obtained on intact cyanobacteria (Foguel et al., 1992). With them, the application of high hydrostatic pressure at room temperature resulted in a strong emission with a red shift to 662 nm when the cells were excited with green light. This result suggested that in these conditions, pressure disconnected the phycobilisomes from the thylakoid membranes resulting in the emission of the terminal phycobilisome component, namely allophycocyanin. The emission vanished when a combination of low temperature and high pressure was used, an indication of dissociation and denaturation of the phycobilisomes components. In agreement with those results, we find here that allophycocyanin does not dissociate at room temperature. Dissociation is only observed when pressure is increased at low temperature. Additional studies with intact phycobilisomes and their isolated protein components are in progress in order to obtain further insights into the assembly process.