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Comparative Analysis of the Effects of a-Crystallin and GroELon the Kinetics of Thermal Aggregation of Rabbit MuscleGlyceraldehyde-3-Phosphate Dehydrogenase
Kira A. Markossian • Nikolay V. Golub • Natalia A. Chebotareva •
Regina A. Asryants • Irina N. Naletova • Vladimir I. Muronetz •
Konstantin O. Muranov • Boris I. Kurganov
Published online: 20 November 2009
� Springer Science+Business Media, LLC 2009
Abstract Effects of a-crystallin and GroEL on the
kinetics of thermal aggregation of rabbit muscle glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) have been
studied using dynamic light scattering and analytical
ultracentrifugation. The analysis of the initial parts of the
dependences of the hydrodynamic radius of protein
aggregates on time shows that in the presence of a-crys-
tallin or GroEL the kinetic regime of GAPDH aggregation
is changed from the regime of diffusion-limited cluster–
cluster aggregation to the regime of reaction-limited clus-
ter–cluster aggregation, wherein the sticking probability for
the colliding particles becomes lower the unity. In contrast
to a-crystallin, GroEL does not interfere with formation of
the start aggregates which include denatured GAPDH
molecules. On the basis of the analytical ultracentrifuga-
tion data the conclusion has been made that the products of
dissociation of GAPDH and a-crystallin or GroEL play an
important role in the interactions of GAPDH and chaper-
ones at elevated temperatures.
Keywords Glyceraldehyde-3-phosphate dehydrogenase �a-Crystallin � GroEL � Aggregation � Dynamic light
scattering � Sedimentation velocity
Abbreviations
DLCA Diffusion-limited cluster–cluster aggregation
DLS Dynamic light scattering
DSC Differential scanning calorimetry
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
RLCA Reaction-limited cluster–cluster aggregation
1 Introduction
The mechanism of thermal denaturation of proteins
involves partial unfolding and conformational modification
of the protein molecule. The interaction of exposed
hydrophobic surfaces during protein unfolding is accom-
panied by formation of aggregates. The molecular chap-
erones, which belong to the family of heat-shock proteins,
can interact with non-native or partially folded polypeptide
chains and prevent irreversible protein aggregation during
heat stress [1, 2]. Chaperonin GroEL from Escherichia coli
[3, 4] and small heat-shock proteins (sHsps) [5, 6] are
among the well-studied chaperones.
GroEL, a 14 subunit, double-ring oligomer of *800 kDa
[3, 4], has some hydrophobic sites exposed to the solvent [7]
and interacts transiently with intermediates of protein fold-
ing, prevents their hydrophobic surfaces from ‘‘incorrect’’
intermolecular and intramolecular interactions, and favors
correct folding of polypeptide chains [2, 8–11].
A range of in vitro studies directly demonstrated that
GroEL can rapidly and efficiently bind to non-native
K. A. Markossian (&) � N. V. Golub � N. A. Chebotareva �B. I. Kurganov
Bach Institute of Biochemistry, Russian Academy of Sciences,
119071 Moscow, Russia
e-mail: [email protected]
R. A. Asryants � I. N. Naletova � V. I. Muronetz
Belozersky Institute of Physico-Chemical Biology,
Moscow State University, 119992 Moscow, Russia
V. I. Muronetz
Faculty of Bioengineering and Bioinformatics,
Moscow State University, 119992 Moscow, Russia
K. O. Muranov
Emanuel Institute of Biochemical Physics,
Russian Academy of Sciences, 119991 Moscow, Russia
123
Protein J (2010) 29:11–25
DOI 10.1007/s10930-009-9217-9
proteins and suppress their aggregation, as has been shown
for ribulose-1,5-bisphosphate oxygenase-carboxylase [12],
a-glucosidase [13], glutamine synthase [14], dihydrofolate
reductase [15], rhodanese, citrate synthase [16] and malate
dehydrogenase [17].
It has been accepted that GroEL prevents protein
aggregation by encapsulating individual chains within the
so-called ‘Anfinsen cage’ provided by the GroEL–GroES
complex [2, 18].
However, GroEL is able to prevent protein aggregation
not only by encapsulating individual protein chains inside
its oligomeric structure [2, 19, 20], but also outside the
cage [21, 22]. In some cases the presence of GroEL either
alone or additionally to ADP or ATP is enough for effec-
tive GroEL-assisted protein folding [23, 24].
It has been suggested that hydrophobic interactions
represent the main factor stabilizing the GroEL complex
with nonnative proteins [25, 26]. However, electrostatic
interactions also play an important role in the complexing
of GroEL with non-native proteins [27, 28].
Recently Semisotnov et al. [29–32] proposed the model
of GroEL functioning as a molecular chaperone. According
to this model, GroEL binds strongly ‘‘hydrophobic’’ states
of proteins or polypeptides decreasing their concentrations
in solution thereby decreasing their non-specific interac-
tions. Bound polypeptides can sometimes dissociate from
the GroEL surface and adopt native conformation in
solution and lose possibility to interact with GroEL.
Chaperonin function consists in ‘‘keeping’’ of hydrophobic
protein intermediates and thereby in decreasing probability
of their non-specific association [31, 32]. According to the
model of the GroEL action proposed by these authors
GroEL-assisted protein folding proceeds out of the chap-
eronin cavity.
Ybarra and Horowitz [33] were the first to demonstrate
the formation of GroEL monomers under conditions
commonly used for preparation of chaperonin. The essen-
tial requirements are the simultaneous presence of nucle-
otides such as Mg-ATP or Mg-ADP and a solid-phase
anion-exchange medium. Surin et al. [34] used the same
procedure to obtain the GroEL monomer. According to the
results obtained by these authors, the change in pH of the
buffer solution from 7.5 to 9.0 resulted in the increase in
the yield of the monomeric form. The monomeric GroEL is
a globular protein with a pronounced secondary and a rigid
tertiary structure. The affinity of the monomeric form to the
hydrophobic probe, 8-anilino-1-naphthalenesulfonic acid,
is an order of magnitude higher than that for the subunit in
the oligomeric particles, suggesting that the monomeric
form is characterized by the high degree of exposure of the
hydrophobic clusters of the protein molecule [34].
Belonging to the family of small heat shock proteins,
a-crystallin is a large oligomeric complex ranging in size
from 200 to 800 kDa and possessing chaperone-like
activity. a-Crystallin binds unfolded proteins and forms
stable, soluble complexes with denatured proteins pre-
venting their aggregation and precipitation [35, 36]. The
ability of a-crystallin to suppress heat-induced aggrega-
tion of proteins is a result of hydrophobic interactions
with these denatured proteins, and this ability increases
when a-crystallin is heated [37–40]. The mechanism of
suppression of thermal aggregation of a number of pro-
teins by a-crystallin has also been studied by Kurganov
et al. [41–47]. It has been shown that suppression of
aggregation is due to the diminution of the size of start
aggregates in the presence of a-crystallin, increase in the
duration of the latent period over which the start aggre-
gates are formed and transition of the aggregation process
from the kinetic regime of ‘‘diffusion-limited cluster–
cluster aggregation’’ (DLCA) into the regime of ‘‘reac-
tion-limited cluster–cluster aggregation’’ (RLCA),
wherein the sticking probability for the colliding particles
becomes less than unity.
Process of thermal denaturation and aggregation of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC
1.2.1.12), a tetrameric enzyme of 144 kDa, is the subject
of a number of studies [48–50]. The secondary structure
of the enzyme is relatively heat stable, showing change
only at pH 8.85. Under these conditions, the enzyme first
dissociates within several minutes, probably into dimers,
and with prolonged heating it becomes irreversibly
aggregated [48]. The dissociative mechanism of thermal
denaturation of GAPDH was supported using differential
scanning calorimetry and analytical ultracentrifugation
[43, 51].
Glyceraldehyde-3-phosphate dehydrogenase has been
used as a target protein to examine the effects of GroEL
[15, 52–54]. Naletova et al. [54] have demonstrated that
GroEL does not prevent thermal denaturation of GAPDH,
but effectively binds the thermally denatured enzyme, thus
preventing the aggregation of the denatured molecules. The
complex GroEL–GAPDH is characterized by increased
thermal stability compared to GroEL.
Earlier we studied the effect of a-crystallin on thermal
denaturation of GAPDH and aggregation at the regime of
temperature elevation with a constant rate [43]. When
aggregation of GAPDH was studied in the presence of
a-crystallin, the start aggregates of lesser size were
observed. In the present work we have compared the
effects of GroEL and a-crystallin on the aggregation pro-
cess of GAPDH monitored at a fixed temperature of 45�C.
We have shown using dynamic light scattering (DLS) that
in the presence of each chaperone GAPDH aggregation
proceeds in the RLCA regime. However, these chaperones
have different effect on the formation of start aggregates
involving unfolded GAPDH molecules.
12 K. A. Markossian1 et al.
123
2 Materials and Methods
2.1 Isolation of GAPDH
Glyceraldehyde-3-phosphate dehydrogenase was isolated
from rabbit skeletal muscles as described by Scopes and
Stoter [55] with an additional purification step using gel-
filtration on Sephadex G-100. GAPDH concentration was
determined spectrophotometrically at 280 nm, using the
absorption coefficient A1%1cm of 10.6 [56].
2.2 Isolation of a-Crystallin
Freshly excised lenses from 2-year-old steers were
obtained from a local slaughterhouse and stored frozen at
-20 �C. Purification of a-crystallin was performed mainly
according to the procedure described earlier [57]. The de-
capsulated lens cortex was homogenized at 0 �C in 40 mM
sodium phosphate, pH 6.8, containing 100 mM NaCl,
1 mM EDTA, and 3 mM NaN3. The homogenate was
centrifuged at 27,000g for 1 h at 4 �C, and the supernatant
containing the soluble crystallin was fractionated by gel
filtration using TSK-gel HW-55 (Sigma) column
(2.5 9 90 cm). The top of the fraction containing a-crys-
tallin was collected by measuring absorption at 280 nm.
a-Crystallin fraction was rechromatographied using TSK-
gel HW-55 columns. a-Crystallin peak corresponding to
molecular mass of 750 kDa was symmetrical. The fraction
containing HMW a-crystallin was eluted in the void vol-
ume. TSK-gel HW-55 column was calibrated with high
molecular mass kit: thyroglobulin (660 kDa), ferritin
(440 kDa), catalase (220 kDa), aldolase (158 kDa) and
BSA (68 kDa) (Sigma). Finally, top of peak corresponding
to a-crystallin was collected and concentrated by ultrafil-
tration (Millipore PTTK disk membrane (Sigma), NMWL
30000). Thus, the procedure used for isolation and purifi-
cation of a-crystallin provided complete separation of
HMW fraction of a-crystallin. Concentration of a-crystallin
was determined spectrophotometrically at 280 nm, using
the absorption coefficient A1%1cm of 8.5 [40].
2.3 Isolation of GroEL
GroEL was expressed in Esherichia coli strain W3110
transfected with the plasmid pOF39. The expression
product was purified as described by Corrales and Fersht
[26] with some modifications [54]. The resulting prepara-
tion was concentrated to 4 mg/ml using YM-10 Centriprep
centrifugal filter units and dialyzed against 10 mM
K-phosphate buffer, pH 7.5. The solution of GroEL was
stored at –70 �C. GroEL concentration was determined
spectrophotometrically at 280 nm, using the absorption
coefficient A1%1cm of 1.8 [58].
2.4 GAPDH Assay
Glyceraldehyde-3-phosphate dehydrogenase activity was
measured by monitoring the increase in the absorbance at
340 nm, caused by the formation of NADH, using a UV
1601 Schimadzu spectrophotometer (Japan). The reaction
was carried out at 20 �C and initiated by the addition of
2–6 lg of the enzyme to the reaction mixture (1 ml) con-
taining 100 mM glycine, 100 mM KH2PO4, pH 8.9, 5 mM
EDTA, 1 mM NAD and 1 mM glyceraldehyde-3-phos-
phate. The dehydrogenase activity constituted 70 U/mg of
the protein.
2.5 Thermal Inactivation of GAPDH
Thermal inactivation of GAPDH was studied at 45 �C in
10 mM Na-phosphate buffer, pH 7.5, containing 1 mM
EDTA and 1 mM dithiothreitol. The inactivation process
was initiated by the addition of a portion of the GAPDH
solution to the buffer preheated for 10 min. At fixed time
intervals, aliquots were withdrawn to determine the enzy-
matic activity in the standard mixture.
2.6 Dynamic Light Scattering Studies
For light scattering measurements a commercial instrument
Photocor Complex (Photocor Instruments Inc., USA
www.photocor.com) was used with a He–Ne laser
(Coherent, USA, Model 31-2082, 632.8 nm, 10 mW) as a
light source. The temperature of sample cell was controlled
by the proportional integral derivative (PID) temperature
controller to within ±0.1 �C. A quasi-cross correlation
photon counting system with two photomultiplier tubes
(PMT) was used to increase the accuracy of particle sizing
in the range from 1.0 nm to 5.0 lm. DLS data have been
accumulated and analyzed with multifunctional real-time
correlator Photocor-FC. DynaLS software (Alango, Israel)
was used for polydisperse analysis of DLS data.
The diffusion coefficient D of the particles is directly
related to the decay rate sc of the time-dependent correla-
tion function for the light-scattering intensity fluctuations:
D = 1/2sck2. In this equation k is the wave number of the
scattered light, k = (4pn/k)sin(h/2), where n is the refrac-
tive index of the solvent, k is the wavelength of the incident
light in vacuum and h is the scattering angle. The mean
hydrodynamic radius of the particles, Rh, can then be
calculated according to the Stokes–Einstein equation:
D = kBT/6pgRh, where kB is Boltzmann’s constant, T is the
absolute temperature and g is the shear viscosity of the
solvent.
The kinetics of thermal aggregation of GAPDH was
studied by DLS in 10 mM Na-phosphate buffer, pH 7.5.
All solutions for the experiments were prepared using
Comparative Analysis of the Effects of a-Crystallin 13
123
deionized water obtained with Easy-Pure II RF system
(Barnstead, USA). The buffer was placed in a cylindrical
cell with a diameter of 6.3 mm and pre-incubated for
10 min at appropriate temperature. Cells with stoppers
were used for incubation at high temperature to avoid
evaporation. The aggregation process was initiated by the
addition of an aliquot of GAPDH. To study the effect of a-
crystallin or GroEL on aggregation of GAPDH, aliquots of
both proteins (protein substrate and chaperone) were
simultaneously added into the cell up to the final volume of
0.5 mL. The scattering light was collected at 90� scattering
angle and the accumulation time of the autocorrelation
function was 30 s.
Analysis of the dependence of the hydrodynamic radius
of protein aggregates on time allows discriminating
between the DLCA and RLCA regimes of aggregation. In
our previous works [41, 42, 51, 59] the conclusion about
the validity of the DLCA regime for thermal aggregation of
proteins was made on the fact that from a certain value of
time (t [ t*) the dependence of the hydrodynamic radius
(Rh) of protein aggregates on time follows the power law:
Rh ¼ R�h 1þ K1 t � t�ð Þ½ �1=df; ð1Þ
where R�h is the Rh value at t = t*, K1 is a constant and df is
the fractal dimension of the aggregates formed as a result
of random aggregation. For DLCA regime, the parameter
df has a universal value: df = 1.8 [60, 61].
The following equation may be used for analysis of the
initial parts of the dependence of Rh on time (see [51]):
Rh ¼ Rh;0 1þ 1
t2Rt � t3ð Þ
� �; ð2Þ
where Rh,0 is the hydrodynamic radius of start aggregates,
parameter t3 is the moment of time at which the start
aggregates appear (we have conserved the notation t3which was used in our previous work [62]) and t2R is the
time interval over which the Rh value increases from Rh,0 to
2Rh,0.
Previously we have shown that when protein aggrega-
tion proceeds in the presence of a-crystallin, the depen-
dence of the hydrodynamic radius on time follows the
exponential law [41–44]:
Rh ¼ Rh;0 expln 2
t2Rt � t3ð Þ
� �� �; ð3Þ
where Rh,0 is the hydrodynamic radius of the start aggre-
gates, t3 is the moment of time at which the start aggregates
appear and t2R is the time interval over which the hydro-
dynamic radius of aggregates is doubled. The reciprocal
value of parameter t2R characterizes the rate of aggregation.
The higher 1/t2R, the higher is the aggregation rate. The
exponential dependence of Rh on time is typical of the
RLCA regime [63].
2.7 Analytical Ultracentrifugation
Sedimentation velocity studies of GAPDH, a-crystallin,
GroEL and the mixture of GAPDH with a-crystallin or
GroEL were carried out at 21 and 45–46 �C in a Model E
analytical ultracentrifuge (Beckman), equipped with
absorbance optics, a photoelectric scanner, a monochro-
mator, a computer on line and special facilities (additional
chamber with heater) for high temperatures. A four-hole
rotor An-F Ti and 12 mm double sector cells were used.
The rotor was preheated at elevated temperature during a
night in the thermostat before the run. Sedimentation pro-
files of GAPDH and a-crystallin were recorded by mea-
suring absorbance at 280 nm. All cells were scanned
simultaneously. The time interval between scans was
3 min. The sedimentation coefficients were estimated from
the differential sedimentation coefficient distribution [c(s)
vs. s] or [c(s, f/f0) vs. s] which were analyzed using
SEDFIT program [64, 65]. The sedimentation coefficient
distribution c(s) of Lamm equation solutions is based on
the approximation of a single, weight-average frictional
coefficient of all particles, determined from the experi-
mental data, which scales the diffusion coefficient to the
sedimentation coefficient consistent with the traditional
s*M2/3 power law. It provides a high hydrodynamic res-
olution, where diffusion broadening of the sedimentation
boundaries is deconvoluted from the sedimentation coef-
ficient distribution. A generalization of c(s) to a two-
dimensional distribution of sedimentation coefficient and
frictional ratio, c(s, f/f0), which is representative of a more
general set of size-and-shape distributions, including mass-
Stokes radius distributions, c(M, RS), and sedimentation
coefficient-molar mass distributions c(s, M) has recently
been proposed [64]. Even though the additional dimension
describing f/f0 (or M, respectively) does not offer high
resolution, the hydrodynamic resolution in sedimentation
coefficient, c(s, *) is usually close to the c(s) resolution
[64]. The c(s, *) distribution can be considered as a more
general version of c(s) distribution, which does not make
any assumptions on the diffusional properties of macro-
molecular ensemble, yet still deconvolutes diffusional
broadening on the basis of the experimentally measured
sedimentation boundary shapes [65].
The c(s) analysis was performed with regularization at a
confidence level of 0.68 and a floating frictional ratio. The
sedimentation coefficients were corrected to the standard
conditions (a solvent with the density and viscosity of
water at 20 �C).
2.8 Calculations
Origin 7.0 software (OriginLab Corporation, USA) was
used for the calculations.
14 K. A. Markossian1 et al.
123
3 Results
3.1 Kinetics of Thermal Aggregation of GAPDH
Effects of a-crystallin and GroEL on thermal aggregation of
GAPDH were studied at 45 �C. First of all, we analyzed the
kinetics of aggregation of GAPDH (0.4 mg/mL, 10 mM Na-
phosphate, pH 7.5) at this temperature using DLS. Figure 1a
and b shows the dependences of the light scattering intensity
(I) and hydrodynamic radius (Rh) of the protein aggregates
on time, respectively. To analyze the dependence of Rh on
time, we constructed the light scattering intensity versus
hydrodynamic radius plot (Fig. 1c). The length cut off on the
abscissa axis by the initial linear dependence of I on Rh
corresponds to the hydrodynamic radius of the protein
aggregates (Rh,0) which are formed in the system at the
moment when the light scattering intensity begins to
increase. We have called such primary clusters the start
aggregates [41]. The Rh,0 value characterizing the size of the
start aggregates is equal to 65.5(±1.3) nm. With a knowledge
of the Rh,0 value we can analyze the initial part of the
dependence of Rh on time using Eq. (2). Parameter t3 (the
moment of time at which the start aggregates appear) was
found to be 4.5(± 0.2) min (Table 1). The reciprocal value
of parameter t2R (t2R is the time interval over which the Rh
value increases from Rh,0 to 2Rh,0) is also given in Table 1.
At rather high values of time (t [ t* = 13 min) the
dependence of Rh on time follows the power law (1) with
df = 1.86(± 0.14). The obtained value of df means that
GAPDH aggregation proceeds in the DLCA regime [63].
This result is consistent with the data we obtained previ-
ously [51].
3.2 Effect of a-Crystallin on Thermal Inactivation
of GAPDH
Since the process of GAPDH thermal aggregation involves
the initial stage of unfolding of the protein molecule, it was
of interest to study the effect of a-crystallin on stability of
GAPDH. Figure 2 shows the kinetics of thermal inactiva-
tion of GAPDH (0.4 mg/mL) at 45 �C. As can be seen
from this Figure, a-crystallin accelerates thermal inactiva-
tion of GAPDH. The accelerating effect of a-crystallin
increases with increasing a-crystallin concentration and
reaches maximum at a-crystallin concentration of 0.2 mg/
mL. At higher concentrations of a-crystallin the shape of
the dependences of the relative enzymatic activity on time
remained practically unchanged. The destabilizing effect of
a-crystallin is consistent with the data obtained by differ-
ential scanning calorimetry (DSC). In our previous work
[43] we showed that the position of maximum on the DSC
profile was shifted towards the lower temperatures in the
presence of a-crystallin.
3.3 Effect of a-Crystallin on Kinetics of Thermal
Aggregation of GAPDH
Figure 3 demonstrates the effect of a-crystallin on the
kinetics of GAPDH aggregation at 45 �C. Addition of
a-crystallin reduced the increment in light scattering
intensity and prevented precipitation of GAPDH.
Fig. 1 Kinetics of thermal aggregation of GAPDH (0.4 mg/mL) at
45 �C (10 mM Na-phosphate buffer, pH 7.5). The dependences of the
light scattering intensity and hydrodynamic radius of the protein
aggregates (Rh) on time (panels A and B, respectively). The solidcurve in panel B was calculated from Eq. (2). Inset in panel B shows
the initial part of the dependence of Rh on time. The horizontal dottedline corresponds to the Rh,0 value (Rh,0 = 65 nm). c The light
scattering intensity versus the hydrodynamic radius plot
Comparative Analysis of the Effects of a-Crystallin 15
123
Calculations of the hydrodynamic radius show that
aggregates of lesser size are formed in the presence of
a-crystallin (Fig. 4a–d). To carry out the quantitative
analysis of the dependences of Rh on time obtained in
the presence of a-crystallin, we estimated the hydro-
dynamic radius of the initial particles (Rh,0) occurring in
the system at the instant when the initial increase in the
light scattering intensity was registered. For this purpose
we constructed the light scattering intensity versus
hydrodynamic radius plots (Fig. 5). The values of Rh,0
determined from the length cut off on the abscissa axis
by the straight line in these coordinates for a-crystallin
concentrations of 0.025, 0.05 and 0.1 mg/mL were
found to be 9.2–9.3 nm (Table 1). It should be noted
that this value is close to the hydrodynamic radius of
a-crystallin measured at 45 �C (10.1(± 0.2) nm) [43].
Therefore, we assumed that the initial particles regis-
tered in the system at the point in time, at which the
light scattering intensity begins to increase (i.e., the
particles with Rh = Rh,0), are the complexes of a-crys-
tallin with denatured GAPDH.
The initial parts of the dependences of Rh on time
obtained in the presence of a-crystallin at the concentra-
tions of 0.025, 0.05 and 0.1 mg/mL follow the exponential
law. Solid curves in insets in Fig. 4a, b and in Fig. 4c are
calculated from Eq. (3). Parameter t3 and reciprocal value
of parameter t2R are given in Table 1. The diminution of
the 1/t2R value characterizes the decrease in the aggrega-
tion rate.
As in the case of thermal aggregation of aL-crystallin
[41], aggregation of GAPDH in the presence of a-crys-
tallin (0.025–0.1 mg/mL) is accompanied by splitting the
aggregate population into two components at rather high
values of time (t [ tcrit). The values of tcrit and the values
of Rh at t = tcrit (Rcrit) are given in Table 1.
It is noteworthy that at higher concentrations of
a-crystallin (0.4 mg/mL) the increase in the Rh value in
time is insignificant and distribution of aggregates by size
remained unimodal over the entire period of process
registration (1,100 min; Fig. 4d).
Fig. 2 Effect of a-crystallin on thermal inactivation of GAPDH
(0.4 mg/mL) at 45 �C (10 mM Na-phosphate buffer, pH 7.5,
containing 1 mM EDTA and 1 mM dithiothreitol). The dependences
of the relative enzymatic activity A/A0 on time (A0 and A are the
initial and current values of the enzymatic activity). Concentrations of
a-crystallin were as follows: (1) 0, (2) 0.1, (3) 0.2 and (4) 0.4 mg/mL
Fig. 3 Effect of a-crystallin on GAPDH (0.4 mg/mL) aggregation at
45 �C. The dependences of the light scattering intensity on time
obtained in the absence of a-crystallin (1) and in the presence of
various concentrations of a-crystallin: (2) 0.025, (3) 0.05, (4) 0.1 and
(5) 0.4 mg/mL
Table 1 Parameters Rh,0, t3, 1/t2R, tcrit and Rh,crit for the dependences of Rh on time in the case of aggregation of GAPDH (0.4 mg/mL) registered
at 45 �C in the absence and in the presence of a-crystallin
Concentration of a crystallin
(mg/mL)
Rh,0 (nm) t3 (min) 1/t2R (min-1) tcrit (min) Rh,crit (nm)
0 65.5(± 1.3) 4.5(± 0.2) 0.53(± 0.06) – –
0.025 9.3(± 0.7) 16.4(± 0.4) 0.0111(± 0.0007) 390 125
0.05 9.2(± 0.8) 16.2(± 1.0) 0.0057(± 0.0009) 650 110
0.1 9.3(± 0.6) 15.7(± 1.4) 0.0017(± 0.0001) 790 25
Equations (2) and (3) were used for calculations of parameters t3 and 1/t2R for GAPDH aggregation studied in the absence and in the presence of
a-crystallin, respectively
16 K. A. Markossian1 et al.
123
3.4 Effect of a-Crystallin on Heat-Induced
Dissociation of GAPDH
To control the oligomeric state of GAPDH and a-crystallin,
we carried out the sedimentation velocity studies of
GAPDH and a-crystallin at 21 �C. The major peak in the
c(s) distribution for GAPDH (Fig. 6a) with sedimentation
coefficient of 8.6(± 0.6) S corresponds to the tetrameric
form of GAPDH. Figure 6b shows the differential sedi-
mentation coefficient distribution c(s) for the mixture of
GAPDH (0.4 mg/mL) and a-crystallin (0.4 mg/mL; dashed
line). The sedimentation coefficient (8.6(± 0.6) S) and the
Fig. 5 The light scattering intensity versus the hydrodynamic radius
plots for thermal aggregation of GAPDH (0.4 mg/mL) in the presence
of a-crystallin at 45 �C. The concentrations of a-crystallin were as
follows: a 0.025, b 0.05 and c 0.1 mg/mL
Fig. 4 Effect of a-crystallin on the size of aggregates formed in the
course of heating of GAPDH (0.4 mg/mL) at 45 �C. The dependences
of Rh on time obtained in the presence of various concentrations of
a-crystallin: a 0.025, b 0.05, c 0.1 and d 0.4 mg/mL. Insets show the
initial parts of the dependences of Rh on time. Solid curves are
calculated from Eq. (3)
Comparative Analysis of the Effects of a-Crystallin 17
123
shape of the main peak in c(s) distribution for mixture were
the same as in the case of GAPDH alone. The similar result
was obtained for the mixture of GAPDH (0.4 mg/mL) and
a-crystallin (1.0 mg/mL; solid line). For clarity sake, inset
in Fig. 5 shows the c(s) distribution for a-crystallin alone
(1 mg/mL).
Comparison of the sedimentation profiles of GAPDH
(0.4 mg/mL; Fig. 7a) and the mixture of GAPDH with
a-crystallin (0.4 mg/mL; Fig. 7b) indicates that levels of
optical densities corresponding to absorbance of GAPDH
are identical. These results suggest that there is no inter-
action of GAPDH with a-crystallin at 21 �C.
Earlier we showed that incubation of GAPDH at 45 �C
induced dissociation of the protein [51]. Differential sedi-
mentation coefficient distributions for GAPDH and mixture
of GAPDH with a-crystallin are presented in Fig. 8. Panel A
shows general c(s, *) distribution obtained for GAPDH at
45 �C and corrected to the standard conditions. Total incu-
bation time of was 1.5 h. The dissociated enzyme forms
with the sedimentation coefficients s20,w = 3.3(± 0.1) and
5.4(± 0.1) S were observed. The main peak (3.3 S) corre-
sponds to the monomeric form and minor peak (5.4 S) corre-
sponds to the dimeric form of the enzyme. Figure 8b–d shows
the c(s) distributions for mixtures of GAPDH (0.4 mg/mL)
and a-crystallin at the concentrations of 0.1, 0.4 or 1.0 mg/
mL, respectively. All distributions were obtained at 45 �C
and corrected to the standard conditions. Total incubation
time was 1.5 h. The disappearance of the peak corresponding
to the monomeric form of GAPDH with s20,w = 3.3(± 0.1)
S (Fig. 8b–d) was observed. The peak with sedimentation
coefficient of 5.8(± 0.1) S registered for the mixture of
GAPDH with a-crystallin (0.1 mg/mL, Fig. 8b), probably,
corresponds to the complex of GAPDH dimer and products
of dissociation of a-crystallin. When the concentration of
a-crystallin was increased, this peak was shifted towards
the higher values of sedimentation coefficient, namely, to
7.2(± 0.4) S at a-crystallin concentration of 0.4 mg/mL
(Fig. 8c) and 13.0(± 0.2) S at a-crystallin concentration of
1.0 mg/mL (Fig. 8d). Simultaneously the peaks with the
higher sedimentation coefficients appear on c(s) distribu-
tions obtained for mixtures containing a-crystallin 0.4 and
1.0 mg/mL (Fig. 8c, s20,w = 12.7(± 0.3) and 14.0(± 0.3)
S; Fig. 8d, s20,w = 14.7(± 0.2) and 16.5(± 0.3) S). These
peaks correspond to free a-crystallin. This conclusion can be
made from the comparison of c(s) distributions presented in
Fig. 8c and d with the corresponding distributions obtained
for a-crystallin heated for 1.5 h at 45 �C (Fig. 9a, s20,w =
12.9(± 0.3) and 14.0(± 0.3) S; Fig. 9d, s20,w = 14.9(± 0.2)
S). It should be noted that Fig. 9a and b demonstrate disso-
ciation of a-crystallin at elevated temperatures. The char-
acter of the c(s) distribution for a-crystallin at the
temperature of 21 �C was indicative of its polydispersity, the
sedimentation coefficients for main peaks being in the range
20–25 S (inset in Fig. 6b). The c(s) distribution for a-crys-
tallin (1 mg/ml) heated for 1.5 h at 45 �C contains two peaks
with s20,w = 10.2(± 0.2) and 14.9(± 0.2) S (Fig. 9b).
Decrease in a-crystallin concentration to 0.4 mg/mL resul-
ted in further dissociation of the protein. This is evidenced by
the increase in the fraction of the peak with s20,w = 10.2 S
and transformation of the peak with s20,w = 14.9 S to two
peaks with lesser values of s20,w (14.0 and 12.9 S). Such a
stimulation of dissociation of a-crystallin at the decrease in
its concentration we have observed earlier at 48 �C [44].
3.5 Effect of GroEL on the Kinetics of Thermal
Aggregation of GAPDH
To compare the effects of a-crystallin and GroEL on
thermal aggregation of GAPDH, we have studied the
kinetics of GAPDH aggregation at 45 �C in the presence of
Fig. 6 Sedimentation behavior of GAPDH (10 mM Na-phosphate
buffer, pH 7.5, containing 10 mM NaCl) in the absence and in the
presence of a-crystallin at 21 �C. a Differential sedimentation
coefficient distribution c(s) of GAPDH, b c(s) distributions of the
mixture of GAPDH (0.4 mg/mL) and a-crystallin at the concentration
of 0.4 mg/mL (dashed curve) or 1 mg/mL (solid curve). Figure near
the curve is the value of sedimentation coefficient. Inset shows c(s)
distribution for a-crystallin (1.0 mg/mL)
18 K. A. Markossian1 et al.
123
various concentrations of GroEL. The addition of GroEL
resulted in the reduction of the initial increment in the light
scattering intensity in the course of GAPDH aggregation
(Fig. 10). Figure 11 shows the time dependences of the
hydrodynamic radius of particles obtained in the course of
aggregation of GAPDH (0.4 mg/mL or 0.5 lM) at con-
centrations of GroEL as follows: 0.15, 0.3, 0.6 and 1.2 mg/mL
(0.031, 0.062, 0.125 and 0.25 lM, respectively). The
distinctive property of these dependences is that the
hydrodynamic radius of protein aggregates remained con-
stant over certain time interval. This circumstance makes it
possible to size the initial protein aggregates. The calcu-
lations show that the average values of Rh of the initial
protein aggregates (52.5, 58.7, 57.1 and 61.9 nm for the
above concentrations of GroEL) are very close to the size
of the start aggregates registered in the absence of GroEL
(Rh,0 = 65.5 nm). Above a certain point in time (t [ t3) the
increase in the Rh value with time follows the exponential
law. Solid curves in Fig. 11a–d were calculated from Eq.
(3). The use of Eq. (3) allows estimating parameters t3 and
t2R. The values of these parameters are given in Table 2.
The t3 value (the moment of time at which the Rh value of
protein aggregates begins to increase) rises with increasing
the concentration of GroEL. The decrease in the 1/t2R value
with increasing GroEL concentration is indicative of the
diminution of the aggregation rate.
3.6 Analysis of Possibility of Interaction between
GAPDH and GroEL
To check a possibility of interaction of GAPDH and GroEL
under non-denaturing conditions, we studied the sedimen-
tation behavior of GAPDH, GroEL and their mixture at
room temperature (0.1 M NaCl, 10 mM Na-phosphate
buffer, pH 7.5; Fig. 12). The main peak in the differential
sedimentation coefficient distribution c(s) for GAPDH
[s = 7.7(± 0.5) S] in Fig. 12a) corresponds to tetramer.
The major peak in c(s) distribution for GroEL
[s = 22.4(± 0.6) S] in Fig. 12b corresponds to the homo
14-mer with molecular mass of *800 kDa. The additional
peaks with s = 5.4(± 0.5), 10.5(± 0.5) and 15.1(± 0.5) S
correspond to the dissociated forms of GroEL. On the basis
of the obtained values of sedimentation coefficients and the
values of the frictional ratio we estimated the molecular
masses for the peaks with s = 5.4 and s = 15.1 S (72 and
402 kDa). One can infer that these peaks correspond to
the monomeric and heptameric forms, respectively. The
appearance of the dissociated forms in the preparation of
GroEL is a not a surprising result, because the procedures
used for isolation and purification of GroEL can promote
the formation of GroEL monomers [12, 13]. Figure 12c
shows the sedimentation coefficients distribution c(s) for
the mixtures of GAPDH and GroEL. Analysis of c(s) dis-
tribution indicates that there is no interaction between
GAPDH and GroEL at room temperature. The major peak
with s = 7.8(± 0.3) S corresponds to the tetrameric form
of GAPDH. The peaks with s = 6.0(± 0.3), 15.0(± 0.2)
and 21.7(± 0.3) S represent the oligomeric forms of
GroEL, which are identical to the corresponding oligo-
meric forms registered for individual GroEl (Fig. 12b).
Figure 13 shows the c(s) distributions for two concen-
trations of GroEL heated for 2 h at 46 �C. When the GroEL
concentration was 1 mg/mL, c(s) distribution included three
peaks (Fig. 13a). The main peak with s20,w = 22.8(± 0.4) S
corresponds to the 14-mer of GroEL. One can assume that
the peak with s20,w = 4.0(± 0.3) S corresponds to the
unfolded form of the GroEL monomer and peak with
Fig. 7 Sedimentation profiles
of GAPDH (0.4 mg/mL) in the
absence (a) and in the presence
(b) of a-crystallin (0.4 mg/mL)
at 21 �C. The rotor speed was
44,000 rpm
Comparative Analysis of the Effects of a-Crystallin 19
123
s20,w = 6.4(± 0.3) S corresponds to aggregates formed by
this species. It is significant that the monomeric form of
GroEL is characterized by markedly lower thermostability
than the tetradecameric form [13]. The increase in the GroEL
concentration to 1.7 mg/mL (Fig. 13b) resulted in the disap-
pearance of the peaks with s20,w = 4.0 and 6.4 S. However,
the higher molecular mass species with s20,w = 10.8(± 0.6),
16.5(± 0.6 S) and 19.8(± 0.4) S appeared. It should be noted
that apart from 23.5 S-peak (14-mer) the c(s) distribution
contained large-sized particles with s20,w = 27.5((0.3) S.
This species probably corresponds to the complex of tet-
radecameric GroEL molecule with the unfolded GroEL
subunits. One can speculate that the appearance of the
peak with s20,w = 19.8 S is connected with the formation
of the complex between single-ring chaperonin and
denatured GroEL. Thus, heating of GroEL may result
in dissociation of the tetradecameric form of GroEL to
monomers, denaturation of the monomeric form and
complexation of the products of denaturation with single-
and double-rings.
Fig. 9 Sedimentation behavior of a-crystallin at 45 �C. Distributions
c(s) were obtained for a-crystallin (0.4 mg/mL (a) and 1.0 mg/mL
(b)) heated for 1.5 h at 45 �C and corrected to the standard
conditions. The rotor speed was 30,000 rpm
Fig. 8 Differential sedimentation coefficient distributions c(s, f/f0) for
GAPDH (0.4 mg/ml; a) and c(s) for the mixture of GAPDH (0.4 mg/mL)
and a-crystallin at the concentrations of 0.1, 0.4 and 1.0 mg/mL (b–d,
respectively) heated for 1.5 h at45 �C. Distributions c(s, f/f0) andc(s) were
obtained at 45 �C and corrected to the standard conditions. The rotor speed
was 30,000 rpm
20 K. A. Markossian1 et al.
123
In the case of GAPDH, heating resulted in more pro-
nounced dissociation of the initial oligomeric form into the
oligomeric forms of lesser size. Figure 14a shows the
sedimentation behavior of GAPDH (0.2 mg/mL) heated for
2 h at 46 �C before the run. The c(s) distribution contains a
single peak with s20,w = 4.6(± 0.3) S. The frictional ratio
was equal to 1.9. This peak corresponds to the dissociated
form of the tetrameric GAPDH (the dimeric form with the
changed conformation).
Figure 14b shows the sedimentation behavior of the
mixture of GAPDH (0.2 mg/mL) and GroEL (1.0 mg/mL).
There are major peaks with s20,w = 6.6 ± (0.6), s20,w =
12.0 ± (0.6) and 19.8(± 0.3) S and also several minor
peaks in the c(s) distribution. When the GroEL concen-
tration in the mixture with GAPDH was 1.7 mg/mL
(Fig. 14c), the c(s) distribution contained the peaks with
s20,w = 6.7(± 0.6), 10.3(± 0.6) and 27.3(± 0.3) S. It
should be noted that the peak corresponding to the 14-mer
GroEL is lacking in both mixtures of GAPDH with GroEL
(Fig. 14b, c; confer with Fig. 13a, b). The peak with
s20,w = 27.5 S registered in the mixture of GAPDH
(0.2 mg/mL) and GroEL (1.7 mg/mL; Fig. 14c) is proba-
bly identical to the corresponding peak in the c(s) distri-
bution obtained for GroEL (1.7 mg/m) heated for 2 h at
46 �C Fig. 13b. As assumed, this species is the complex of
the 14-mer GroEL and products of GroEL denaturation.
One can assume that the species with s20,w = 27.5 in the
mixture of GAPDH with GroEL (Fig. 14c) is not a result of
interaction of the 14-mer GroEL with denatured GAPDH.
The c(s) distribution shown in Fig. 14c does not contain the
peak corresponding to dissociated form of GAPDH
(s20,w = 4.5; Fig. 14a). Thus, denatured GAPDH becomes
in complex with the dissociated forms of GroEL and is
represented by 6.6 S-peak or 10.3-peak (Fig. 14c). It
Fig. 10 Effect of GroEL on thermal aggregation of GAPDH (0.4 mg/
mL) at 45 �C. The dependences of the light scattering intensity on
time obtained without GroEL (1) and in the presence of various
concentrations of GroEL: (2) 0.15, (3) 0.3, (4) 0.6 and (5) 1.2 mg/mL
Fig. 11 The dependences of the hydrodynamic radius of the protein
aggregates on time for aggregation of GAPHD (0.4 mg/mL) heated at
45 �C in the presence of GroEL at the following concentrations:
a 0.15, b 0.3, c 0.6 and d 1.2 mg/mL. Solid curves are calculated from
Eq. (3)
Comparative Analysis of the Effects of a-Crystallin 21
123
should be noted that the c(s) distribution for heated GroEL
contains 10.8 S-peak (Fig. 13b), whereas 6.7 S-peak is
lacking. Taking into account this result we can assume that
6.7 S-peak (Fig. 14c) corresponds to the main complex of
dissociated forms of GAPDH and GroEL. This conclusion
is supported by the observation that 6.6 S-peak is a major
Table 2 Parameters Rh,0, t3 and 1/t2R for the dependences of Rh on time for aggregation of GAPDH (0.4 mg/mL) registered at 45 �C in the
absence and in the presence of GroEL
Concentration
of GroEL (mg/mL)
Rh,0 (nm) t3 (min) 1/t2R (min-1)
0 65.5(± 1.3) 4.5(± 0.2) 0.53(± 0.06)
0.15 52.5(± 1.9) 7.7(± 0.4) 0.22(± 0.02)
0.3 58.7(± 2.3) 12.3(± 1.3) 0.132(± 0.017)
0.6 57.1(± 1.3) 49.2(± 1.5) 0.069(± 0.007)
1.2 61.9(± 1.5) 99(± 2) 0.015(± 0.001)
Equations (2) and (3) were used for calculations of parameters t3 and 1/t2R for GAPDH aggregation studied in the absence and in the presence of
GroEL, respectively
Fig. 12 Differential sedimentation coefficient distributions c(s) for
GAPDH (0.4 mg/mL; a), GroEL (1.4 mg/mL; b) and their mixture at
the same concentrations (c) obtained at 21 �C. The rotor speed was
48,000 rpm
Fig. 13 Sedimentation behavior of GroEL at 46 �C. Distributions
c(s) for GroEL at the concentrations: a 1.0 mg/mL and b 1.7 mg/ml.
The solutions of GroEL were heated for 2 h at 46 �C before the run.
The rotor speed was 44,000 rpm
22 K. A. Markossian1 et al.
123
component in the c(s) distribution of the mixture of
GAPDH (0.2 mg/mL) and GroEL (1.0 mg/mL; Fig. 13b).
The fact that the monomeric form of GroEL is character-
ized by the higher degree of exposure of the hydrophobic
clusters of the protein molecule in comparison with the
tetradecameric form [12, 13] can serve as an explanation of
the preferential binding of the denatured GAPDH with the
GroEL monomer.
4 Discussion
One of the functions of molecular chaperones is stabilization
of unfolded proteins [66]. Complexation of chaperones with
unfolded protein substrates prevents aggregation of the lat-
ter. The challenge now is to carry out a comparative analysis
of the anti-aggregating ability of chaperones of different
classes. In the present work we compared the anti-aggre-
gating ability of chaperones of two classes. One chaperone
(a-crystallin) is a representative of the family of small heat
shock proteins, and the other chaperone (GroEL) belongs to
the family of Hsp60 chaperones, also termed chaperonins.
Our experiments on thermal aggregation of GAPDH have
shown that the kinetic mechanisms of suppressive action of
a-crystallin and GroEL are similar. The exponential char-
acter of the dependence of the hydrodynamic radius of pro-
tein aggregates on time obtained in the presence of a-
crystallin and GroEL indicates that these chaperones induce
the transition of the kinetic regime of GAPDH aggregation
from the DLCA regime, wherein the rate of aggregation is
limited by diffusion of the interacting particles (the start
aggregates and aggregates of higher order) and sticking
probability for the colliding particles is equal to unity, to the
RLCA regime, wherein the sticking probability for the col-
liding particles becomes lower the unity. As for a-crystallin,
this conclusion is in agreement with the results of our pre-
vious papers [41, 42, 67]. However, there is a distinction
between a-crystallin and GroEL in their influence on the first
stage of thermal aggregation of GAPDH, namely the stage of
formation of the start aggregates. Complexation of denatured
form of GAPDH with a-crystallin blocks the formation of the
start aggregates. Sticking of the complexes denatured
GAPDH-a-crystallin proceeds in the RLCA regime. The
denatured GAPDH-a-crystallin complex is the initial parti-
cle participating in the aggregation process. When the sys-
tem contains GroEL, the start aggregates are identical in size
to those registered in the absence of GroEL. However,
incorporation of GroEL in the start aggregates results in such
alterations of their surface which diminish their sticking
probability.
Sedimentation velocity and DSC analysis show that at
the elevated temperatures a-crystallin or GroEL can
interact with the intermediates of unfolding of GAPDH.
Recent investigations show that, when interpreting the anti-
aggregating function of the chaperones, one must take into
account dissociation-association processes both for protein
substrates and chaperones. The data obtained in the present
work support the idea that the products of dissociation of
both proteins play an important role in the interactions of
these proteins at elevated temperatures. The similar results
were obtained for interaction of a-crystallin with glycogen
phosphorylase b at 48 �C [44].
Fig. 14 Sedimentation behavior of GAPDH (0.2 mg/mL) and the
mixture of GAPDH with GroEL at 46 �C. a Differential sedimenta-
tion coefficient distribution c(s) for GAPDH. b and c c(s) distributions
for the mixture of GAPDH (0.2 mg/mL) with GroEL at the
concentrations of 1.0 and 1.7 mg/mL, respectively. All samples were
heated for 2 h at 46 �C before the run. The rotor speed was
44,000 rpm
Comparative Analysis of the Effects of a-Crystallin 23
123
Scheme 1 demonstrates the interaction of a-crystallin
with the denatured form of GAPDH. In this scheme, D is
the denatured form of GAPDH, A is the start aggregate
containing n denatured GAPDH molecules, C is a-crys-
tallin and A is the start aggregate containing GroEL.
Strictly speaking, this scheme should be a complex one.
The fact that a-crystallin accelerates thermal inactivation
of GAPDH (the data of the present work) and reduces
thermostability of the enzyme (according to the DSC data
presented in our previous work [42]) is indicative of its
ability to interact with the intermediates of GAPDH
unfolding. As for GroEL, this chaperone does not affect
thermal inactivation and denaturation of GAPDH [68]. It is
interesting to note that according to the DSC data GAPDH
enhances thermostability of GroEL: in the presence of
GAPDH the position of maximum on the DSC profile for
GroEL is shifted from 60.6 to 63.6 �C (the ratio for molar
concentrations [GAPDH]:[GroEL] was varied from 1:1 to
4:1) [68]. Our results show that when GAPDH denaturation
proceeds in the presence of GroEL the latter becomes
incorporated in the start aggregates formed by the dena-
tured molecules of GAPDH. One may imply that it is not
simply complexation of GroEL with denatured GAPDH,
but incorporation of GroEL in the start aggregates, which is
responsible for stabilization of GroEL in the presence of
GAPDH. The multiple contacts of GroEL molecule with
the denatured molecules of GAPDH may be realized in the
start aggregate. It should be noted that diminution of the
size of the start aggregates, i.e., their destruction in the
presence of a-crystallin, was also observed when studying
thermal aggregation of aL-crystallin from bovine lens [41],
yeast alcohol dehydrogenase I [42], glycogen phosphory-
lase b from rabbit skeletal muscles [44, 67] and aspartate
aminotransferase from pig heart mitochondria [45]. The
fact that a-crystallin and GroEL have different effects on
thermostability of GAPDH is indicative of different char-
acter of interaction of these chaperones with the protein
substrate under study. The interaction of a-crystallin with
unfolded GAPDH blocks the formation of the start aggre-
gates. As for GroEL, the interaction of this chaperone with
unfolded GAPDH does not affect the formation of the start
aggregates. However, GroEL incorporated in the start
aggregates reduces the sticking probability for the colliding
start aggregates. Thus, one can assume that a-crystallin and
GroEL interact with different regions of unfolded GAPDH
molecule.
Acknowledgments This study was funded by the Russian Foun-
dation for Basic Research (grants 08-04-00666-a, 08-04-00231_a and
08-08-00540_a), the Program ‘‘Molecular and Cell Biology’’ of the
Presidium of the Russian Academy of Sciences and CNTP (Russia,
02.512.11.2249).
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The stage of formation of start aggregates
nD →
The stage of aggregate growth
+ → 2
. . . . . . . . . . . . .
i + jDLCAregime⎯⎯⎯→ i+j
. . . . . . . . . . . . .
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(D⋅C)i + (D⋅C)jRLCAregime⎯⎯⎯→ (D⋅C)i+j
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The stage of formation of start aggregates
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The stage of aggregate growth
+ → 2
. . . . . . . . . . . . .
i + jRLCAregime⎯⎯⎯→ i+j
. . . . . . . . . . . . .
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