(Received for publication, April 27, 1995; and in revised form, August 22, 1995)
From the
Transparency of the lens of the eye is the result of a short
range order in the packing of crystallin molecules within the fiber
cells. Short range order depends on crystallin-crystallin as well as
water-crystallin interactions. Light scattering measurements can
provide information on the hydration of crystallins. Light scattering
intensities were obtained as a function of scattering angle,
concentration, and temperature on dilute solutions of
,
, and
fractions of bovine
lens crystallins. The temperature dependence of the second virial
coefficient was negative for the
crystallin fractions and
positive for the
fraction as well as that for
crystallin
(Wang, X., and Bettelheim, F. A.(1989) Proteins Struct. Funct.
Genet. 5, 166-169). The partial molar enthalpy values of the
solutions were negative for the
crystallin fractions, indicating
a tendency for homo- and heterodimer and -oligomer association. The
enthalpy values were positive for the
and
fractions. The
negative values of the enthalpy of solutions differentiate the
crystallins from the other crystallins. The partial molar entropy
values of solutions of
and
fractions were
identical, those of the oligomeric
fraction were
higher, whereas those of
crystallin were a magnitude larger than
those of the the smaller crystallin molecules.
,
, and
crystallins are the major structural
proteins in mammalian lenses. Their function is to provide a
transparent assembly with a refractive index gradient that is capable
of focusing light onto the retina(1, 2, 3) .
crystallins are heteropolymers with a molecular weight of about
600,000-1,000,000(4, 5, 6) .
crystallins, on the other hand, are a family of compact monomers with a
molecular weight of 20,000 having two similar globular
domains(7, 8, 9, 10) . They are
located mostly in the regions of the lens with the highest refractive
index (11) .
crystallins are of intermediate size between
and
crystallins forming oligomers from a variety of
subunits(12, 13, 14) .
Thus, the lens
crystallins can be subdivided into heterogeneous groups on the basis of
their apparent size. The largest group is that of crystallin,
which is made of aggregates of two subunits
A and
B (each of
them with a mass of about 20 kDa). These subunits are individual gene
products exhibiting a very low rate of evolutionary
change(15, 16) . They exist as polydisperse high
molecular weight aggregates.
The crystallins are also
aggregates of many subunits with extensive
polydispersity(12, 13) . Sequencing studies showed
that bovine
crystallins have three acidic (
A2,
A3/A1,
and
A4) and three basic (
B1,
B2, and
B3) subunits
with apparent mass of 23, 25, and 23 kDa and 32, 26, and 27 kDa,
respectively(14) . Bovine
crystallin with
apparent molecular mass of 160-200 kDa contains all the
subunits in different combinations, whereas the
crystallin fraction with apparent molecular mass of 46-70
kDa lacks
B1 subunits(14) . The subunits form homo- and
heterodimers and -oligomers. The structure of the
B2 homodimer
have been studied extensively(17, 18) . Each domain is
formed from two ``Greek key'' motifs, and the connecting
peptide is extended. The secondary structure is made of
sheets(18) . The dimer stability is provided by the
intersubunit
sheet interfaces and the C-terminal extension in
B2 dimerization (18) and the N-terminal extension in the
A3 dimerization(19) .
The crystallin fraction
contains the monomeric
crystallins (7, 8, 9, 10) and also monomeric
s crystallin, which in contrast to the other
crystallins is
completely denatured in 8 M urea at room
temperature(20) . These have a mass of approximately 20 kDa.
The three-dimensional structures of
crystallins have been
elucidated from x-ray diffraction data for
B (7, 10) ,
C(8) , and
E (9) crystallins. Each is made of a two domain structure in
which each domain has two Greek key motifs.
These crystallins are
the products of different genes, although and
crystallins
may have had a common ancestral gene. The various crystallin families
and their individual members are differentially expressed during
development (15) leading to different mixtures of crystallins
along the optical axis. The particular packing of these crystallins
depends on the size as well as on the interaction of the crystallins
with themselves and with the aqueous surrounding. The combination of
these effects results in a protein gradient along the optic axis, and
because each crystallin family has its individual refractive index
contribution(21) , it also results in a refractive index
gradient.
In addition to the structural features of crystallins, their solvation properties and their specific interactions in homo- and heteroaggregations are of importance(22, 23, 24, 25, 26) . The transparency of the lens depends on its hydration(2, 3) . The hydration of the lens is a complex phenomenon(27, 28, 29, 30) . It involves interaction of the crystallins with water, protein-protein interactions, crystallin distribution, and gradient in the lens. The hydration of the lens can be better understood if the role of the individual factors in the hydration process are known. The present study was designed to probe the stability of the crystallins in aqueous solutions by calculating the thermodynamic parameters of solution from light scattering measurements. These parameters are important in assessing the interaction of water with single crystallin molecules, one aspect of the total hydration process.
In Fig. 1typical light scattering data are presented
in the form of a Zimm plot(33) . The sample in this case was
crystallin at 19.0 °C. This
crystallin was concentrated at room temperature under a vacuum to
a 5% concentration and sequentially diluted for a light scattering
study. Another
fraction was lyophylized and dissolved
in buffer. It gave a Zimm plot with identical parameters, indicating
the fact that
does not undergo low temperature
denaturation(31) . In the Zimm plot the light scattering
intensity is presented in the y axis in the form of c/R`
, where c is the concentration
of the protein in g/cm
and R`
is
the Rayleigh ratio for unpolarized light (34) given by the
equation
Figure 1:
Zimm plot of light scattering
measurements on crystallin at 19.0
°C.
where is the scattering angle, I
is the intensity of the scattered beam at angle
, I
is the intensity of the incident beam (at a
scattering angle of 0), and r is the distance between the
scattering volume of the sample and the detector in cm. The x axis contains both concentration and scattering angle. K` is an arbitrary constant (100 in Fig. 1) to spread the
diagram, giving about equal weight to the concentration and to the
scattering angle. The intercept of the two extrapolated lines with the y axis is inversely related to the M
,
weight average molecular weight of the sample; the slope of the 0
concentration line is related to the R
, radius of
gyration, a size parameter. Finally the slope of the 0 scattering angle
line yields the second virial coefficient from which the enthalpy and
entropy of solution of the crystallins can be calculated.
The weight
average molecular weights calculated from the intercept of the Zimm
plots were as follows: 200,000 for , 81,200 for
(indicating mostly tetrameric form), and 19,800 for
crystallin. These are in agreement with literature
values(7, 14) .
The second virial coefficients
obtained from the Zimm plots are presented in Fig. 2as a
function of the reciprocal temperature. The straight line plot
corresponds to the prediction of Flory's theory(35) . It
is interesting to note that in both fractions the second virial
coefficient decreases with temperature while in
crystallin(32) , and in
crystallin it increases with
temperature.
Figure 2:
Second virial coefficients of (
),
(
), and
(
)
crystallins as a function of temperature.
The second virial coefficient A is
expressed as
where V is the partial specific volume, V
is the molar volume of the solvent, N
is Avogadro's number, and
is an interaction parameter related to the partial molar free
energy of the solution. The
interaction parameter
has enthalpy (
) and entropy (
)
contributions(34, 35) .
where T is the absolute temperature and is the Flory temperature; at this temperature the interaction
parameter becomes ½, and the second virial coefficient equals
zero. A plot of A
against [1/T]
such as in Fig. 2yields the entropy parameter as an intercept
and the enthalpy parameters and Flory's temperature as the slope.
From these the partial molar enthalpy (
H) and the partial
molar entropy (
S) can be calculated.
where R is the gas constant and V is the volume fraction of the solute.
The partial molar
enthalpies of solution for ,
,
,
and
crystallins are presented in Table 1as a function of
concentration of crystallins. The
H of
crystallin
is taken from a previous publication (32) and is presented for
comparison. Both
and
crystallins possess positive enthalpy
of solution (endothermic), whereas the
crystallin fractions have
small negative enthalpy of solutions (exothermic).
The partial molar
entropy values of solutions of ,
,
, and
crystallins are also given in Table 1as a function of crystallin concentrations. It is
interesting to note that
and
fractions have
identical partial molar entropies of solutions.
The present study was intended to illuminate the interaction
of crystallins with the aqueous environment. We calculate the partial
molar enthalpy and entropy of solutions from the temperature dependence
of the second virial coefficients of light scattering measurements.
These are extrapolated values, and as such the numerical values of A had a standard deviation of ±2.5%, and
the temperature dependence of A
is statistically
significant at the 90% confidence level. Thus, it is better to focus on
the relative trends shown by the different crystallins than on the
absolute values of partial molar enthalpies and entropies.
The
strong concentration dependence of the partial molar enthalpy of
crystallin indicates polydispersity. The partial molar enthalpy
represents the energy expended when 1 mol of crystallin is dissolved in
a solution of specified concentration having infinitely large volume. A
positive
H value means that in order to solvate the
crystallin, to surround it with a bound water layer, energy input is
necessary. This is what happens in
and
crystallins. The
crystallin fractions have small but negative partial molar
enthalpy values. This implies that the water-
crystallin
interaction is less energetic than the average of water-water and
crystallin-crystallin interaction. This indicates a tendency for
dimeric and oligomeric aggregation.
The partial molar entropy values
are positive for all crystallin fractions. Thus the randomness on the
dispersion of a crystallin in water and the gain in segment mobility of
the polypeptide chain upon solvation are greater than the contribution
of water immobilization in the bound solvation layer. The entropy of
solution values follow the molecular size of the crystallins.
crystallin has entropy values 1 order of magnitude greater than the
other groups. The monomeric
fraction and the mainly tetrameric
fraction have identical partial molar entropy of
solutions indicating the compact nature of these proteins. The
fraction that is largely made of oligomeric
aggregates of subunits has larger albeit the same order of magnitude of
partial molar entropy values as those of the smaller molecular weight
compounds.
The combination of enthalpy and entropy of solution may
explain the behavior of the different crystallins in immobilizing water
in their solvation layer. The crystallin has greater
bound (nonfreezable) water content than
crystallin(24) .
Even though the mass of
is three to five times
smaller than that of
crystallin and thus does not gain that much
in segment mobility in solubilization, it still binds more water than
crystallin. The negative enthalpy of solution implies strong
solute-solute interactions. A strong attraction, possibly among the
Greek motifs, would trap more water in the bound form around the
extended connecting peptides. The hydration of
crystallin is
largely entropy driven. The
and
crystallins
immobilize water to about the same degree in their solvation layer (26) in the form of nonfreezable water. However, when the same
tendency is probed by water vapor sorption, the
fraction has much
less bound water in the solvation layer(24) . This may be the
result of the negative enthalpy of solution of the
crystallin, which enables strong interactions in the dimerization
trapping more water around the dimer than around the monomeric
crystallins.
Bovine lens uses three different families of crystallins to
build a protein concentration gradient along the optic axis. For
transparency and hence for optimal packing all three crystallins are
necessary. The main finding of this study was that the slope of the
temperature-dependent second virial coefficients and hence the enthalpy
of hydration of crystallins are negative, whereas that of
and
crystallins are positive. Thus, one could propose that
although the
crystallins by their size may provide an optimal
packing, the
crystallins by their tendency of strong
intermolecular association and water immobilization also enhance close
packing.
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