The soluble proteins of the lens
Abraham Spector
A review of recent work upon lens proteins suggests that Morners concept of three soluble
lens proteins may still be tenable. This conclusion is based upon consideration of physical and
chemical studies. Ultracentrifugal investigations of alpha crystallin indicate that it is composed
of a number of different-sized aggregates of subunits held together by noncovalent forces.
Studies at various pH's and toiih a number of solvents indicate that the size of the aggregates
is dependent upon their environment. An average unit size of approximately 4 x 10* was
observed in 7M urea. The dissociation may be reversed by returning the protein preparation
to neutral aqueous conditions. Amino acid analyses of fractionated alpha crystallin aggregates
indicates that they have the same or very similar composition. Amino acid analyses of the
isolated subunits of alpha crystallin lead to a similar conclusion. Studies upon beta crystallin
also indicate that it is composed of aggregates of smaller units. The amino acid compositions
of isolated beta aggregates also appear to be very similar. The amino acid composition, N
terminal sequence, and immunochemical reactivity of the gamma crystallins reported by
Bjo'rk12 support the concept that the gamma crystallins are very closely related. Thus each
of the crystallins appears to be composed of a group of species very similar in composition.
I
t is exactly seventy years since Morner1
suggested that the lens protein is composed of three soluble fractions: alpha
crystallin, beta crystallin, and albumin
(gamma crystallin), as well as insoluble
material called the albuminoid fraction.
It is therefore appropriate to review some
aspects of the progress which has been
made in our understanding of the proteins
of the lens at this time.
Originally alpha crystallin was obtained
by isoelectric precipitation, beta crystallin
by salting-out procedures, and gamma
crystallin comprised the remaining soluble
protein.2"1 These proteins could also be dis-
tinguished by differences in size and
charge. Electrophoresis5"7 at alkaline pH's
indicated that alpha crystallin had the
greatest mobility, gamma crystallin the
slowest mobility, and beta crystallin an
intermediate mobility. Ultracentrifugal
studies of alpha crystallin suggested homogeneity and a molecular weight in the order of 1 x 10(1.s> 9 Studies with gamma
crystallin in the ultracentrifuge also suggested homogeneity and a molecular
weight of approximately 2 x 104.10'12 Ultracentrifugal analyses of beta crystallin10'1X
indicated some heterogeneity with a size
range between those of gamma and alpha
crystallins. While the relative proportions
of the crystallins appear to vary, it seemed
clear that beta crystallin represented the
major fraction of the soluble lens protein
and that the proportions of alpha and
gamma crystallin changed markedly with
species and age.
Thus in the mid 1950's the simple pic-
From the Howe Laboratory of Ophthalmology,
Harvard Medical School, and the Massachusetts
Eye and Ear Infirmary, Boston, Mass.
This investigation was supported by Grant No.
B-1900 from the National Institute of Neurological Diseases and Blindness, United States
Public Health Service.
579
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Investigative Ophthalmology
August 1965
580 Spector
ture of the soluble lens proteins first suggested by Morner still appeared acceptable. Alpha crystallin was the largest and
most acidic protein, the major protein of
the lens, beta crystallin was of intermediate size and acidity, and the gamma crystallin was the smallest and most basic
protein.
While alpha crystallin could be separated from the other lens proteins without
undue difficulty, considerable trouble was
encountered in purifying the other crystallins. To resolve these difficulties new techniques of separating protein mixtures were
employed. A surprising finding revealed
by these investigations was that instead of
the expected three crystallins being obtained, a much larger number of soluble
lens protein components were observed.13"10
Another approach to the study of lens
proteins also revealed inconsistencies with
the simple three protein concept. Ultracentrifuge studies with alpha crystallin
suggested that this protein might be heterogeneous. This suspicion arose because
of the unusually wide spectrum of molecular weights ranging from 0.80 to 3.3 x 10°
which were observed by different investigators.8' °>17-'21
Thus the suggestion of a large number
of different proteins and the indication of
size heterogeneity in alpha crystallin have
raised the question of whether Morner's
simple picture is an adequate explanation
of the state of the lens proteins.
The major objective of this paper is to
demonstrate that, despite these findings,
the soluble lens proteins are basically composed of the three crystallins as originally
proposed by Morner. This conclusion has
been arrived at by correlating the many
newly found lens protein fractions with
the classical crystallin fractions on the basis
of their chemical and physical properties.
The first problem was to obtain pure
alpha, beta, and gamma crystallins which
could then be used to identify the many
new fractions which had been demonstrated. Such a purification has recently
been accomplished by a relatively simple
procedure22 combining zinc glycinate and
isoelectric precipitation together with fractionation upon Sephadex 75.
It was then possible to correlate these
purified alpha, beta, and gamma crystallins with the newly observed fractions.22
For this purpose DEAE cellulose column
chromatography was used and by this procedure 10 to 12 fractions were obtained
from a soluble calf lens protein preparation (Fig. 1). By fractionating the purified
alpha, beta, and gamma crystallins by
DEAE cellulose chromatography it was
then possible to relate the DEAE fractions
to the classical crystallins. Thus, when the
isolated gamma crystallin was fractionated
on the DEAE cellulose column, almost all
the material was recovered in fraction 1
of the ten fractions arising from fractionation of a whole soluble lens preparation.
The purified beta crystallin corresponded
to fractions 2, 3, 4, and 5 of the fractions
obtained from the whole lens preparation
and the last group of fractions was related
to purified alpha crystallin. Since both
alpha and beta material was eluted with
0.05M PO4 (fraction 6), this fraction was
not assigned to either group. Thus almost
all the fractions obtained from a whole
soluble lens protein preparation could be
assigned to alpha, beta, or gamma crystallins.
Since the DEAE fractions could be correlated to one of the three crystallins, it
was now possible to relate the physical
and chemical characteristics of the unfractionated crystallins to their respective
DEAE fractions. Let us first consider the
question of the size heterogeneity of the
total alpha crystallin and of its DEAE
fractions.
This problem has been investigated by
means of sedimentation velocity and sedimentation equilibrium techniques.21 This
latter approach is particularly well adapted
for revealing the extent of size heterogeneity present in a particular preparation.
Fig. 2 illustrates the results obtained by
high-speed sedimentation equilibrium techniques with an alpha crystallin prepara-
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Soluble proteins of lens 581
Volume 4
Number 4
40
20
0
60
80
FRACTION
Fig. 1. Fractionation of calf lens proteins. The protein was fractionated on a 15 Cm. DEAE
cellulose column by stepwise elution. All buffers but the final one were prepared by dilution
of 0.50M (NaHJPO,, K,HPO4) pH 6.85 ± 0.05. The final buffer was 0.2M NaH2PO«, pH 6.85
0.2M NaCl. The brackets indicate tlie aliquots used for amino acid analyses.
ALPHA
CRYSTALLIN
pH 7.5
3.00
1000
M, =1.090.000
2.50
log A F
100
2 .00
50
I .50
C o =0.032 %
C
49.5
50.0
50.5
51.0
25
= 0.016 %
51.5
X2 in Cm2
Fig. 2. Molecular weight determinations of alpha crystallin by high speed sedimentation
equilibrium experiments. The experiments were performed at 5° C. in 0.15M KC1, 0.15M
Tris, pH 7.5. The abscissa represents the square of the distance from tlie center of rotation.
The right ordinate gives fringe displacement. The left ordinate gives the logarithm of tlie
fringe displacement. Similar results were obtained at initial concentrations of 0.032 and
0.016 per cent. M« is the weight average molecular weight determined from tlie slope at
the upper end of tlie cell and ML is tlie weight average molecular weight determined from
the slope at the lower end of tlie cell.
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liwcsligatioc Ophthalmology
August 1965
582 Spector
tion which appears homogeneous according to conventional ultracentrifugal and
electrophoretic investigations.21 The plot
of AF VS. x2 would give a straight line
for homogeneous material but an upward
curvature was observed indicating heterogeneity. The alpha crystallin species are
distributed so that they become progressively heavier from the upper boundary
to the lower boundary of the cell. From
these data it was apparent that an average
molecular weight distribution from 0.72
x 10° to greater than 1,1 x 10G was present
in the alpha preparation. By an extrapolation procedure the weight average molecular weight for the entire content of the cell
M\V was estimated to be 1.2 x 10°. This
suggests that the heaviest species were
considerably larger than those detected
at the base of the cell.
Thus it is apparent from the sedimentation equilibrium experiment that native
alpha crystallin does not consist of a singlesized species as had been previously assumed, but was composed of a group of
species varying considerably in size.
In view of such experiments which indicate that lens protein is made up of
many fractions, it can be argued that the
size heterogeneity of alpha crystallin is
basically a reflection of the heterogeneity
demonstrated by the fractionation of the
protein on DEAE cellulose columns. Therefore, in order to investigate this hypothesis, analyses of the two major fractions of
the alpha crystallin group, the O.OSM and
the 0.4M material, was undertaken.23
Sedimentation velocity patterns of the
O.OSM material (Fig. 3) indicate a very
considerable heterogeneity. In order to
ascertain the molecular weight distribution of the preparation, sedimentation
equilibrium experiments were performed.
These experiments indicated a very considerable size heterogeneity with weight
average molecular weights of 6 x 10s to
more than 2 x 10c being observed. Thus
the size heterogeneity of the O.OSM alpha
fraction is greater than that of the original alpha crystallin.
m
L.
0.08M
fraction
0.4M
fraction
Fig. 3. Sedimentation velocity patterns of the
O.OSM (left)- and 0.4M (right) alpha crystallin
fractions. The experiments were performed in
0.15M KC1, 0.05M Tris, pH 7.4, 10-* mercaptoethanol at 4° C , 59,780 r.p.m. The O.OSM material was studied at concentrations of 0.62 and
0.31 per cent and gave S2o, w = 23 S and
S i'of^ of 19.7 S. The 0.4M material was studied
at a concentration of 0.55 per cent and gave approximate values of S2o, w — 18.5 S and 1.5 S-
When the alpha crystallin fraction eluted
with 0.4M PO4 was examined by sedimentation velocity techniques, an unusual
schlieren pattern was obtained (Fig. 3).
The more rapidly moving peak showed
marked asymmetry and a high degree of
convective disturbance. Schachman21 has
shown that such convection results from
the slow re-equilibration of monomers and
polymers. The 0.4M material gave the
classical pattern of a polymerizing system
whose re-equilibration kinetics are very
slow. Surprisingly a slow-moving peak was
also observed.
It is clear from these experiments that
fractionation of alpha crystallin by DEAE
chromatography results in the isolation of
fractions having a much greater molecular
heterogeneity than the original material.
Thus the observed heterogeneity of alpha crystallin does not appear to be a
result of its being composed of a mixture
of DEAE cellulose separable fractions but
appears to be a characteristic of the protein system. A perhaps more surprising
observation is that alpha crystallin may
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Volume 4
Number 4
Soluble proteins of lens 5S3
be composed of an aggregating-deaggregating system. Recent work has now made
it apparent that alpha crystallin is in fact
made up of subunits which are held together by noncovalent forces. It is probably this system of aggregates of subunits
which makes alpha crystallin appear so
heterogeneous upon ultracentrifugal analyses. Let us then consider the evidence
which lends support to the subunit concept.
In recent years there has been an increasing number of observations which
suggested that alpha crystallin was composed of a number of polymers or aggregates. Dissociation of alpha crystallin into
two or three components was observed at
acid pH's by sedimentation velocity techniques.7' 20> -5 Deaggregation10'2C as well as
aggregation20 was also observed at pH's
in the range of 8 to 9.8. At both acid
and alkaline pH's the aggregation-deaggregation reaction appeared to be very
sensitive to ionic strength. Recently Spector and Katz21 have examined the effect
of a number of reagents upon the dissociation of alpha crystallin. The effect of
increasing pH upon alpha crystallin is il-
lustrated by sedimentation velocity schlieren patterns in Fig. 4. The results suggest
that with increasing pH, alpha crystallin
dissociates into progressively smaller aggregates until at pH 12.2 only one boundary with an S2Oj w of 3 S remains. When
the pH 12.2 material was readjusted with
acid to pH 8.1 and then examined in the
ultracentrifuge, a pattern similar to that
of the original material was observed.
Thus the alkaline dissociated material can
be reassociated at neutral pH, although
a greater degree of heterogeneity is apparent. Sedimentation equilibrium studies
at pH 12.8 indicated considerable heterogeneity and a weight average molecular
weight, Mw, of 6.6 x 10*, a molecular
weight about 15 times less than that of
the original preparation.
Recently it has been shown17* — that
alpha crystallin will dissociate in 7M urea.
In view of this observation Spector and
Katz21 studied the effect of urea upon alpha crystallin by sedimentation equilibrium techniques. The results are shown in
Fig. 5. It is important to note that heterogeneity was again observed with an M w
of 3.99 x 10*, for the entire contents of
¥t 1 P
PH
s 20.
PH
'20,w
L
I
II
7.4
8.1
10.4
19.0 S
19.4 S
17.6S
MM
10.9
3.1 S. 6 7 S. 13.4 S
ited
3.0 S
19 S
Fig. 4. Sedimentation velocity patterns of alpha crystallin in the presence of increasing concentrations of alkali. The last picture shows the pattern of alpha crystallin which was brought
back to pH 8.1 after being at pH 12.2. The alpha crystallin concentration was 0.55 per cent.
All runs were made at 52,640 r.p.m. at temperatures between 5.0° and 7.0° C. All pictures
were taken 20 to 60 minutes after attaining speed at schlieren diaphragm angles of 55 to
70 degrees. The pH 7.4 run was made in 0.1M Tris, all other runs were performed in 0.2M
KC1, 0.05M borate, except for the pH 12.2 ran which was made with 0.15M borate, 0.1M
KC1.
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Investigative Ophthalmology
August 1965
584 Spector
ALPHA
1 .45
~~
CRYSTALLIN
IN
n
7 M UREA
f>
1.40
1.35
Mb
=48,510
_
— 26
/
M
log
— 28
w
= 39,900
24 <O
&
—
—
C
22
/
1.30
—
— 20
1.25
i
on
Mm
1
50.0
—
•= 3C,910
I
I
I
51.0
52.0
53.0
X2 in
18
Cm2
Fig. 5. Molecular weight determination from sedimentation equilibrium experiment of alpha
crystallin in 7M urea, 0.11M Tris, 0.11M KC1, pH 7.4, 20° C , Co = 21.77 fringes. M,,, is
the weight average molecular weight determined from the initial slope. M& is the weight
average molecular weight determined from the final slope.
the cell with an average spread of molecular size from 3.09 x 10l to 4.8 x 104
being observed. When the urea was removed17- 21> 27 from the alpha crystallin,
reaggregation was found to occur.
The fact that since such mild and diverse reagents as hydrogen ion, hydroxyl
ion, and urea all cause the dissociation of
alpha crystallin clearly indicates that these
aggregates must be held together by noncovalent bonds. However, the extent of
deaggregation was dependent upon the
deaggregating agents. The dissociation by
acid and base can be attributed to the
large increase in electrostatic repulsion
between the polypeptide chains.2S As for
urea, it is still not clear how this reagent
disrupts the secondary forces which maintain molecular stability.28"31 The results
also indicate that reaggregation of the alpha crystallin will occur when the reagent
used for deaggregation is removed. Thus
deaggregation caused by changes in H+
ion concentration or urea can be reversed
by a removal of the deaggregating reagent.
However, a greater degree of size heterogeneity was observed in the reaggregated
material in all experiments.
It is now clear from these physical
studies that alpha crystallin is a heterogeneous preparation composed of a number of different-sized aggregates. These
aggregates appear to be composed of a
large number of subunits which fall apart
and recombine, depending upon their environment. The actual size of a given aggregate is dependent upon its prior history, pH, and ionic strength. At present
there is no evidence to suggest that the
ultimate subunits from which all aggregates are derived has been liberated even
in 7M urea. In fact, it is probable that
the dissociation of alpha crystallin is not
complete in 7M urea. The ultracentrifugal
experiments clearly indicated that alpha
crystallin was composed of aggregates
made up of subunits, but it did not reveal
how many subunits or whether the alpha
DEAE cellulose fractions were composed
of the same or different subunits.
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Volume 4
Number 4
Soluble proteins of lens
peak 5 of the 0.08M material, were analyzed. The column aliquots used for the
analyses are indicated above the peaks
(Fig. 6).
From Table I it is apparent that the
analyses obtained with comparable urea
peaks from the two alpha fractions are
remarkably similar. Thus with the exception of an occasional discrepancy in the
serine, threonine, and methionine results,
excellent agreement was obtained. It can,
therefore, be concluded that, on the basis
of chromatographic behavior and amino
acid analyses, at least four of the urea
peaks from the 0.08M and 0.4M fractions
are the same.
Since these alpha fractions have urea
components with similar amino acid compositions, the unfractionated 0.08M and
0.4M alpha fractions might also be expected to be similar. From Table II it
can be seen that, with the exception of
serine, excellent agreement was obtained
for all amino acids of the two fractions
which were analyzed. On the basis of this
work, it was of interest to compare the
amino acid composition of all DEAE fractions attributed to alpha crystallin. Amino
acid analysis of these fractions (Table II)
again indicates excellent agreement with
To answer these questions it was necessary to isolate the subunits of a number
of alpha fractions and analyze them for
their amino acid content. For this purpose the two major alpha fractions isolated by DEAE cellulose fractionation of
a purified alpha preparation, the 0.08M
and 0.4M fractions, were utilized.23 These
fractions comprise approximately 65 per
cent of the isolated alpha crystallin material. Since ultracentrifugal studies indicated no significant difference in the extent of deaggregation with 6M or 7M urea,
the former concentration was used in these
studies. The DEAE cellulose chromatography profiles obtained when the 0.08M
and 0.4M fractions were chromatographed
in a 6.2M urea buffer system are shown
in Fig. 6. The patterns obtained with the
two alpha fractions are remarkably similar. In the case of the 0.08M fraction, 6
components are clearly evident while the
0.4M fraction contains five definitive peaks
and a suggestion of a sixth. In order to
determine whether the comparable urea
fractionated components from the two alpha fractions are related to each other,
amino acid analyses were performed. Peaks
1, 3 and 3a combined, and 4 of the 0.08M
and the 0.4M alpha material, as well as
-1
1
H
O.O8M
FRACTION
0.3
So
0.2 -
\
\ \
0.1 -
I 1
H
A/I
i 11
N
J
4 ^J 5^"»
3 3o
20
40
585
60
0
20
40
60
FRACTION
Fig. 6. Fractionation of the 0.08M and the 0.4M alpha fractions on DEAE cellulose columns
with 6.2M urea buffers. The buffers in the order used were 0.001M Tris, pH 7.9, 0.04M,
0.05M, and 0.1M Tris, pH 7.4 and finally 0.1M borate, 0.4M KC1 pH 11.0. All buffers contained 6.2M urea. The arrows in the figure indicate where the buffers were changed.
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Investigative Ophthalmology
August 1965
586 Spector
Table I. Amino acid composition of urea-fractionated alpha crystallin fractions
Fraction (fimoles amino acid/1,000 residues)
Methionine
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
17
79
35
59
103
101
53
57
64
57
89
12
73
60
54
88
17
77
32
56
105
106
52
57
63
55
91
12
75
61
53
86
13
100
29
79
105
76
67
42
63
54
89
30
81
45
44
85
18
97
25
76
107
77
65
40
64
54
87
32
84
45
44
84
22
100
26
75
106
74
64
39
64
54
87
33
85
45
17
101
23
100
30
27
81
110
73
66
39
64
53
87
31
83
43
61
116
68
72
53
66
52
93
30
73
50
42
42
37
82
83
76
The indicated peaks obtained by urea DEAE fractionation of 0.08M and 0.4M alpha fractions were analyzed for their
amino acid composition. No values for cysteine or tryptophane are included.
the infrequent exception of two or three
amino acids. Even the material stripped
from the column with 0.2M borate at pH
12.2 gave an amino acid composition similar to that of the other alpha DEAE fractions. Thus all alpha DEAE fractions
have closely related if not similar amino
acid compositions and are probably composed of the same subunits.
Further confirmation of the great similarity between the alpha fractions was obtained by the following experiment.23 When
the 0.08M alpha fraction was deaggregated
in 7M urea and then reaggregated by the
removal of the urea, DEAE chromatography of the reconstituted material gave but
one fraction which was eluted with 0.08M
PO4. Thus the reaggregated 0.08M material behaved in similar fashion to the material from which it arose. However, if the
same experiment were performed with the
0.4M alpha fraction, approximately 70 per
cent of the reaggregated material was
eluted with 0.08M PO4. It should be noted
that rechromatography of the original 0.4M
material gave only one peak eluted again
with 0.4M PO4. This experiment clearly indicates that the 0.4M eluted material can be
transformed into the 0.08M eluted material
by a deaggregation-reaggregation process.
If the DEAE alpha fractions have similar
amino acid compositions and similar subunit
composition, what then is the difference between them? The experiment described
above suggests a possible answer. During
the experiment the deaggregated material
was dialyzed against 7M urea. Possibly
small dialyzable components are associated
Table II. Amino acid composition of alpha
crystallin fractions
Amino acid
Methionine
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Fraction (fimoles amino acid/1,000
residues)
Alkaline
0.4M
buffer
0.08M 0.1M
22
17
21
14
99
27
60
109
83
64
45
90
94
31
72
110
78
64
49
63
53
87
28
77
51
88
31
66
108
87
68
50
64
64
54
87
54
88
24
75
51
28
83
49
44
80
44
82
42
79
28
60
105
83
66
48
65
57
89
29
80
52
45
87
The fractions obtained when a purified alpha crystallin
preparation was chromatographed upon DEAE cellulose
were analyzed for their amino acid composition. Values
for cysteine and tryptophane are not included.
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Volume 4
Number 4
with alpha fractions other than the 0.08M
peak. When these components are removed, the material reverts back to the
0.08M species. It is interesting to note
that the presence of a low molecular
weight component was observed with the
0.4M alpha fraction but not with the 0.08M
fraction.
Bjork18 has reported the isolation of two
alpha DEAE fractions starting with alpha
crystallin prepared by zone electrophoresis.
These components had similar amino acid
compositions but exhibited a real difference
in free electrophoresis at pH 8.0. This difference in mobility could not be explained
by the amino acid composition of the alpha
crystallin fractions but might be explained
by the presence of a small charged dialyzable component.
These results might also be explained by
a difference in the kind or extent of aggregation of the subunits in the various fractions. This latter explanation may account
for the material eluted from the column at
high pH where dissociation of highly aggregated components will occur. While both
the 0.08 M and 0.4M fractions show considerable heterogeneity, the degree of aggregation is markedly greater in the 0.4M
material. Differences in the extent of aggregation may cause differences in the apparent charge of the macromolecule and
result in markedly different chromatographic behavior. It is interesting to note
that Bjork18 observed that the relative
proportions of his two DEAE cellulose
fractions varied with the age of the lens.
It is possible that these changes may reflect a relationship between the state of
aggregation of the alpha crystallin and the
age of the tissue from which it was isolated.
The question of how many different subunits alpha crystallin contains cannot be
definitely answered at this time. However,
from the results obtained by DEAE cellulose urea fractionation (Table I) it is clear
that there are at least three subunits represented by peak 1, peaks 3 and 4, and peak
5. Bjork27 has recently isolated seven frac-
Soluble proteins of lens 587
tions by urea-DEAE chromatography of
unfractionated alpha crystallin. Analyses of
five of these fractions gave somewhat different amino acid compositions. However,
it must be remembered that in 7M urea,
alpha crystallin still shows size heterogeneity which is probably due to the presence of a certain degree of aggregation.
This heterogeneity increases the difficulty
in resolving the problem of the number of
different subunits since it may account for
some of the observed urea-DEAE cellulose
fractions. Stronger deaggregating agents
such as guanidinium. hydrochloride may be
more effective in eliminating all aggregation of the alpha crystallin.
While there are some significant differences in amino acid composition between
the urea-DEAE cellulose fractions, it is the
similarity of these fractions which is most
striking. It is possible that alpha crystallin
is an aggregate of one unique polypeptide
chain. The differences in amino acid compositions that have been detected in the
urea-DEAE fractions may be due to alterations in the messenger RNA template
which codes for the alpha crystallin subunits. Recent observations32 suggest that
some messenger RNA's may have an exceedingly long life. Certainly the synthesis
of protein in areas of the lens which do
not contain DNA would require the presence of an abnormally stable messenger
RNA. Such an RNA molecule might be
expected to change with time, thus producing an altered alpha crystallin polypeptide. If this condition does exist in the lens,
then the degree of chemical heterogeneity
of the alpha crystallin subunits might be
expected to increase with age.
Immunochemical techniques have also
been used to ascertain the degree of heterogeneity of lens proteins. Manski, Plalbert, and Auerbach33 have observed that
alpha crystallin is composed of a number
of distinct immunochemically active components. However, it has now been shown20
that purified alpha crystallin contains but
one immunochemically active component,
although reaggregation of the individual
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Investigative Ophthalmology
Atigust 1965
588 Spector
urea DEAE fractions resulted in the formation of aggregates which gave reactions
ranging from complete to no immunochemical identity with the original alpha
crystallin. Thus it is clear that the observed
differences in amino acid composition of
the polypeptide chain or a change in the
aggregation pattern of identical subunits
is sufficient to produce immunochemically
different components. The earlier results
showing immunochemical heterogeneity of
the intact alpha crystallin may in part be
due to contamination with beta and
gamma crystallin as well as to reaggregation reactions.
Thus our present concept of alpha crystallin suggests a macromolecule made up
of approximately 35 or more subunits.
These subunits are either identical or
closely related to each other in both size
and chemical constitution and are held together by noncovalent forces. The fractionation of alpha crystallin by numerous techniques into a number of fractions is probably due either to alterations in the aggregation pattern or to the binding of small
charged molecules to the alpha macromolecule. The present view is essentially
consistent with Momer's classification of
alpha crystallin as one of the three soluble
lens proteins.
Let us now consider beta crystallin.
While relatively little work has been done
with the beta crystallin group, a pattern
similar to that for alpha crystallin is beginning to emerge. Beta crystallin appears
to be physically heterogeneous,22'34 giving a number of distinct peaks in sedimentation velocity experiments. It appears to
have a molecular weight range in between
that of gamma and alpha crystallin. That
beta crystallin is composed of noncovalently linked polymers is apparent since
deaggregation to subunits comparable in
size to the alpha subunits occurs in 6M
and 7M urea.17'22> 35 Reaggregation of the
beta subunits has also been observed by
Bloemendal and co-workers.17
As mentioned earlier in this paper it is
now possible to define which of the DEAE
Table III. Amino acid composition of beta
crystallin fractions
Amino acid
Methionine
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Fraction (fimoles
amino acid/1 000 residues)
0.015M
0.03M
Peak
Peak
Peak
Peak
1
2
2
1
31
22
28
20
98
99
100
108
30
29
28
29
53
60
51
50
166
170
153
169
62
59
62
56
112
108
106
103
64
59
63
55
64
61
63
61
33
38
34
43
57
55
57
60
41
40
45
52
46
50
46
44
42
44
47
47
36
37
37
35
72
70
71
72
The beta crystallin fractions isolated by DEAE column
chromatography were analyzed for their amiino acid
composition. Values for cysteine and tryptophane are not
included.
cellulose fractions obtained from the fractionation of soluble lens protein are beta
crystallin fractions (Fig. 1). Recently the
amino acid compositions of these DEAEcellulose beta fractions have been determined30 (Table III). The second 0.015M
peak and the two 0.03M peaks give very
similar amino compositions with the exception of major differences in methionine
and occasional minor differences in a few
of the other amino acids. As for the first
0.015M peak, even though this material is
slightly contaminated with gamma crystallin,22 an amino acid composition not
very different from the other beta fractions
was found. The similarity in the amino
acid composition of the beta DEAE column fractions suggests that the same subunits are involved in all the aggregated
beta fractions. The differences between
the beta DEAE fractions may be due to
-factors similar to those involved in the differentiation of the alpha fractions. Thus, as
with alpha crystallin, beta crystallin can
be considered to be basically a single protein entity as proposed by Morner.
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Volume 4
Number 4
Soluble proteins of lens 589
Table IV. Amino acid composition of
gamma protein fractions
Fraction (pinoles per 1,000 ftmoles)
Amino acid
Alanine
Arginine
Aspartic
acid
Glutamic
acid
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Tyrosine
Proline
Serine
Threonine
Valine
Total
gamma
nib
13
125
112
Ilia
27
131
107
114
IVb
19
127
116
115
125
121
116
131
87
83
49
45
94
39
49
90
48
93
37
42
13
67
6
98
13
72
6
44
54
25
55
25
31
25
57
92
93
50
85
18
37
99
50
73
12
35
95
47
81
31
35
11
32
37
79
58
70
28
37
19
122
37
32
111
116
81
24
37
48
76
58
42
22
48
The amino acid compositions obtained by Bjork12 for his
gamma crystallin fractions have been recalculated and are
compared to a total gamma
preparation isolated by DEAE
column chromatograp'hy.30 Values for cysteine and tryptophane are not included.
Relatively few investigations of gamma
crystallin have been reported. Ultracentrifugal studies indicated homogeneity and
an M w of approximately 2 x 104.10"12'10 No
evidence that gamma crystallin is composed of aggregates has been observed.22
While gamma crystallin is almost completely eluted as a single fraction from
DEAE cellulose (Fig. 1), it can be separated into a number of fractions on carboxymethyl cellulose.80 Recently Bjb'rk has
fractionated gamma crystallin into four
major fractions upon sulfoethyl Sephadex.
Amino acid analyses of some of the fractions gave somewhat similar results (Table
IV), although differences in a few amino
acids were noted. While the amino acid
analyses of the gamma fractions appear to
vary more than those of the alpha and beta
fractions, other observations support the
conclusion that the gamma crystallin fractions are closely related. Thus the N terminal amino acid sequence was found to be
NHa-grycine-glutamic acid-(leucine or isoleucine) for all four gamma fractions investigated.12 Furthermore, all fractions but
one gave reactions of complete immunochemical identity with each other and with
unfractionated gamma crystallin.
It is interesting to find that although
the alpha crystallin fractions are perhaps
more closely related to each other in amino
acid composition than those of the gamma
fractions, immunochemically only partial
identity was obtained with the alpha fractions while complete identity was obtained
with the gamma fractions. Of course the
finding of similar amino acid compositions
for a number of proteins only suggests that
the amino acid sequences of the proteins
are similar. It may be that there is a somewhat greater divergence in the amino acid
sequences of the alpha fractions. However,
the investigations of alpha crystallin clearly
show that the lack of immunochemical
similarity is primarily due to differences
in the conformation and spatial relationship of the aggregates. Thus, dissociated
and reaggregated alpha crystallin in some
instances gave partial or no immunochemical identity with the original alpha crystallin. This clearly indicates that changes
in the conformation of the antigenic sites
grossly alter the immunochemical properties of the protein. Great care must therefore be exercised in the interpretation of
immunochemical experiments.
On the basis of these investigations,
gamma crystallin appears to consist basically of a single polypeptide chain with a
molecular weight of approximately 2 x 10*.
The small differences in the amino acid
composition which have been noted may
be due to degeneration in the coding system for the polypeptide as has already
been suggested for beta and alpha crystallins.
The amino acid compositions of the
three crystallins are markedly different as
indicated in Table V. (Note that the values for gamma crystallin are based on
analyses of unfractionated gamma crystallin30 isolated by DEAE chromatography
[see Table IV]). These differences in composition suggest that the subunits which
make up the alpha and beta aggregates
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590 Spector
Investigative Ophthalmology
August 1965
Table V. Comparison of the amino acid
compositions of the soluble lens proteins
Amino acid
Aspartic acid and
•glutamic acid
Arginine and lysine
Isoleucine, leucine
and valine
Phenylalanine and
tyrosine
Proline
Clycine and alanine
Serine and threonine
Histidine
Crystallin fraction
(nmoles per 1,000 nmoles)
Alpha \ Beta \ Gamma
201
267
247
servations. Thus, on the seventieth anniversary of Momer's epic observations of
the lens, there is sufficient evidence to believe that his three protein concept is still
acceptable.
I should like to express my appreciation to Dr.
J. H. Kinoshita for reading the manuscript and
making some suggestions which have improved
the clarity of the presentation.
134
207
114
154
135
171
106
89
125
Addendum
83
114
95
44
61
171
83
37
58
125
65
37
Recent experiments in 5M guanidine hydrochloride indicate that the average-sized alpha
unit in this medium is approximately 2 x 104.
Thus an average of fifty such units is necessary
to form an average-sized alpha aggregate.
This summary is based upon the average amino acid
values for alpha and beta crystallin and the analysis of
the gamma crystallin isolated by DEAE cellulose chromatography.
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gamma crystallin. The question of how
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chain occurs independently of the template. Such a viewpoint suggests that the
amino acid sequence is the primary factor
in directing the aggregation reactions. If
alpha aggregates contain different subunits
than the beta aggregates, the respective
subunits must either be able to exclude
each other as well as the gamma crystallin
or they must be aggregated in the absence
of the other species. A recent experiment30
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units are excluded during the reaggregation of alpha and beta crystallin subunits.
The situation with respect to the exclusion
of alpha and beta subunits from the aggregates of each other is still obscure.
From the evidence presented in this
paper it appears likely that there are only
three basically different proteins comprising the structural soluble protein of the
lens. This conclusion is based upon a consideration of size, amino acid composition,
behavior of aggregates and subunits upon
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