Download The Purification and Characterization of the Highly Labeled

The purification and characterization of the
highly labeled protein fraction from
calf lens
Abraham Spector, Thaddeus Wandel,* and Lu-Ku Li
The highly labeled (HL) protein fraction of calf lens has been purified and characterized.
The purified protein has a specific activity eight to ten times greater than any other isolated
lens protein fraction. Amino acid analyses and immunochemical experiments indicate that it
is essentially identical to alpha crystallin. A study of the effect of temperature upon the SH
reactivity to para-hydroxymercuribenzoate reveals an identical profile for the HL protein and
the <x,-protein. However, there are a number of differences between the two proteins. Ultracentrifuge experiments demonstrate that the HL protein in contrast to alpha crystallin appears
to be physically homogeneous in both the aggregated and deaggregated state. The aggregate
molecule has an apparent molecular weight of (6.8 - 0.2) x 105 and the subunits an apparent
molecular weight of (18.0 - 0.2) x 10s. Independent determinations of the Z average molecular
weight give slightly higher values. Based on the summation of the tveights of the amino acid
residues in a subunit, a molecidar weight of 20.0 x 10s has been calculated. The ninhydrin
color yield per unit protein is 40 per cent greater with the HL protein than with alpha
crystallin. Differences in electrophoretic mobility were also observed. The relationship between
the HL protein and alpha crystallin and the basis for the observed differences between these
proteins is discussed.
I
n 1964, Spector and Kinoshita1 reported
that when calf or rabbit lenses were maintained in a tissue culture medium, a minor
protein fraction incorporated labeled amino
acids three to four times more rapidly than
any other soluble lens protein. This fraction, which was called the highly labeled
(HL) protein, represented approximately
10 per cent of the total protein but contained more than 36 per cent of the total
incorporated radioactivity. Similar labeling
patterns were obtained from whole lens,
the equatorial region, and the anterior section. Moreover, incorporation studies with
14
C-labeled neutral, acidic, and basic amino
acids gave comparable results.
Investigation of the material clearly indicated that it was not associated with the
gamma crystallin fraction. However, since
the protein was obviously impure, determination of whether it was an independent
component or related to either the alpha
or beta crystallins could not be made. In
this communication, the purification of the
HL fraction is reported. It is shown that
From the Department of Ophthalmology, College
of Physicians and Surgeons, Columbia University, New York, N. Y.
Supported by grants from the National Institute
of Neurological Diseases and Blindness and by
the John A. Hartford Foundation, Inc.
A preliminary report of this work was presented
at the Ophthalmic Biochemistry Meeting at
Woods Hole, Mass., June, 1967.
* Post-Doctoral Fellow of the National Institute of
Neurological Diseases and Blindness.
179
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Investigative Ophthalmology
April 1968
180 Spector, Wandel, and Li
this protein is very similar to alpha crystallin.
Methods and materials
Calf lenses weighing approximately 1 gram were
removed from the eye within 90 minutes after
death. The lenses were immediately placed in a
300 ml. Erlenmeyer flask containing 80 ml. of
a modified Difco T.C. 199 medium2 with 40 units
of penicillin. After the addition of 70 y-c of 1J C
histidine the preparation was incubated at 37° C.
with gentle shaking for 6 to 8 hours. Following
incubation the lenses were washed with cold
distilled water and then frozen. The frozen lenses
were separated into nuclear and peripheral regions
with a 1 cm. (I.D.) borer. The periphery accounted for 40 per cent of the total lens. Nuclear
and peripheral materials were treated in a similar
manner. Since protein isolated from the periphery
had a higher specific activity than nuclear protein,
in most experiments the peripheral fractions were
utilized. However, similar results were obtained
with either the periphery or nucleus.
The thawed periphery was homogenized in iced
distilled water to give a 20 per cent (wet weight)
solution. The insoluble material was removed by
a 20 minute centrifugation at 23,000 x g. Dialysis
of the supernantant was then conducted against
a fiftyfold excess of 0.002M PCX, pH 6.8 at 4° C.
with two changes of solution during an 18 hour
period.
DEAE-cellulose column chromatography was
carried out in the usual manner1 on a column
5 by 55 cm. with a flow rate of 1 drop per second. Aliquots of 20 ml. were collected. As much
as 3 Cm. of protein could be fractionated on a
column of this size. The protein was followed by
monitoring the 280 m/t absorption with a Beckman DU or a Zeiss PMQ III spectrophotometer.
Aliquots were taken for determination of radioactivity by techniques previously described.1
Material isolated from the DEAE-cellulose column was dialyzed against a twentyfold excess of
water at 4° C. with one change of water over
an 18 hour period and then lyophilized. The dried
protein was dissolved in no more than 1.8 ml. of
a solution containing 0.001M EDTA, 0.01M Tris,
pH 7.8, and 0.1M KC1. This preparation was then
fractionated on an agarose column (agar gel
A-5M 100 to 200 mesh, Bio-Rad Corporation, 5
by 40 cm.) with the above mentioned buffer as
eluant. Aliquots of 4 ml. were collected.
The high specific activity fractions from this
procedure were again pooled, dialyzed against a
twentyfold excess of H2O with one change over
an 18 hour period and then lyophilized. The protein was then prepared for a second pass through
the agarose column by the procedure described.
After chromatography, the fractions with constant
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specific activity (± 5 per cent) were pooled,
dialyzed, and lyophilized. All further experiments
were performed with this material.
Immunoelectrophoresis was performed on a 2
per cent agar gel with a veronal buffer pH 8.2
according to procedures described by Manski and
co-workers.3
Sedimentation velocity and equilibrium experiments were performed with a Spinco Model E
ultracentrifuge equipped with Rayleigh interference optics. Photographic plates were analyzed
with a Nikon Model 6C optical microcomparator.
The experiments were performed with and without 5M guanidine. The calculated sedimentation
coefficients were extrapolated to infinite dilution
at 20° C. to obtain S°=o, w values.
Sedimentation equilibrium experiments were
performed by two different techniques. For the
aggregated HL protein the high-speed method of
Yphantis4 was employed as well as the methods
of Van Holde and Baldwin,5 utilizing both
schlieren and Rayleigh optics.0 For the deaggregated HL protein in 5M guanidine, only the latter approach was used. All experiments were performed with liquid columns 2.0 to 2.5 mm. in
height. Initial concentrations were determined
with a synthetic boundary cell. Before sedimentation, all preparations were dialyzed against a
two hundred-fold excess volume for at least 24
hours with 3 changes of the dialysate. Dilutions
of the initial preparation to give concentrations
varying from 0.025 to 0.25 per cent were prepared with the last dialysate.
The apparent weight average molecular weight
(Mw) was determined at different positions in
the cell from the slope of log C versus r2 according to the equation,
2 RT
d In C
(1-Vp) u)2
d r2
where R is the gas constant, T the absolute temperature, C the concentration in fringes, w the
angular velocity, r the distance from axis of rotation, p the density, and V the partial specific
volume. A value of 0.73 was used for V. This
value was calculated7 from the amino acid composition of the HL protein.
Two different average molecular weights are
reported, the weight average molecular weight
Mw, and the Z average molecular M z s where
S W,M,
2 Wi
M,
=
2 Wi
MI2
2 Wi
MI
WI is the total weight of the species with molecular weight Mi.
A constant value for the slope of log C versus
Volume 7
Number 2
HL protein fraction 1S1
r2 is indicative of physical homogeneity. In such
a situation M , = M,, Mz was also determined
independently by Method 2 of Van Holde and
Baldwin5 with modification of their equation No.
46, so that
RT
r (1 - Vp)
M, =
where z = vertical deflection of the
wire image on the photographic plate
refractive index gradient) and n' = J z
b represent given positions in the cell.
tion
schlieren
(i.e., the
dr, a and
The frac-
is equal to the slope of — versus n'. Again in
this case a constant slope is indicative of physical
homogeneity. Calculations of some of the data
were carried out utilizing an IBM 7090 computer.
Amino acid analyses were carried out with a
Technicon Amino Acid Analyzer according to the
method of Spademan, Stein, and Moore.9 Tryptophane was determined independently by the spectrophotometric method of Bencze and Schmid.10
Amino groups were determined by the method
of Moore and Stein,11 Sulfhydryl groups were
2.8
determined by the para-hydroxymercuribenzoate
method of Boyer,1- as modified by Spector and
Zorn.13
Results
A typical elution pattern of protein from
the peripheries of calf lenses incubated
with 14C histidine and then fractionated on
the DEAE-cellulose column is shown in
Fig. 1. Only the relatively minor 0.05M
PO., peak, the HL material, showed a high
level of incorporation, as indicated by the
high specific activity. All the other protein
fractions incorporated much less histidine
and to about the same level of specific
activity. The first peak, the gamma crystallin fraction, has an average specific activity
of about 500 c.p.m. per absorbancy unit,
the 0.03M peak, the beta fraction, 700
c.p.m. per absorbancy unit, the 0.08M and
0.4M peaks, the alpha fractions about 600
to 800 c.p.m. per absorbancy unit and the
NaOH wash 900 c.p.m. per absorbancy
unit. In contrast to these values the 0.05M
fraction has an average specific activity of
3,200 c.p.m. per absorbancy unit. Some of
r
2.4 -
I C.RM. /ABSORBANCE
-i 5000
1/
150
200
250
300
350
FRACTIONS
Fig. 1. Fractionation of protein from calf lens periphery. The starting buffer was 0.002M P d ,
pH 6.8, followed by increasing concentrations of POt buffer. The final eluting solution was
0.5M NaOH (see Methods).
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182 Spector, Wanclel, and Li
Table I. Summary of the fractionation of
the protein from calf lens periphery on
DEAE cellulose
Specific
% of
% of
activity
recovered recovered
c.p.m./
ahsorbance counts
Fraction
absorbance
13
23
530
0.002M PO4
37
690
48
0.03M PO,
31
3,200
8
0.05M PO.,
9
0.08M PO,
5
580
2
0.04M PO,
3
770
10
0.5M NaOH
11
900
the material in this HL fraction has a specific activity more than five times greater
than that of any other component. From
Table I which summarizes these observations, it can be seen that the HL fraction
contains 8 per cent of the 280 m/x absorbance recovered from the column but
31 per cent of the recovered radioactivity.
Thus, these results confirm the earlier report.1
Although previous work has related most
of the DEAE-cellulose column fractions to
the classical alpha, beta, and gamma crystallins, no unambiguous definition of the
HL fraction could be made.14 In fact, the
earlier work suggested that the 0.05M POd
peak was composed of protein from both
the beta and alpha crystallin groups.
Examination of the individual fractions
in the HL peak indicated that the material
was indeed highly impure. A fivefold variation in specific activity was noted. Thus,
while the average specific activity of the
HL peak was 3,200 c.p.m. per absorbancy
unit, values of isolated aliquots varied
from 1,000 to 5,000 c.p.m. per absorbancy
unit. Significant variations were also found
in the 280/260 m^ absorption ratio and in
the ratio of the ninhydrin color to 280 m/x
absorbance. Further purification of the HL
protein was therefore undertaken with the
fractions of the 0.05M PO., peak containing the highest specific activity. These
aliquots represented approximately 50 per
cent of the peak material and 67 per cent
of the radioactivity and had a specific ac-
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Inocstigativc Ophthalmology
April 1968
tivity of about 4,200 c.p.m. per absorbance
unit. After dialysis and lyophilization the
material was passed through a column of
A-5 M agarose, which is capable of fractionating molecules in the molecular weight
range of 5 x 105 to 5 x 10G, the smaller
components requiring a larger volume for
elution. From the elution profile (Fig. 2)
it is apparent that the HL fraction contains a significant amount of a smaller
molecular weight component with a very
low specific activity of 600 c.p.m. per absorbancy unit. Although this material is
separated by the agarose fairly well, the
HL peak is still heterogeneous, as shown
by the variation in specific activity throughout the peak. Moreover, the heterogeneity
is found primarily in the early fractions of
the peak while the rest of the peak appears
homogeneous. Thus, the fractions of highest specific activity which also varied little
in specific activity (5,800 to 6,100 c.p.m.
per absorbancy unit) are found just after
the absorbancy curve reaches a maximum.
These fractions were isolated, concentrated and again passed through the agarose column. The results are shown in
Fig. 3. Now, only one peak with a constant
specific activity of 6,000 c.p.m. per absorbancy unit ± 2 per cent was obtained.
The 280/260 m^ ratio was 1.61 for all
fractions in the peak. Furthermore, the
ratio of the ninhydrin absorbancy to 280
m^, absorbancy which showed wide variation, in the initial preparation also had
a constant value throughout the peak.
These results suggest that a pure material has been isolated.
A summary of the over-all fractionation
procedure is shown in Table II. The per
cent recovery was calculated on the basis
of the fraction of the starting material recovered. The data indicate that about 12
mg. of material can be isolated from approximately 1.4 Gm. of protein. Recoveries
in the order of 1 per cent are usually obtained. The difference between the specific
activity of the purified HL protein and
the starting homogenate is sixfold, but
the difference is even greater when the
HL protein fraction 183
Volume 7
Number 2
6000
5000
CPM./ABSORBANCE
V
0.4
4000
£0.3
<
3000
m
a:
O
4 BSORBANCE- 280 mju
4
i 2000
1000
0.1
40
50
60
70
FRACTION
80
90
100
110
Fig. 2. First fiactionation of the HL material upon Agar Gel A-5 M column (see Methods).
0.4
Ninhydrin
Absorbance
3 ?
0.3
o
< 0.2
7000
oo
o:
O
to
co O.I
6000
Absorbance
280 m/j
20
50
30
40
FRACTION
60
70
5000
Fig. 3. Second pass of HL protein through Agar Cel A-5 M column (see Methods).
Table II. Summary of the isolation of the HL protein
Fraction
Starting material
DEAE column chromatography
0.05M PO4 fraction
High specific activity fractions
from 0.05M peak
First agarose column
Second agarose column
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% recovery of starting
material
280 mti
Activity
Specific activity
280 nip
absorbancy c.p.m. x 10-4 c. p.m. /absorba nee absorption
Activity
141
1,000
1,360
111
56.0
16.1
12.0
35.2
3,200
8.2
25.0
23.4
9.7
4,200
6,000
6,000
4.1
1.2
0.9
16.6
6.9
5.1
7.2
184 Spector, Wanclel, and Li
HL protein is compared to the other lens
proteins. Thus, the HL fraction has a
specific activity approximately eight to ten
times greater than most of the other lens
proteins.
It is difficult to quantitatively determine
how large a fraction of the total material
is represented by the HL protein. However, results such as those shown in Table
II suggest that with consideration for losses
of material caused by the procedure, approximately 2 to 3 per cent of the total
soluble lens protein is represented by the
HL protein.
To further confirm the homogeneity of
the HL protein, immunoelectrophoresis
was carried out, as illustrated in Fig. 4.
A 1 per cent solution of the HL protein
was placed in the upper well and a bovine
lens homogenate in the lower well. After
electrophoresis in 0.05M veronal acetate
buffer, ionic strength 0.05, pH 8.2 for 1.5
hours with a potential of 6 v. per centi-
Innestigativc Ophthalmology
April 1968
meter, rabbit antibovine lens serum was
added to the trough and diffusion allowed
to proceed for 2 or 3 days. A typical lens
protein pattern of many precipitin lines
was obtained in the lower section of the
plates. Only one precipitin line was produced by the HL protein. The center of
the HL precipitin arc was located on the
cathodic side of the alpha crystallm arc
as seen in the reaction with the soluble
lens proteins. The identity of HL protein
with alpha crystallm, purified by procedures previously described,13 was confirmed
in another experiment (see Fig. 5). The
HL protein was placed in the upper well
and alpha crystallin in the lower well. After
electrophoresis under similar conditions to
those described, alpha crystallin was placed
in the upper trough and rabbit antibovine
lens serum in the lower trough. The pattern in the upper section of the figure
shows a complete blending of the alpha
precipitin band with the HL protein pre-
Fi<> 4
Fig. 5
Fig. 4. Jmmunoelectrophoresis of purified HL protein and soluble proteins. The HL labeled
protein, 10 nig. per milliliter was placed in the upper well and bovine soluble lens protein in
the lower well. After electrophoresis at pH 8.2 (the positive pole is on the left side of figure)
rabbit anti-bovine lens sera was placed in the trough and diffusion allowed to proceed for
3 days (see text).
Fig. 5. Immunoelectrophoresis of HL protein and purified alpha crystallin. The HL protein
was placed in the upper well and the purified alpha crystallin in the lower well. Following
electrophoresis, purified alpha crystallin was placed in the upper trough and rabbit anti-bovine
lens sera in the lower trough (the positive pole is on the left side of figure) (see text).
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Volume 7
Number 2
HL protein fraction 185
cipitin band. No spurs were observed. Such
patterns indicate that the HL protein has
a complete identity with alpha crystallin.
However, it is interesting to note that the
HL precipitin arc corresponds to only a
section of the alpha crystallin arc. Since
comparable concentrations of both materials were utilized, this observation suggests that the HL protein is more homogeneous than the alpha crystallin. Furthermore, the position of the HL precipitin arc
in comparison to that of alpha crystallin
suggests that the HL protein is less negatively charged and therefore does not have
as great a mobility toward the positive
pole.
In order to gain further information concerning the relationship of the HL protein
and alpha crystallin, amino acid analyses
were undertaken. Following acid hydrolyses of the proteins for 24, 48, and 72
hours, the amino acid compositions were
determined and the results extrapolated to
zero time. Tryptophane was determined
independently.10 The results shown in
Table III indicate that the HL protein and
alpha crystallin are indeed almost identical.
Small variations beyond experimental error
were observed with only aspartic acid,
glutamic acid, and serine. Independent
studies utilizing para-hydroxymercuribenzoate (HMB) indicated the presence of
one SH per 20 mg., a value similar to that
obtained with alpha crystallin. Since the
molecular weight of the HL protein and
alpha crystallin subunit is approximately
2 x 10l (see ultracentrifuge results), we
can assume with reasonable certainty that
there is but one SH/subunit. Experiments
reported by Waley15 support such a value
for alpha crystallin. On this basis, an amino
acid composition can then be determined
for the subunit (see Table III). Of course,
it should be noted that such a compilation reflects the average amino acid composition of a subunit. Previous work1G"ls
indicates that there are probably two or
more different subunits in the alpha aggregate.
Recently, Spector and Zorn13 reported
that there may be three packing arrangements of the alpha crystallin subunits in
the aggregate macromolecule which can
be observed under normal environmental
conditions. This conclusion was based upon
the reactivity of the SH groups of the alpha
Table III. The amino acid composition of HL protein and alpha crystallin
Amino acid
95
34
125
105
77
59
42
60
12
54
86
30
80
48
44
80
10
6
—
-
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91
33
118
114
78
60
42
60
12
52
84
30
78
48
42
77
10
6
—
-
Residues/sub unit
HL
Alpha
cri/stallin
protein
16
6
21
18
13
10
7
10
2
9
14
5
13
8
7
13
2
15
6
20
19
13
10
7
10
2
9
14
5
13
8
7
166
13
2
1
165
20,038
19,965
i—i
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Clycine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Tryptophane
Cysteine
2 residues
2 residue weights
Residues/1,000 residues
HL
Alpha
cri/stallin
•protein
1S6 Spector, Wandel, and Li
Investigative Ophthalmology
April 1968
1.10
.7. 1-00
chO.90
£
O
5 0.80
0.70
0.60
10
20
30
40
50
60
TEMPERATURE (°C)
70
80
Fig. C. The eflect of temperature upon the reactivity of the SH groups of the HL protein to
para-hyclroxymercuribenzoate at pH 8.0.
Fig. 7. Schlieren pattern of sedimentation velocity
run of HL protein. The protein concentration was
0.95 per cent, in 0.1M Tris, pH 7.7, Speed
48,000 r.p.m., temperature 4° C, picture was
taken 24 minutes after attaining speed.
macromolecule to HMB as a function of
temperature. In the aggregate structure
many of the SH groups are masked at
room temperature but become available to
HMB in a characteristic manner as the
temperature is increased. Studies with the
HL protein gave a reactivity profile (Fig.
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6) identical to that previously reported for
one of the alpha structures, ax. With this
material more than 70 per cent of SH is
available for reaction in the 20° C. range.
No change is observed until 34° C. at
which point a linear increase in SH available to HMB occurs until 50° C. when all
SH groups have reacted. Since a greater
proportion of the SH groups are available
at room temperature in this ax form than
any other, it is assumed that the subunits
are more loosely packed in this aggregate
structure.
To further characterize the HL protein,
ultracentrifuge investigations were carried
out. Sedimentation velocity studies with
the aggregated HL protein indicate a single well-defined boundary (Fig. 7) even
after relatively long experimental periods.
Experiments were run at concentrations
between 0.13 and 1.1 per cent. After extrapolation to infinite dilution an S°20,w =
18.3 was obtained. These results indicate
that the HL protein has a Svedberg constant in the range reported for alpha crystallin. Hie schlieren pattern, however, suggests a greater homogeneity than is found
with alpha crystallin.
To determine the molecular weight and
examine the homogeneity of the preparation more critically, high speed sedimentation equilibrium experiments over a wide
HL protein fraction 187
Volume 7
Number 2
2.5
r
2.0
I .5
HL PROTEIN
in Aq.ueous Solution
o
o
_J I . 0
M W (M Z ) = 6.9xl0 5
0.5
48.8
49.0
49.5
50.0
IN CM 2
r2
50.5
Fig. 8. Sedimentation equilibrium run of HL protein. Speed 6,400 r.p.m., temperature 4° C.
protein concentration 0.05 per cent in 0.1M Tris, pH 8.0.
1.3
1.2
HL PROTEIN
in 5M Guanidine
M W ( M Z ) = I.SOxlO 4
Co=l5.33fringes
1.0
48
49
r2
50
IN CM 2
51
52
Fig. 9. Sedimentation equilibrium run of HL protein in 5M guanidine. Co = 15.33 fringes,
speed 15,000 r.p.m., in 0.1M Tris, pH 7.35, 0.1M KC1, 0.01M mercaptoethanol, 5M guanidine,
speed 15,000 r.p.m., temperature 20° C.
range of concentrations varying from 0.025
to 0.13 per cent were undertaken. A typical
run with an 0.05 per cent solution of the
HL protein is shown in Fig. 8. The linear
relationship throughout the cell between
log C and r2 is indicative of molecular
homogeneity. On the basis of a number of
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experiments, an apparent molecular weight
Mw, Mz = (6.8 ±0.2) x 10s, was obtained.
Experiments run by classical techniques'5'G
in the concentration range of 0.1 to 0.25
per cent also gave comparable results. The
Z average molecular weight, Mz, was also
determined independently by Method 2
Inoestigativc Ophthalmology
April 1968
188 Spector, Wanclel, and Li
of Van Holde and Baldwin5 with 0.1 to
0.25 per cent protein solutions. Such experiments gave an average value of (7.4 +
0.5) x 105 and again indicated no molecular
heterogeneity. In all experiments, no concentration dependence was observed. At
present, no satisfactory explanation for the
small but significant difference between
the Mw and independently determined Mz
values is available. Possibly this variation
may be due to a small amount of undetected higher molecular weight species
which would cause Mz values to be greater
than Mw values.
Since 5M guanidine has been shown to
effectively deaggregate alpha crystallin,19
the effect of this material upon the HL
protein was examined. Sedimentation velocity experiments indicated a single well-
defined boundary with an S°o0jW of 1.5.
Such results clearly indicate that the HL
protein is an aggregate of much smaller
units. To ascertain the size and homogeneity of the subunits, equilibrium experiments
were performed under classical conditions.5' G A typical weight average molecular weight experiment is shown in Fig. 9.
Since no physical heterogeneity was observed, Mw = Mz, and values of (18.0 +
0.2) x 103 were obtained from the average
of four experiments over a concentration
range of 0.125 to 0.25 per cent. Independent determination of Mz by Method 2 of
Van Holde and Baldwin5 gave a somewhat
higher value of (20.0 ±0.5) x 103. Once
again, no apparent heterogeneity was detectable. The actual computer readout of
the data from such an experiment is shown
n'X I 0 3
Fig. 10. Sedimentation equilibrium run to determine M, in 5M guanidine. Figure shows actual
computer readout of the data. Protein concentration 0.25 per cent, speed 17,000 r.p.m., buffer
0.1M Tris, pH 7.35, 0.1M KC1, 0.01M mercaptoethanol, 5M guanidine, temperature 20° C.
(see Methods).
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Volume 7
Number 2
in Fig. 10. Computer analysis of the individual points indicated no significant variation from linearity.
Discussion
It is evident from this report that the
HL protein is an alpha crystallin. This
conclusion is supported by the essentially
identical amino acid compositions, the immunochemical identity, and the similarity
in physical properties. Indeed, the experiments reported here indicate that the differences between these preparations are
minor.
Fractionation of alpha crystallin by
DEAE-cellulose chromatography in urea
suggests that this protein is composed of
two or more subunits with different amino
acid compositions.1C"18 The identity in
amino acid composition of the HL protein
and alpha crystallin would therefore indicate that the proportion of different subunits in these proteins is probably the
same. Despite the probable presence of
more than one subunit, the sedimentation
equilibrium experiments in 5M guanidine
with the HL protein indicate a homogeneity in subunit size. Calculation of the
subunit molecular weight based upon the
amino acid composition of the isolated
alpha crystallin subunit fractions obtained
from DEAE cellulose-urea experiments17
give values which only vary from 19.5 to
20.0 x 10'. Thus, in spite of the considerable difference in the amino acid composition of the subunits, their molecular weights
are very similar. These results confirm the
observation of physical homogeneity obtained by sedimentation equilibrium analyses. It should also be noted that the subunit molecular weight values obtained by
sedimentation experiments upon HL protein are very similar to the subunit molecular weights which are calculated from the
amino acid compositions of the alpha crystallin and the HL protein (see Table III).
One of the basic differences between the
two materials is the apparent physical
heterogeneity of the alpha crystallin macromolecule in contrast to the homogeneity
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HL protein fraction 189
noted for the HL protein. Previous work
with alpha crystallin10 indicated a broad
molecular spectrum ranging from 6.6 x 105
to values greater than 2 x 10G. The minimum M«- detected in the alpha preparations is about the same as the M»- of the
HL protein. Such data suggest that alpha
crystallin may be composed of a number
of macromolecular species arising from the
HL protein.
There are some other interesting differences between the HL protein and alpha
crystallin which should be noted. Although
the amino acid compositions of the two
proteins are almost identical, the HL protein gives a ninhydrin color which is about
40 per cent greater than alpha crystallin.
Since the ninhydrin color is a measure of
amino groups, this observation suggests
that the HL protein contains more free
amino groups than alpha crystallin. Furthermore, electrophoresis at pH 8.1 indicates that alpha crystallin has fewer positively charged groups than the HL protein.
This result can also be attributed to an
excess of amino groups in the HL protein.
How can these observations be reconciled
with the finding that the proteins have the
same amino acid composition? Recently,
Waley20 and Bloemendal21 have shown that
the N-terminal amino acid of the alpha
subunits does not contain a free amino
group but is acetylated. Possibly then, the
fundamental difference between the subunits of alpha crystallin and the HL protein is that the N-terminal groups of the
HL protein are not acetylated. Preliminary
experiments suggest that this may be the
case. If so, the HL protein can be looked
upon as a newly formed alpha crystallin
whose synthesis has not been completed,
i.e., the final step in the synthesis of the
protein may be the acetylation of the end
group. Since recent reports suggest that
the beta crystallins also have their N-terminal groups acetylated,20 nonacetylated precursors for these proteins may also be
present in the lens.
We express our appreciation to Dr. Julian
Manski for conducting the imniunochemical ex-
190 Spector, Wanclel, and Li
periments and to Mr. Roy Kidwell for technical
assistance.
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