Studies on lens proteins. I.
Subunit structure of beta crystallins
of rabbit lens cortex
M. K. Mostafapour and V. N. Reddy
A method has been developed to isolate and characterize (3-crystallins of rabbit lens cortex.
Chromato graphic separation of water-soluble structural proteins of rabbit lens cortex on a
Sephacryl S-200 gel column yielded four (3-crystallin peaks ((3h /32, /33 and jSj, all elating
between a- and y-crystallins. Their molecular weights were estimated to be 250,000, 130,000,
60,000, and 37,000 daltons, respectively. SDS-gradient gel electrophoresis of these
(3-crystallins gave rise to characteristic polypeptides; (3h two polypeptides of 30,000 and 23,000
daltons; fi2, one major polypeptide of 33,000; /33, two polypeptides of 28,000 and 26,000; and
(34, two polypeptides of 22,500 and 11,200 daltons. From a knotoledge of the molecular weights
and the ratio of the polypeptides in each crystallin, their oligomeric structure was calculated to
be 5:5, 4, 1:1, and 1:1. The relative abundance of these four (3-crystallins was found, to be
25.6%, 7.2%, 27.7%, and 2.8% of the total water-soluble proteins of the lens cortex.
Key words: Sephacryl S-200, water-soluble lens proteins, /3-crystallins,
column chromatography, SDS-gradient acrylamide electrophoresis,
subunit, native protein, molecular weight, oligomeric
structure, rabbit lens cortex
ie soluble structural proteins of the eye
lens have been classically divided into three
groups of a-, /3-, and y-crystallins.1' 2 With
the advent of better separation techniques a
steady progress has been achieved in isolation and characterization of these proteins.
The a- and y-crystallins have been purified
and their amino acid composition and sequences determined. 3 " 12 But, despite a
considerable number of attempts, the /3crystallins, which constitute the major portion of the lens crystallins, have been more
From the Institute of Biological Sciences, Oakland University, Rochester, Mich.
This study was supported by National Institutes of
Health grants EY-00484, EY-02027 and EY-07044.
Submitted for publication March 20, 1978.
Reprint requests: Dr. V. N. Reddy, Institute of Biological Sciences, Oakland University, Rochester, Mich.
48063.
660
difficult to purify and characterize. 13 16 Reports on better separation of /3-crystallins
have appeared in the literature only in the
last few years. 17 " 21 The Bloemendal group
has fractionated the /3-crystallins of the calf
lens into a heavy (^H) and a light (^L) protein.
The component polypeptides of each of these
two proteins have been resolved electrophoretically and shown to contain a number of
polypeptide chains in common among the
two proteins. 17 ' 21
We wish to report here on our attempts to
fractionate the water-soluble structural proteins of the rabbit lens cortex and to determine the constituent polypeptides of the
major /3-cry stall ins. Our results indicate that
there are a minimum of four major native
/3-crystallins, each one composed of unique
polypeptides. The molecular weights of these
proteins, their polypeptide subunits, and
0146-0404/78/0717-0660$00.70/0 © 1978 Assoc. for Res. in Vis. and Ophthal., Inc.
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Volume 17
Number 7
Beta crystallins of rabbit lens
20
30
40
so
60
70
80
661
90
Fig. 1. Elution profile of water-soluble proteins of rabbit lens cortex. Proteins were separated
by descending chromatography on a Sephacryl S-200 column. Column size was 3.3 by 101 cm.
Chromatography was performed at 4° C with a constant pressure head of 90 cm, and fractions
of 8 to 8.5 ml were collected.
oligomeric (native) structures have been determined.
Materials and methods
Sephacryl S-200 superfine and blue dextran
were purchased from Pharmacia Fine Chemicals,
Piscataway, N. J. All marker proteins were obtained from Worthington Biochemical Corp.,
Freehold, N. J., or through Sigma Chemical Co.,
St. Louis, Mo. Electrophoretic grade sodium
dodecyl sulfate (SDS), Tris, glycine, acrylamide,
/tts-acrylamide, and TEMED were obtained from
Bio-Rad Laboratories, Richmond, Calif. All other
chemicals used were reagent-grade stock material.
Preparation of water-soluble proteins of lens
cortex. Either fresh or frozen lenses obtained from
young rabbits were decapsulated and weighed,
and the cortex was solubilized by stirring the
lenses in a hypo tonic buffer made up of 0.05M Tris
HC1, pH 7.0, containing 0.1 mM EDTA and 1
mM dithiothreitol. Four lenses were placed in 2
ml of this buffer and stirred on a magnetic stirrer,
in the cold, for about 30 min, at which time the
cortical fibers separated from the clear lens nuclei.
The nuclei were removed, and the cortical material was further stirred for up to 4 hr. This was
centrifuged at 30,000 x g for 30 min. The clear
supernatant was removed and used as water-soluble proteins of lens cortex. Protein concentration
was determined by the Bio-Rad protein assay solution.
Table I. Molecular weights of crystallins
of rabbit lens cortex*
Peak
I
II
III
IV
V
VI
VII
Crystallin
a
/3,
&
ft
ft
y
—
Molecular weight
(daltons)
> 400,000
250,000
130,000
65,000
37,000
22,000
13,000
*Molecular weights were determined on a column of Sephacryl
S-200 superfine.
Chromatographic separation. Sephacryl S-200
superfine was packed in a column with an I.D. of
3.3 cm to a height of 101 cm and equilibrated with
the elution buffer consisting of 0.05M Tris HC1,
pH 7.0, containing 0.1 mM EDTA, 1 mM dithiothreitol, and 0.2M NaCl.
Routinely about 100 to 150 mg of the soluble
protein solution were loaded on top of the column
and eluted at a constant pressure head of 90 cm by
gravity flow (descending elution); 8 to 8.5 ml fractions were collected. The elution profile was
monitored at 280 nm either automatically with an
ISCO monitor (Instrumentation Specialties Co.,
Lincoln, Nebr.) or by manual absorbance measurement on a Gilford spectrophotonieter (Gilford
Instrument Laboratories, Inc., Oberlin, Ohio), or
both.
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Invest. Ophthalmol. Visual Sci.
July 1978
662 Mostafapour and Reddy
10
20
30
40
SO
60
70
80
90
Fig. 2. Elution profiles of rechromatographed proteins shown in Fig. 1. Peak fractions of/3- and
•y-crystallins were concentrated and rechromatographed individually under the same conditions (solid lines). Dotted line shows the contour of the composite profile which is similar to
that shown in Fig. 1.
CATAIASE
CREATING
KINASE
HEMOGLOBIN
E LUTION
VOLUME
(ml)
Fig. 3. Calibration curve for the determination of
molecular weights. Ten milligram quantities of
proteins with known molecular weights were
applied to the Sephaciyl column and their elution
volumes determined. Void volume was measured
by determining the exclusion volume of blue dextran.
For molecular weight determinations 10 mg
quantities of catalase, aldolase, creatine kinase,
bovine serum albumin, pepsin, chymotrypsinogen, hemoglobin, myoglobin, lysozyme, and cytochrome C were used as markers, either separately
or in groups. The void volume of the column was
established with blue dextran.
SDS-polyacrylamide gel electrophoresis. Stock
solutions of SDS, acrylamide, and buffers were
prepared according to the method described by
Laemmli.22 A 5% to 20% gradient slab gel of 1.5
mm thickness was poured and stacked with a 5%
gel. The gel was p re run for about 1 hr before
sample application.
The samples were prepared by precipitating a
known amount of protein with cold 10% trichloraceticacid(TCA). After centrifugation at 4000 X g
for 15 min the precipitate was washed twice by
resuspension and centrifugation in ethylalcohol : acetone (1:1).
The final pellet was dissolved in an appropriate
volume of the sample buffer (1% SDS, 10% sucrose, 1% glycerol, 20 mM dithiothreitol in 0.05M
Tris, pH 6.8) to yield afinalprotein concentration
of 5 to 20 ing/ml. This solution was then kept in a
water bath at 100° C for 2 to 3 min to dissociate the
proteins into their polypeptide constituents. After
addition of a drop of bromophenol blue solution
(0.05% in 0.05M Tris, pH 6.8), about 5 to 10 /ul of
the sample were underlayed on each slot on the
gel. Constant current of 10 mA/gel was applied.
Electrophoresis was terminated when the indi-
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Volume 17
Number 7
Beta crystallins of rabbit lens
663
4a
Fig. 4. Subunit composition of the crystallins of rabbit lens cortex. Crystallins isolated by
column chromatography were dissociated into their constituent polypeptides and subjected to
electrophoresis on SDS-acry!amide gradient slab gel (5% to 20%). The polypeptide subunits of
total water-soluble crystallins of the rabbit lens cortex are marked "T."
cator dye reached to about 5 mm from the lower
(anodic) end of the gel. Gels were stained for at
least 2 hr in 0.2% Coomassie brilliant blue R 250
in H2O :methanol: acetic acid (46:46:8). Destaining was carried out both electrophoretically and by
repeated changes of a 7.5% acetic acid-5% methanol destaining solution.
Densitometric measurements were performed
with a Gilford spectrophotometer equipped with a
linear gel transport system. Scanning was done at
600 nm wavelength. The percentage of each constituent polypeptide was calculated from the peak
height ratios.
Table II. Molecular weight and percent
abundance of polypeptide chains present in
the total water-soluble proteins of rabbit
lens cortex*
Polypeptide
Molecular weight
(daltons)
jS 4 a
a*.
An
fiu\
£a»
Results
Fig. 1 shows the elution profile of the
soluble structural proteins of lens cortex
separated on the Sephacryl S-200 superfine
column at a pH of 7.0 and 0.2M salt concentration. The peaks are numbered by Roman
numerals I through VII. As shown in Table I
and referred to in the text, peak I is acrystallin, and peaks II to V are identified as
/3,- to j34-crystallins; in the order of their
emergence from the column. Peak VI is
y-crystallin, and peak VII appears to be non-
fit
(%)
1.4
11,200
19,500
20,000
22,500
22,500
23,000
26,000
28,000
30,000
33,000
Density
8.5
16.2
10.9
1,4
12.8
13,9
13.8
12.8
•
7,2
T h e polypeptides were separated on SDS-acrylamide gel slabs
and their relative amounts determined by densitometry.
protein in nature. The width of most of the
peaks at the base causes an overlap with the
adjacent peaks, and it is for this reason that
the baseline absorbance does not reach to
zero in between the peaks. In order to obtain
a cleaner separation, main fractions from
each peak were concentrated by precipitation
with ammonium sulfate (90% saturation) and
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Invest. Ophthalmol. Visual Sci.
July 1978
664 Mostafapour and Recldy
Table i n . Oligomeric structure of /3-crystallins of rabbit lens cortex*
Cnjstallin
a
&
ft
y
Native protein
(mol. wt. X 10~3)
(chitons)
>400
250
130
65
37
22
Polypeptide chain
(mol. tot. X 10~3)
(daltons)
Ratio of
polypeptides
Oligomeric
structure
20, 22.5
3:2
?
30, 23
33
28, 26
22.5, 11.2
1:1
5:5
4
1:1
1:1
1:1
1:1
1
19.5
*The structure was deduced from the molecular weights of native proteins, the subunit composition, and the relative abundance of the
subunits in each crystallin.
reapplied in a volume of 2 ml to the column.
Fig. 2 is a composite elution profile of the
proteins chromatographed separately under
identical conditions. It can be visualized (dotted line) that the contour of these profiles
closely resembles the elution pattern of
Fig. 1.
Fig. 3 demonstrates the useful range of the
Sephacryl column for the determination of
molecular weights of proteins. Although the
middle part of the curve fits a straight line, it
falls off rapidly on both ends, thus making the
determination of molecular weights above
200,000 and below 15,000 less accurate.
Table I shows the molecular weight of native soluble proteins of lens cortex as determined on Sephacryl column. The values for
/3-crystallins, except (3U fall within the useful
range of the curve. The molecular weight of
a-crystallin, which elutes at the void volume,
cannot be determined.
In order to determine the constituent
polypeptides of each peak, the peak fractions
of each native protein eluting from the Sephacryl column (Fig. 1) were processed for gel
electrophoresis as described under Materials
and methods. Fig. 4 shows the polypeptide
composition of each of the six protein peaks
separated on the Sephacryl column. The protein in peak I dissociates into the classic A
and B bands of a-crystallin. Peak II shows
one major and at least one minor band. Peak
III shows one heavily stained band (which is
also the heaviest polypeptide among all the
/8-crystallin polypeptide chains) and two
minor bands. The two minor bands appear to
be contaminants from peak IV, which is composed of only these two bands. Peak V also
shows two bands, and peak VI is clearly composed of a single type of polypeptide and corresponds to y-crystallin. The right-hand side
of Fig. 4 shows the polypeptide pattern of the
total soluble proteins resolved on the SDSgradient gel. The bands in this profile account for all of the polypeptides obtained
from proteins of individual peaks.
Table II shows the amount of the constituent polypeptides relative to each other,
on the basis of the intensity of Coomassie
blue staining and calculated from the densitometric tracings. The molecular weights of
resolved polypeptides are also shown in this
table. They were estimated by comparison
with known marker polypeptides of chymotrypsinogen, pepsin, myoglobin, and cytochrome C.
Table III shows that each pair of polypeptides occurring in proteins of peaks II, IV,
and V are present in a 1:1 ratio. Only the
protein of fraction III (/32) appears to be made
up of one major type of polypeptide with a
molecular weight of about 33,000. From
these data the subunit structure of the native
proteins can be estimated. Because the polypeptides of each protein occur in equal
quantities (with the exception of protein III),
dividing the molecular weight of each native
protein by the sum of the molecular weight of
its constituent polypeptides yields the number of each polypeptide chain present in that
protein (column 6). For example, it can be
seen that protein II (j3x) with a native molec-
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Volume 17
Number 7
ular weight of 250,000 is composed of five
polypeptides of 23,000 and five polypeptides
of 30,000 molecular weight.
Discussion
There are numerous reports concerning
the number and the molecular weights of
the constituent polypeptides of the /3-crystallins.16- 17- 20> 23> 24 Recently Jedziniak et
al. (personal communication) have determined the molecular weight and concentration of total water-soluble proteins of the
human lens. But to our knowledge there
have not been any reports on the ratio and
the number of the polypeptide chains of the
native soluble proteins of the lens. Thus the
oligomeric structure of j8-crystallins has remained unresolved.
Utilizing Sephacryl S-200 column, we have
fractionated the /3-crystallins of the rabbit
lens cortex into four major proteins which
elute between the a- and the y-crystallins.
Operationally, we define these proteins to be
0-crystallins. /3-Crystallins have been the
least well defined structural proteins of the
lens. The difficulty with purifying these proteins has been that on gel filtration columns
frequently used for their separation, they
elute with a wide profile at the base of the
peaks and thus tend to cross-contaminate the
adjacent peaks. More purified proteins can
be obtained by concentrating and rerunning
of the main peak fractions of each protein
(Fig. 2). This, of course, will result in a final
smaller yield, but such small amounts will be
sufficient for electrophoretic characterization
of the constituent polypeptides of the purified proteins provided that the thickness of
the acrylamide gel is small. In our work, we
have used a gradient slab gel with a thickness
of 1.5 mm, but it is possible to reduce this
even further by a factor of 2 or 3.
We have resolved the subunit(s) of each of
these proteins according to their molecular
weights and have identified the corresponding band(s) in the total soluble proteins into
polypeptide subunits on the same gel. Further, we have determined the relative abundance of each of these polypeptides of the
Beta crystallins of rabbit lens 665
total soluble proteins. These ratios, determined by densitometry, are a reflection of
the actual ratios in the soluble proteins of the
lens cortex. On the basis of such data it became possible to determine the oligomeric
composition of the rabbit lens /3-crystallins.
Since the percent quantity of each of the
subunits is known (third column of Table II)
and since each polypeptide band can be assigned to a certain native protein (Fig. 4), the
relative abundance of each protein in the
total soluble proteins of the cortex can be calculated by simply adding up the percent
densities of bands belonging to specific native
crystallins. Thus the relative abundances of
Pu /32, 03, /34 crystallins are 25.6%, 7.2%,
27.7%, and 2.8% of the total water-soluble
proteins, respectively.
Admittedly, the molecular weights for the
native /3-crystallins and their subunit polypeptides reported here may not be very
exact. Also, the number of polypeptides reported is the minimal number based only on
the molecular weight determinations. Polypeptides with similar molecular weights but
with differing amino acid composition and
net charge cannot be distinguished on SDSacrylamide gels. Thus the oligomeric structure proposed in this paper is based solely on
the molecular weight parameters. These uncertainties notwithstanding, we consider it a
worthwhile attempt in the right direction,
hoping that this report will be an impetus for
more intensive work on the elucidation of the
structure of/3-crystallins.
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