Methods for separation of proteins

Albert M. Potts

Most of the viethods available for general separation of proteins have been applied to the
proteins of the crystalline lens. The classical work of Mb'rner, in 1894, suggested that there were
three soluble lens proteins tohich he labeled a-, j6- and y-crystallin and an insoluble residue
which he called albuminoid. Subsequent attempts at fructionation using differential solubility,
low-voltage electrophoresis, and ultracenirifugation appeared to confirm this finding. However,
utilization of high-voltage electrophoresis immunodiffusion, immunoelectrophoresis, and chro-
matography on modified cellidose columns showed a vmch larger number of fractions, i.e., ten
or more. It has been recently demonstrated that separation procedures on high molecular weight
proteins conducted in solutions with a high concentration of urea can cause dissociation of the
proteins into smaller molecidar weight components which on removal of the urea reassociate
once more. It has been shown that these phenomena obtain for a- and (3-cnjstallins. These find-
ings suggest that Morner's original three soluble proteins are still the major protein components
of the lens. Whether the subcomponents demonstrated by the newer separation methods have
an independent biological existence is yet to be proved.

I t is appropriate that the lens, the tissue

with the highest protein content of any in
the body, should have its proteins studied
by most of the available methods of pro-
tein separation. The questions to be an-
swered were and are:
How many proteins are there in the lens,
and what is their nature?
Solubility methods
The giant step in the study of lens pro-
teins was taken by Morner in 1894.x Using
classical methods of differential solubility,
he separated four components of lens pro-
tein. Although even today the theoretical
aspects of protein separation by solubility
are not completely worked out, consider-
able study on the subject has been in-
vested, particularly by Cohn and co-work-
ers.2' 3> l A concise treatment of the em-
From the Eye Research Laboratories, the Uni-
versity of Chicago, 111.
531
pirical theory is given by Dixon and Webb5
in a recent review. These authors describe
the salting-in effect at low ionic strength,
presumably due to increase in dielectric
constant of the medium caused by the
added salt. If pH and temperature are held
constant and ionic strength is increased
continuously, the salting-out effect then
takes over and this portion of the curve can
be described by Cohn's equation where log
solubility varies inversely as the ionic
strength over a wide range of ionic
strengths, and a straight line plot results.
p
This is expressed as log s = /?'—K's —.
Here the constant K's represents the slope
of the straight line, and /?' represents the
intercept on the y axis—a hypothetical
solubility at zero ionic strength. The posi-
tion of the straight line varies with the
nature of the protein, of course, and for
any given protein varies with the temp-
erature and the pH. As might be ex-

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 532 Potts
pected the pH-solubility relationship passes
through a minimum at the isoelectric point.
The theoretical explanation for salting-out
has not been worked out in a satisfactory
way, but one useful model which has been
suggested is the notion that hydration re-
quired by the inorganic salt ties up enough
water molecules to decrease effectively the
water available for solution of the large
protein molecule. A survey of the basic
theory of salting-in and salting-out is given
by Edsall and Wyman.G
If less polar organic solvents such as
ethanol are added to the aqueous medium,
the dielectric constant of the solvent is
reduced and small changes in ionic strength
exert large effects on the solubility of
polyvalent, large molecule ampholytes such
as the proteins. If temperature is kept low,
in the neighborhood of -3° to -5° C, de-
naturation of proteins does not occur, and
these systems may also be used for separa-
tion of proteins.
The original work of Morner used a num-
ber of different aspects of these solubility
parameters to separate the proteins of lens.
Albuminoid was insoluble in quarter-sat-
urated saline. Alpha crystallin was pre-
cipitated isoelectrically at pH 5.2. Beta
crystallin was precipitated at pH 7 with
additional salt, and the albumin fraction,
later called gamma crystallin, remained in
the supernatant. Low temperature and al-
cohol were employed by Francois, Rabaey,
and Wieme7 to separate lens protein frac-
tions. They distinguished three fractions:
«i, a2, and /?, the latter being resolved
into two components by paper electro-
phoresis. By and large, however, the indi-
viduals who worked with precipitation
methods found only four fractions in very
much the same way as Morner did.
Ultracentrifuge methods
A second major method of protein sep-
aration is ultracentrifugation. It was shown
by Svedbergs that large molecules sediment
in an ultracentrifugal field at a rate which
is dependent upon a number of variables.
These are expressed in the classical Sved-
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Investigative Ophthalmology
August 1965
RTs
berg equation M = -. Where M
8 4
D(l-pv)
equals molecular weight, R equals uni-
versal gas constant, T equals absolute
temperature, s is sedimentation velocity, D
is the diffusion coefficient, v is the partial
specific volume of material being studied,
and p the density of the solution. It is
evident that if sedimentation rate can be
measured and the other factors are known
with sufficient accuracy, the method may
be used for determining molecular weights.
It should be said from the outset that ultra-
centrifugal preparation has never played a
major role in the separation of lens pro-
teins. However, the ultracentrifuge has
been used on occasion to examine fractions
separated by other methods and for es-
timation of molecular weight. In general,
ultracentrifugal examination of native lens
protein has shown the same three soluble
factors obtained with precipitation meth-
ods.9- 10
The practice of solving the Svedberg
equation for sedimentation and expressing
this as "Svedberg units" at 20° C. has now
become standard. Usually an arbitrary vol-
ume is used for the partial specific volume
of the anhydrous protein. The Svedberg
unit is the measured value divided by 10*13,
an easily handled figure. Determinations
by various authors have placed the sedi-
mentation velocity of a-crystallin between
15 and 20 Svedberg units and calculated
molecular weights have been from 400,000
to 1.2 million. The mode lies between
800,000 and 1 million molecular weight,
and this gives an adequate estimate of
order of magnitude. The molecular weight
of y-crystallin has been well established at
19 to 20,000." In the ultracentrifuge, /?-
crystallin has given at least two fractions
which are intermediate in molecular weight
between a and y.12
Despite the valuable information on
molecular weights contributed by ultra-
centrifugation and despite new and sophis-
ticated advances in the state of the art,
including density gradient sedimentation,
new methods of computation, and advanced
 Volume 4
Number 4
optical systems,13 there is little likelihood
that this technique by itself will contribute
to the solution of the immediately pressing
problems of lenticular proteins.
Electrophoresis
The modern use of the technique of
electrophoresis begins with the innovations
of Tiselius.14 The thermostated cell with
optical quality windows, and the pneu-
matic hammer for separating sections of
the cell containing wanted protein frac-
tions, together with the Topler-schlieren
optical system made electrophoresis for the
first time a quantitative and effective
method for separation of proteins. The
modified recording systems of Longsworth
and Mclnnes15 and of Philpot10 and Swens-
son17 added elegance to the technique.
Early work on the free electrophoresis of
lens proteins, such as that of Hesselvik,is
served simply to confirm the excellence of
the Morner-type separation. Electrophoresis
of soluble proteins of lens showed two
electrophoretically defined components
which correspond to a- and /?-crystallin,
and after a Morner-type separation was
done the a and /? fractions each showed
a single boundary which was distinctive
for the substance. Repetition ten years later
by Siillmann and co-workers10 showed no
essential difference, although in calf lens
a second component in the (3 group was
identified.
This was the situation, then, in the early
1950's. The three soluble protein fractions
of the lens, described by Morner more than
half a century before, had held up under
examination by methods as sophisticated
as the Svedberg ultracentrifuge and the
Tiselius electrophoresis apparatus. All this
was due for sudden change and the change
was brought about by proliferation of new
techniques for protein separation. Out-
standing among these was electrophoresis
done on some support material plus dem-
onstration of fractions by the use of dyes,
the protein still remaining on the support.
The first type of support used was filter
paper, and this poor man's electrophoresis
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Methods for separation of proteins 533
at one blow eliminated the careful thermo-
stating and the elaborate optical system re-
quired for free electrophoresis. Although
paper electrophoresis was first applied to
amino acids and proteins in 1939,20 its
enthusiastic adoption occurred in the years
just surrounding 1950 (for review, see
Cooper21). Simple paper electrophoresis of
the lens proteins suggested that there was
a bit more complexity to the Morner pic-
ture, for the /?-crystallin fraction was
frequently resolvable into two compo-
nents.7' 22> 23
In free electrophoresis the chief factors
determining migration of the protein ap-
peared to be net charge on the molecule
(which in turn is a function of the pH and
the isoelectric point of the protein in ques-
tion), the mass and volume of the hydrated
molecule, and the current density. Electro-
osmotic effects were minimized by the
volume of solution used and the nature of
the buffers. As a first approximation in
paper electrophoresis, similar considera-
tions apply. The attraction of protein for
the paper support is hopefully negligible,
although because of the smaller volumes
involved electroosmotic effects become sig-
nificant.
In other and highly useful developments
of supports for electrophoresis of proteins,
a further factor conducive to separation
was introduced. Gel-type supports such as
agar,21' 24~20 starch,27'2S and polyacrylam-
ide20> 30 all exhibit this property in vary-
ing degrees. Apparently the pore size of
such supports is sufficiently small so that
the support additionally retards the protein
according to its molecular volume. The
result is sharper separation of the indi-
vidual fractions.
With the use of agar electrophoresis,
Francois and Rabaey arranged conditions
so that only the a-crystallin migrated to
the anode from the point of application,
whereas the other fractions were drawn
toward the catliode by the electroosmotic
flow of fluid and would string out accord-
ing to their charge and size. Varying num-
bers of protein fractions were identified,
 534 Potts
Fig. 1. Separation of soluble proteins of calf lens by agar electrophoresis.
depending partly upon the age of the lens,
and in an infant at least ten different pro-
tein components were distinguished. When
the method was modified, using higher
tension up to 50 v. per centimeter and
cooling, as many as seventeen fractions
were isolated from the lens of a child and
varying numbers in the same order of
magnitude for the lenses of lower ani-
mals.3:l"3a However, if one uses milder
conditions with ordinary preparations of
beef lens, the usual result with agar is
shown in Fig. 1. This brings up the ques-
tion of what the high-tension electro-
phoresis actually accomplishes in the way
of degrading protein, and the subject will
be dealt with below.
Starch-gel electrophoresis of native lens
protein was done by Bloemendal and Ten
Care.3* Here a-crystallin separated as a
unit band which was ultracentrifugally
homogenous and /?-crystallin gave two
bands. The use of polyacrylamide gel in
electrophoresis of lens proteins was intro-
duced by the Bloemendal group33 at a
1962 meeting in Bruges. With the standard
amount of gel cross-linking it was found
that a-crystallin did not move into the
polyacrylamide gel at all, but with lower
degrees of cross-linking (that is 1 to 150
rather than 1 to 99) at least six zones were
visible of which the least rapidly migrating
one was a-crystallin. Again one must won-
der whether the procedure was responsible
for alteration in the properties of the pro-
teins used.
Electrophoresis procedures using support
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Investigative Ophthalmology
August 1965
materials, but which were much closer in
nature to free electrophoresis, were utilized
for lens proteins by Wood and co-workers30
and by Bjork.37 Wood used continuous flow
electrophoresis in which gravity flow along
a filter paper is at right angles to an im-
posed electric field. Five separate fractions
were found in rabbit lens. The second
procedure employed the preparative zone
electrophoretic procedure of Porath38 in
which cellulose powder was the solid agent.
By this technique three soluble fractions
of beef lens were collected, the middle one
of which was proved to be dual by ultra-
centrifuge and free electrophoresis.
Immunological techniques
Related to the previous methods are two
immunological techniques which at about
the same time as agar-gel electrophoresis
indicated that there were more potential
proteins in the lens than was suggested by
the original precipitation experiments. One
of these, immunoelectrophoresis, utilized
the same kind of agar electrophoresis de-
scribed above, but identified the protein
fractions by the precipitin reaction which
they made with antiserum placed in a
trough cut in the agar parallel to the path
of the electric current. The usual technique
was that defined by Grabar and Wil-
liams.39- 40 This technique was first applied
to the lens by the Ghent group41 in 1956
and Witmer.42 Between five and eight
separate components were identified by this
method. An older method of protein separa-
tion also with an immunological basis but
 Volume 4
Number 4
without using electrophoresis is the agar-
plate method of Ouchterlony.43 In this
method the protein to be examined is
placed in one well in an agar plate, and
antiserum to the protein is placed in an-
other well. As the two proteins diffuse
toward each other in the agar the differ-
ences in rate of diffusion allow individual
precipitin bands to be formed. This tech-
nique was first applied to the lens proteins
in 1955 and the years following shortly
thereafter.'11'4i> 45 By this means, too, be-
tween five and eight antigens were demon-
strable in beef lens protein.
Column separations
The idea of cascading various types of
physicochemical separations by the use of
a column has proved so productive in other
areas that numerous efforts were made to
adapt these types of procedures to protein
separation. Protein denaturation by organic
solvents and low partition coefficients be-
tween aqueous and immiscible nonaqueous
solvents made partition columns and paper
partition chromatography impractical. The
difficulty with adsorption chromatography
and ion exchange was that the usual ad-
sorbents used for small molecules were
inappropriate for the extremely large num-
bers of polar groups possessed by proteins.
The result was that the protein could not
be eluted from the column by nondestruc-
tive reagents. Tims, successful column
chromatography of proteins had to await
the development of special materials par-
ticularly adapted to the ion exchange char-
acteristics of protein. This occurred when
Sober and Peterson40-47 introduced two cel-
Fig. 2. Separation of subfractions of calf a-crystallin by starch gel electrophoiesis.
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Methods for separation of proteins 535
lulose-based ion exchangers. Their cation
exchanger, carboxymethyl cellulose, was
made by reacting cellulose powder with a
very small amount (1 mEq. per gram) of
chloracetic acid.- The anion exchanger, di-
ethylaminoethyl cellulose (DEAE), was
prepared by reacting similarly small quan-
tities of 2-chlorotriethylamine with cellu-
lose powder. The relatively small number
of exchangeable groups so created were
ideal for many types of protein separations
and have been utilized with effect for the
separation of lens protein fractions. Spec-
tor18 was able to use DEAE cellulose first
alone and then in conjunction with- zinc
glycinate preliminary fractionation.''9 At
least fourteen fractions could be separated
from the beef lens. An additional column
technique, "gel filtration," based on differ-
ential rate of diffusion in dextran gel has
been used for isolation of y-crystallin and
for fractionation of /3-crystallin by Bjork.50'51
Discussion
It would seem on the basis of this brief
review that the number of components
isolated from lens protein is strictly a func-
tion of the sophistication of the isolation
method. On this basis one might assume
that further developments of protein sep-
aration would give an infinite number of
lens protein fractions with resultant con-
fusion in the field beyond all hope of re-
trieval. Fortunately, a recent set of experi-
ments based on newer knowledge of pro-
tein denaturation promises to unify our
concept of the state of the lens proteins in
a veiy satisfactory manner. The reports of
Bloemendal and co-workers,5- of Bjork,53
& *• • • %
\ \
 536 Potts
and of unpublished work in our own lab-
oratory54 substantiate the fact that a-crys-
tallin, when treated with high molarities
of urea, fragments into as many as thirteen
separate portions which can be separated
by starch-block electrophoresis or poly-
acrylamide electrophoresis, but that when
the urea is removed the original high
molecular weight a-crystallin is restored
(Fig. 2).
The same realization about other proteins
has permeated all divisions of the field of
protein chemistry and has given new im-
petus to the study of the nature of de-
naturation. A highly complete and most
recent review on the subject is that of
Reithel.55 On the basis of evidence culled
from all fields of protein chemistry, it ap-
pears that there is none for single poly-
peptide chains greater than 66,000 in
molecular weight and that more commonly
chains are one half to one third this size.
The determining factor in this respect is
perhaps the size of the genetic control
units responsible for protein synthesis.
Thus, all proteins which behave as a unit
and show higher molecular weight must
be the result of intramolecular association.
This tertiary structure as well as the sec-
ondary helix formation is apparently dis-
rupted reversibly by the presence of urea
or guanidine in high concentration. How
this works is still a matter of conjecture.
There is some theoretical objection to the
suggestion that hydrogen bonds are dis-
sociated reversibly by the amides, and there
is little reason for believing that nonpolar
secondary linkages can be broken by these
substances. Nevertheless the empirical fact
remains that all large protein molecules
are degraded, usually in a reversible man-
ner by this kind of treatment.
Here, then, is the explanation for the
results on lens protein that have appeared
so curious to begin with. Alpha crystallin
is, indeed, a single large molecule as orig-
inally suggested by Morner and as sub-
stantiated by ultracentrifuge and free elec-
trophoresis studies. However, treatment
with urea fragments the a-crystallin revers-
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Investigative Ophthalmology
August 1965
ibly into as many as thirteen subunits
which reassociate readily to form the orig-
inal single molecule. A similar event occurs
with beta crystallin, but because of its
smaller initial size the number of sub-
fragments is fewer. Gamma crystallin may,
indeed, be a single substance of molecular
weight in the vicinity of 20,000. This is the
explanation for the highly variable number
of substances separated by the various
methods. For the first time in a decade the
subject of lens protein chemistry begins
once more to make sense. To sum up the
present state of the art, one could do no
better than to modify the motto of these
United States and have it read E pluribus
tres.
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Number 4
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 538 Potts
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Investigative Ophthalmology
August 1965
51. Bjork, I.: Fractionation of /J-crystallin from
calf lens by gel filtration, Exper. Eye Res. 3:
10, 1964.
52. Bloemendal, A., Bont, W. S., Jongkind, J. F.,
and Wisse, J. H.: Splitting and recombination
of a-crystallin, Exper. Eye Res. 1: 300, 1962.
53. Bjork, I.: Studies on the subunits of a-crystal-
lin and their recombination, Exper. Eye Res.
3: 1, 1964.
54. Potts, A. M.: Unpublished results.
55. Reithel, F. J.: The dissociation and association
of protein structures, Adv. Protein Chem. 18:
123, 1963.