149
SUB mapillust
C O L O R 7: x$ = C H R $ ( 1 3 4 )
L O C A T E 23, 22: P R I N T "pH": L O C A T E 2, I: P R I N T '~-Lo~'; M e $
L O C A T E 2, 41: P R I N T " F R E E E N E R G Y M A P F O R A T P H Y D R O L Y S I S "
L O C A T E 3, 46: P R I N T " ( p H v s "; Me$; ")"
L O C A T E 5, 43: P R I N T " - 8 . 2 k c a l < n; : C O L O R 0: P R I N T x$: C O L O R 7
L O C A T E 6, 43: P R I N T " - 8 . 2 - 8.4 "; : C O L O R I: P R I N T x$: C O L O R 7
L O C A T E 7, 43: P R I N T " - 8 . 4 - 8.6 "; : C O L O R 4: P R I N T x$: C O L O R 7
L O C A T E 8, 43: P R I N T " - 8 . 6 - 8.8 "; : C O L O R 5: P R I N T X$: C O L O R 7
L O C A T E 9, 43: P R I N T " - 8 . 8 - 9.0 "; : C O L O R 2: P R I N T x$: C O L O R 7
L O C A T E 10, 43: P R I N T " - 9 . 0 - 10 "; : C O L O R 3: P R I N T x$: C O L O R 7
L O C A T E 11, 43: P R I N T "-10 - 11 "; : C O L O R 6: P R I N T x$: C O L O R 7
L O C A T E 12, 43: P R I N T "-11 > "; : C O L O R 7: P R I N T x$: C O L O R 7
F O R i = 46 T O 322 S T E P 46
L I N E (51, i ) - ( 5 6 , i): L I N E (300, i ) - ( 3 0 5 , i)
L O C A T E i / 16 + .5, 5: P R I N T U S I N G "@"; i / 46
NEXT
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L I N E (i + 56, 0 ) - ( i + 56, 4): L I N E (i + 56, 3 3 2 ) - ( i + 56, 336)
L O C A T E 22, 8.5 + i / 8: P R I N T U S I N G "#"; i / 48 + 4
NEXT
END SUB
Size E x c l u s i o n C h r o m a t o g r a p h y and Size Exclusion
H P L C o f Proteins
S A A D T A Y Y A B , S E E M A Q A M A R and MOZAFFARUL
ISLAM
Interdisciplinary Biotechnology Unit
Aligarh Muslim University
Aligarh 202002, UP
India
Introduction
Size exclusion chromatography (SEC) is also known as gel
filtration, gel permeation or molecular sieve chromatography. A
number of articles on gel filtration of proteins have appeared 1-4
but none of them dealt with all aspects of SEC. In this paper we
describe the methodology and applications of SEC including size
exclusion HPLC ( S E - H P L C ) with the aim of familiarizing
people how to use this powerful technique without the need for
sophisticated instrumentation.
SEC may be used to fractionate and characterize proteins
according to size. The sieving medium is a porous gel. Molecules
much smaller than the pore diameter will have more probability
of penetrating the gel and will pass through the column more
slowly. The actual speed of movement of each component in a
mixture is dependent on the ease with which molecules can pass
into the gels and be retarded. Molecules with diameter much
larger than the pore size will have less probability to penetrate
the gel particles and will be excluded from the gel and will pass
through the column unimpeded. Intermediate size molecules can
pass into some of the gel particles but compared to very small
molecules, a greater proportion of the intermediate size mol-
ecules will be outside the gel at any time. The most widely used
types of the gels are cross-linked dextrans (Sephadex), cross-
linked agarose (Sepharose), cross-linked polyacrylamide (Bio-
gel), cross-linked allyldextran (Sephacryl) and controlled pore
glass beads. These are graded according to pore size making it
possible to vary the range of molecular weight which can be
fractionated. The upper limit of the fractionation range is the
exclusion limit which means that the molecules with molecular
weight greater than this will have less probability of penetrating
the gel and will be completely excluded.
SEC may be carried out on a large scale but since large
columns are rather time-consuming to run and the gel media
required to fill them expensive, the method finds most appli-
cation in the later stages of protein purification.
Packing the column
Dry gels are allowed to swell in water or buffer under conditions
specified by the manufacturer. If gel is preswollen, then it is
directly processed for the removal of fines which is important
because the presence of fines can block the column. Fines are
BIOCHEMICAL EDUCATION 19(3) 1991
removed by repeated decantation. A glass column of uniform
diameter previously washed with chromic acid, detergent and
water is mounted in a vertical and vibration-free position. The
outlet of the column is connected to tubing with a screw stop-
cock for regulation of the flow rate. The diameter of the column
may be determined at several places along the height of the
column by collecting a known amount of water in to preweighed
weighing bottles whose volume can be taken as the volume of a
cylinder. A small amount of glass wool previously boiled in
water is placed at the bottom of the column and its surface is
covered with few glass beads. After filling one-third volume of
the column with the operating buffer, degassed gel slurry is
poured slowly into the column with the help of a glass rod. The
column should be packed in a single step to avoid discrimination
among the beads during settling and an extension should be used
if necessary. The gel is left to settle under gravity overnight.
When the gel has formed a smooth upper surface, the outlet is
opened with a flow rate of 5 mi/h. As the gel settles down, the
flow rate is increased gradually. The gel bed should be stabilised
by passing three bed volumes of eluent.
Experimental determination of chromatography parameters
The three parameters Ve, Vo and Vi are used to describe the
behaviour of a molecule on a gel filtration column and these
must be determined experimentally.
The elution volume (Ve) is the volume of eluent collected
from the start of loading the sample to the point of its maximal
elution. The behaviour of a solute is described by its Kd
(distribution coefficient) value which is the fraction of inner
volume (Vi) accessible to a solute molecule:
K d = (V e - Vo)/Vi
Value of K d will be zero for solutes totally excluded from the
column and 1.0 for solutes to which the solvent both within the
pores and in the void volume is equally accessible. A Kd >1.0
indicates adsorption or ionic interactions between solute and the
gel material. It is difficult to determine the exact value of V~
since some inner volume is occupied by the gel matrix (Vm) and
bound water to it. Therefore it is usual to take the available
value of Ka, ie Kav:
~:~ = (vo - V o ) / ( v , - Vo)
The void volume (Vo) is the volume of interstitial liquid.
Molecules with diameter larger than pore size are completely
excluded from the gel and their elution volume is equal to void
volume of the column. Blue dextran (a dextran with a blue dye
chemically linked to it, Mr 2 x 106) is completely excluded from
Sephadex, polyacrylamide gels and some agarose gels and may
be estimated by extinction measurements at 625 nm and is
widely used for the determination of the void volume. Caution is
necessary when blue dextran is mixed with proteins, however,
since it forms complexes with some proteins. With spherical
beads, Vo is 30-35% of the total volume (Vt) depending on how
tightly the column is packed. Thus the useful range for resolving
proteins lies within about 80% of the remaining volume (Vt -
Vo) ie, about 55% of Vt. Blue dextran is also used for checking
the column packing. A symmetrical peak of elution indicates
homogeneity of packing.
The inner volume (Vi) of the column can be determined by
subtracting the void volume from the elution volume of small
molecules such as glucose or tyrosine having K d -- 1.0.
The total volume (Vt) of the column is the sum of void volume
and inner volume. However, the total volume accessible to
liquid is slightly less than the total volume due to a finite volume
occupied by the gel matrix plus tightly bound water. Vt is equal
to the volume of a cylinder whose height and radius are same as
the dimensions of the column.
 18791468, 1991, 3, Downloaded from https://iubmb.onlinelibrary.wiley.com/doi/10.1016/0307-4412(91)90060-L by Iraq Hinari NPL, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
150

Sample size 2.0

This should not exceed 3% of the total volume and a smaller
volume than this will give slightly better results down to about
1% of Vt. The starting protein concentration should ideally be in
the range 10-20 mg/ml. A n increase in sample size will make the
resolution poor. However, for desalting application of SEC 1
where the matrix has been chosen such that the desired protein ~1.5
elutes in the void volume while the elution volume of the salt
approaches the total volume of the column, it is possible to use
sample size as large as 20% of Vt.

Sample application 1.0

The operating buffer above the column bed is drained off and
I I
the clamp on the outlet tubing is closed. Then sample is applied 418 s.o s.4
gently to the column with a Pasteur pipette, allowing it to run LogM~
down the side of the column while moving the pipette around the
column. The stop-cock is then opened slowly and the sample Figure 1 Plot of V e / V o v e r s u s log Mr for various marker proteins
allowed to pass down the upper surface of the gel. When all the obtained on Sephadex G-150 column (52 x 1.7 cm) equilibrated
protein sample has passed into the gel, buffer is applied in the with 0.06 M sodium phosphate buffer, p H 7.0. About 1.2 ml of
same way and elution is performed with a constant flow rate. the sample containing 10 mg of each of these marker proteins was
The column dimensions are important for resolution. Squat applied. Elution was performed at a flow rate of 20 ml/h and
columns have fast flow rates and less turbulence leading to less fractions of 3 ml were collected. The column was monitored for
spreading, but resolution is poor because the length covered will protein using the method of Lowry et al. 6 The different marker
be smaller. In addition, it is difficult to achieve homogeneous proteins were: (1) a-chymotrypsinogen, (2) ovalbumin, (3) BSA
sample application and buffer flow. On the other hand, a long monomer, (4) BSA dimer and (5) ~,-globulin
thin column (20-40 times longer than its diameter) will give
better results since the sample occupies a greater column depth. 60
Fractions of appropriate size are collected and the column is
monitored for protein using different methods (eg spectrophoto-
metric, a dye binding method 5 or the method of Lowry et al. 6)
Carbohydrate may be monitored using the method of Dubois et
al. 7 Enzymatic activity may also be monitored. 40
Eluting buffers should be of high enough ionic strength (eg
>20 mM) to counteract the few charges which may be present
on the gel. Apart from that, the only criterion for the buffer is
that the proteins are stable in it.

Applications of SEC 2O
Determination of Mr SEC does not require pure, homogeneous
protein. All that is needed is a method for detecting the protein
4" 014 0.6
' o'.8 1'.o
as it comes off the column. For the determination of the Mr of
any protein, a column is calibrated by passing proteins of known
M r through the column. The elution volume of each protein is Figure 2 Treatment of gel filtration data (as shown in Fig 1)
determined. These values of elution volume (Vc) are divided by according to Porath 9
void volume (Vo) to obtain (Ve/Vo), comparable on using
different columns, for a particular protein. From the data Determination of hydrodynamic parameters For the determi-
obtained, a graph may be drawn between Ve/Vo and log Mr nation of the Stokes radius, a column is calibrated with marker
(Figure 1). The protein of unknown molecular weight is then proteins of known Stokes radii. The elution volumes of marker
passed through the column and its Ve/Vo used to estimate its Mr proteins are normalised into Kd and Kay values and these data
from the calibration curve. 8 are analysed according to Laurent and Killander 1° and Ackers 11
SEC data can also be treated according to Porath. 9 Values for using the following equations:
the elution volumes of marker proteins are normalised in terms
of Kd values. A graph of Kd 1/3 and Mr v3 may be obtained using (-log Kay) 1/2 : A a + B
the values of marker proteins (Figure 2). Then the Kd value of and
t h e unknown protein is determined and hence its M r may be a = ao + bo erfc -1 Kd
estimated.
One source of error in this procedure is that it takes no where a is the Stokes radius of the protein with distribution
account of molecular shape. In fact a protein molecule which is coefficient Kd. A, B, ao and bo are calibration constants and
somewhat elongated will pass down the column faster than a erfc -1 Kd is the inverse error function complement of Kd. ~2 Both
spherical one of same molecular weight. Also it has been known the above treatments yield straight lines (Figs 3 and 4). By
for some time that glycoproteins, particularly those rich in substituting the values of ( - l o g Kay) v2 and erfc -1 Ks of the
carbohydrate, show anomalous behaviour on SEC and this will unknown protein, its Stokes radius can be determined.
also be the case with proteins which are not globular. Nor is the Values of the diffusion coefficient, D, and frictional ratio fifo
technique applicable to proteins which have affinity towards of a protein may be calculated from the value for Stokes radius
carbohydrate. Nevertheless, the Mr derived by this technique by using the following relationship 8
with globular proteins seems to be accurate to about +10%. It is
also possible to measure molecular weights under denaturing D = k T/(61r-qa)
conditions provided appropriate standard curves are con-
structed. fifo = a/(392 Mr/4"rrN) I/3

BIOCHEMICAL EDUCATION 19(3) 1991

 18791468, 1991, 3, Downloaded from https://iubmb.onlinelibrary.wiley.com/doi/10.1016/0307-4412(91)90060-L by Iraq Hinari NPL, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
151

1.2
its major drawback is that it is time consuming. Separation times
are long resulting in poor resolution. This is because of eddy dif-
fusion, mass transfer problems and extra column effects. Speed-
ing up the solvent flow by applying high pressure results further
decrease in column efficiency and resolution. Application of high
=> pressure causes the compaction of the bed due to the soft nature
v
3 4
co 0.8 of the gel and results in lower flow rates. High pressure or
O high performance liquid chromatography (HPLC) involving the
v
I principle of size exclusion chromatography also known as size
exclusion HPLC or S E - H P L C is an alternative approach for
getting good resolution of macromolecules within a short time.
Though primarily used for analytical purposes, the technique has
0A
now become a powerful purification technique. The funda-
mental principle remains the same for both SEC and SE-HPLC.
The advantages offered by HPLC are good resolution and speed
Stokes radius (nm) of analysis, reusability of column without repacking and regener-
ation, high reproducibility due to the close control of parameters
Figure 3 Treatment of gel filtration data (Fig 1) according to effecting the efficiency of the separation, easy automation of
Laurent and Killanderw instrument operation and data analysis and its adaptability for

/
large-scale preparative procedures. These advantages of HPLC
are the result of two major advances: (i) the development of new
column packing material which is packed in narrow columns and
which increases column efficiency 10-100-fold, and (ii) the
A improvement of elution rates achieved by applying high pressure
E (up to 300 atm). In general, S E - H P L C employs an immobile
c 4
phase bonded onto a porous silica which allows high flow rates to
"lO
.E
be used. Semirigid gels can be used for fractionation of
molecules up to a molecular weight of 10 000 000 under aqueous
conditions. Rigid silica beads have several advantages over
o.9 semirigid gels including ease of packing and compatibility with
(n 2 water and organic solvents. The new packings are typically
spherical beads consisting of a solid non-porous core (40 I~m in
diameter) with a thin, porous outer shell of absorbent (silica gel,
1 I alumina resin). Recently microparticulates (porous particles
0.4 1.0
with diameters in the range 20-40 I~m and 5-10 Ixm) have been
e r f c -~ Kd widely used because they offer greater resolution and faster
separation with lower pressures. A variety of bonded phases
Figure 4 Treatment of gel filtration data (Fig 1) according to the have been used to mask the cationic surface of silica and prevent
method of Ackers 11 nonpermeation effects. These include glycerylpropyl diol and N-
acetylaminopropyl silane. Although a number of nonsilica based
where k is the Boltzmann constant (1.386 x 10 -16 ergs/degree), support materials have been used, most work has involved the
T is the absolute temperature, -q is the coefficient of viscosity of use of silica based material. Silica has the disadvantage of being
the medium (0.01 poise for water and dilute aqueous solutions at unstable at pH values above 8.0. This can be overcome either by
20°C), a is the Stokes radius of the protein, Mr is the molecular using a polymer coating or by surface stabilisation with
weight of the protein, 92 is the partial specific volume and N is zirconium which results in the development of rigid, cross-linked
Avogadro's number. polymeric supports such as monobeads (Pharmacia) or TSK-PW
(Toyo-Soda Company). Some of the column materials and their
SE-HPLC properties are listed in Table 1.
Although size exclusion chromatography has several advantages, For a successful HPLC separation, selection of both column

Table 1 S E - H P L C column materials available for protein purification*

Particle size Pore size Fractionation range

Column (~m) (nm) (Ka) pH stability

Superose - 12 10 25 1-300 1-14

Superose - 6 13 40 5-5000 1-14
TSK 2000 SW 10 13 1-50 2.5-7.5
TSK 3000 SW 10 24 5-400 2.5-7.5
TSK 4000 SW 13 45 40-1000 2.5-7.5
Zorbax GF-250 4 15 10-250 3-8.5
Zorbax GF-450 6 30 25-800 3-8.5
Polyol=Si 300 5,10 30 10-500 2-8.5
Polyol=Si 500 10 50 40-900 2-8.5
SynChropak GPC 100 5 10 5-200 2-8
SynChropak GPC 300 5 30 10-670 2-8
SynChropak GPC 500 7 50 10->670 2-8
*Taken from ref 13

BIOCHEMICAL EDUCATION 19(3) 1991


152
packing as well as solvent system is required. The mobile phase
should consist of a very high purity solvent which should not
react with sample or column packing and there should not be any
interference with the detector. It has long been known that the
polarity of a solvent dictates its ability to displace adsorbed
solutes from the column ie eluting power, E, which increases
with an increase in polarity. Sometimes the use of a single
solvent does not give better resolution of solutes. This difficulty
known as general elution problem is due to the wide range of Ka
values of different components present in a multicomponent
system. In such cases, solvent binary mixtures provide an extra
advantage in resolving complex mixtures. Alternatively, the
composition of the mobile phase can be varied starting with a
weakly eluting solvent (low E) and gradually increasing the
concentration of strongly eluting solvent (high E) in order to
have gradient elution. The solvent are chosen for such type of
gradient on empirical basis. In S E - H P L C where elution is
carried out by using physiological buffers, the speed of S E -
HPLC can result in the quantitative purification of enzymes with
full activity. However, for labile enzymes, S E - H P L C can be
performed in the cold and at the highest possible flow rate. For
those proteins which show aggregation, detergents generally
0.1% SDS can be included in the mobile phase.
References
Wallach, J M (1989) In 'Practical Biochemistry for Colleges' (edited by
Wood, E J) pp 63-64, Pergamon Press, Oxford
eDixon, H B F (1985) Biochem Educ 13, 181-183
3Versee, V (1985) Biochem Educ 13, 33-34
4Malhotra, O P and Kumar, A (1989) Biochem Educ 17, 148-150
5Bradford, M M (1976) Anal Chern 72, 248-254
6Lowry, O H, Rosebrough, N J, Farr, A L and Randall, R J (1951) J
Biol Chem 193, 265-275
7Dubois, M, Gilles, K A, Hamilton, J K, Rebers, P A and Smith, F
(1956) Anal Chem 28, 350-356
8Andrews, P (1970) Methods Biochem Anal 18, 1-53
9porath, J (1963) J Pure Appl Chem 6,233-234
mLaurent, T C and Killander, J (1964) J Chromatog 14, 317-330
1~Ackers, G K (1967) J Biol Chem 242, 3237-3238
t2In 'Tables of the error function and its derivative', NBS Applied
Mathematics Series 41, United States Government Printing Office,
Washington, DC, 1954
X3Welling, G W and Welling-Wester, S (1989) In 'HPLC of Macro-
molecules - - A Practical Approach', pp 77-89, IRL Press/OUP,
Oxford
Disulphide Groups in Proteins
W L BAKER and A PANOW
Chemistry Department
Swinburne Institute o f Technology
John Street
Hawthorn 3122, Melbourne
Australia
Introduction
Disulphide groups in proteins may have a role in metabolic
activity and control ~ or in the interaction of hormones with their
receptors. 2 However, teaching at the undergraduate level tends
to concentrate primarily on their stabilising role on the tertiary
structure of proteins and peptides. In either case it is necessary
to have adequate analytical methods to determine the number of
disulphide groups in proteins. The final year biochemical
practical unit in this Institute contains an experiment which
compares two methods of analysis of disulphide groups. The
BIOCHEMICAL EDUCATION 19(3) 1991
18791468, 1991, 3, Downloaded from https://iubmb.onlinelibrary.wiley.com/doi/10.1016/0307-4412(91)90060-L by Iraq Hinari NPL, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
procedure involves fluorescence using fluorescein mercuric
acetate 3 and a reduction with sodium borohydride. 4
The fluorescence procedure was chosen to complement the
very strong instrumental bias which students get in their applied
chemistry practical course. Students are told about the great
sensitivity of fluorescence, the requirements of separate excit-
ation and emission wavelengths and the phenomenon of
quenching which are involved in fluorescent assays. The
experiment is run in the final year of the course because of the
potential toxicity of fluorescein mercuric acetate 3 and the care
required with the powerful reducing agent sodium borohydride.5
As described, the work may be performed in less than four hours
by a pair of students.
Materials and Methods
Solutions All protein solutions are made up in distilled water at
a concentration of 1 mg/ml. Fluorescein mercuric acetate is
maintained at 10 -4 M in 0.01 M NaOH but before use is diluted
to 10 -5 M with 1 M NaOH. Ellman's reagent, 5,5'-dithiobis-(2-
nitrobenzoic acid), is made at 10 mM in 0.05 M phosphate
buffer pH 8, and a 1 M solution of KH2PO 4 containing 0.2 M
HC1 is also prepared and kept as a stock solution.
Fluorescence Assay A solution of crystalline RNase is used as a
standard and the solution containing 1 mg/ml is diluted 1 + 30.7
with distilled water to contain 10 nmol of disulphide groups per
ml (2.5 nmol or 31.5 ~g of protein). Standard 1 ml solutions are
then prepared from this dilution to contain 1-10 nmol of
disulphide groups.
Other protein solutions are diluted to contain approximately
5 nmol of disulphide per ml. The dilutions used are: 1 + 57 for
lysozyme (M r 13 930), 1 + 49 for trypsin (Mr 23 800), and 1 + 54
for bovine serum albumin (Mr 65 900). One ml volumes of the
final dilution are used.
One ml of 10-SM fluorescein mercuric acetate solution is
added to each tube, followed by 8 ml of 1 M NaOH. Tubes are
vortexed and left for 1 h prior to determination of fluorescence
intensity in each tube.
Fluorescein mercuric acetate shows intense fluorescence.
Thiol groups react with this reagent under both alkaline
conditions and at neutral pH whereas disulphides only react at
alkaline pH. A correction is required when thiol groups are
present in the protein. The reaction is presumed to involve a
relatively slow alkaline scission of disulphide bonds and a very
rapid formation of a complex between the thiol groups formed
and fluorescein mercuric acetate to quench the colour. There are
probably irreversible side reactions as well.
(CH~COO)Hg~ v
~-CJ~'~.~'~,.Hg(OOCCH0
COOH
Students are required to determine the wavelengths of
maximum emission and excitation, which are about 525 and
480 nm respectively, and draw the appropriate curves. Although
procedures vary with the instrument the principle is general for
all instruments. The emission wavelength is determined using a
fixed excitation wavelength (usually 300 nm) and variation in
sensitivity or attenuation of the instrument. The excitation
wavelength is determined by scanning from the wavelength of
maximum emission.
Results are obtained from the standard curve of RNase and
the number of moles of disulphide per mol of protein are
determined by working back through the dilutions.
Reducing assay Lysozyme is diluted to a concentration of 0.5