Affinity Chromatography
Affinity Chromatography
Affinity Chromatography
Affinity Chromatography
5.1 INTRODUCTION
416
Affinity Chromatography 41’7
cl
HIXTURE OF PROTE INS
SPACER ARH,
SUCH AS HEXAMETHYLENE ATTACHMENT OF
DIAMINE DESIRED ENZYME
WASHING BUFFER
REMOVAL OF
CONTAMINATING PROTEINS
ELUTION BUFFER
RECOVERY OF
PURIFIED ENZYME A
cells
t
Disrupt
t
Extract
t
Centrifuge
A \
Affinity column Acetone precipitation
t t
Dialysis or Protamine treatment
molecular sieving
t
(NH,I$O, Fractionation
t
Dia/ysis
Hydroxyapatite
chromatography
t
Dialysis
t
Ion-exchange cellulose
chromatography
t
Molecular sieving
K
as
=-=
k
a IEL I (5.1)
kb
for the interaction of the ligand [L] and the ligate [El:
+ L
!a._ EL
E - (5.2)
kb
The adsorption of the ligate by the ligand can be represented
by the Langmuir-type isotherm (10):
(5.3)
where
(5.4)
IE I =
1
+-------II
E
(5.5)
IEL I K
as ILclI ’ KED l Lo I
- (5.6)
Where for [E] and [EL] are obtained from the elution
curve (Figure 5.3). The shaded area in this figure represents
the amount of enzyme E bound to the ligand L. The values
for [E] and [EL] from experiments with different
concentrations of E at constant concentrations of D can be
plotted as in Figure 5.4 to give straight lines whose abscissa
values can be used to obtain values for K,, and K,n.
Elution volume
Figure 5.3. Elution profile of enzyme B applied to columns
of immobilized A. The hatched area represents the amount
of B bound to A. This is displaced from the column by some
competing ligand C at concentration D (Reference 12).
Affinity Chromatography 421
rTDi:SHES 1 - ELUTIONS
El 3 5 7 9 II I3 15 17 19 21 23 25 27 29 30
TOTAL WASHES AND ELUTIONS, (XV’+ CM I/v
dC aC dc a2c
u-- +c----- +
%
--
DZ
___2 = 0 (5.8)
dZ dt at i3Z
where X is a constant.
Kf a (c - C*) (5.10)
“bt =
* (5.11)
tE - tB =
'b Go XE B
In- * ___ In (5.12)
'E - ‘Ii = fi c 1-o
f 0 xB
Example 5.2
The breakthrough curve data for trypsin on Sepharose
4B columns with soybean trypsin inhibitor ligand (Figure 5.7)
and arginine peptide ligand (Figure 5.8) have been used with
Equation 5.12 to calculate the average values of the
volumetric coefficients of the overall trypsin transfer. These
values are shown in Table 5.2: The fact that changes in
trypsin concentration or in ligand caused little change in K>
indicates that the reaction rate of trypsin with the ligand
was not rate limiting. The finite difference method of Crank
and Nicolson (19) was used to generate the calculated
breakthrough curves. Note that the measured breakthrough
curves are somewhat steeper than the calculated curves at
the beginning of breakthrough. This has been attributed to
the variation of the intraparticle mass transfer coefficient
with the progress of adsorption.
I I
0cm.-------- O
I,0 - 0
//
/
N :
27 (DEFF/DnULK) L (5.14)
pore
NP(T-1) = -1.69
I
ln(l - X)2 + 0.61
1 (5.16)
where
where
OCX(E (5.21)
Nf (T - 1) = -mNp(T-1) =a
where
m = - Nf/N (5.23)
and
a = l/(1 + N /N ) (5.24)
f P
Example 5.3
Typical breakthrough curves for the adsorption of
bovine serum albumin conjugated to arsanilic acid are shown
in Figure 5.9, on controlled pore glass and in Figure 5.10, on
Affinity Chromatography 429
1.0
X
0.5
5.3.1 Supports
0’
OH 0-CeN
t CNBr -
OH
t
\
t i?
OCNH,
C-NH
0
NH H II H
0-t-N-R
trourea Carbamrtc
Imtdocarbonace
A, EPOXY ACTIVATION
\
Ad..Fccr*uliq
J
cy--.-- c;d/"' o.cM, CM
t I
OH-yO7
I” L
/"t
cr*rr-
C-W_
iti-
A’ I”
+
ALSO COUPLING OF LIGAND-SH
‘;3:I
DIRECTLY WITH: YJU
t-sH
m
B
.C.O+l
REDUCTION
CH2 0
II
cti- C - NH,
CH- C-NH,
II
0 I
I
CH, CH,
H Y 1
CH -C-r;- Ct$ - N-C-CH
II k
0
t
C.N.CH, .CH, NH*
t k
Aminoethylated
Derivative
Hydrolysis IOH- 1
- CONH,
Carboxylated Derivative
CDNHNH,
t
Hydrazide
:, OH
I I
0-~i-O-S,iKH2)n R
:, 0 0
I I
R (CH2), Si(OCH2CH,), + HO-ii-O- )----_* -0-Si - 0- SiKH21n R
:, :, :,
I I
HO-ii-0 -0-Si - 0-Si (CH21n R
b :, dH
I
:,
6 0
CARRIER -O-ii (Ctiz 13 NH: 0 NO2 SODIUM DITHIONITE
:,
:, :, 0
0+ O &
CARRIER -0- .di (CHZ), NH2 t f I) -O-&CH~)3NH~(CH2), COOH
b
I
CHO
bH0
53.2 Spacers
Hydrocarbon spacers between ligand molecules and the
support matrix are used extensively in affinity
chromatography. The usefulness of these spacers has been
attributed to the relief of steric restrictions imposed by the
matrix backbone and to an increased flexibility and mobility
of the ligand as it protrudes farther into the solvent (35).
Shaltiel (36) has pointed out, however, that hydrocarbon
extensions by themselves may bind proteins through
mechanisms unrelated to specific recognition of the ligand.
Such binding is more likely to occur when the affinity
chromatography material is prepared by first coating the
matrix with hydrocarbon arms and then attaching the specific
ligand to those extensions. Those hydrocarbon spacers which
do not have an attached ligand may bind other proteins
through hydrophobic interactions, thereby reducing the
specificity of the system. Therefore, when spacers are
employed between the matrix and the ligand, it is advisable
first to synthesize the ligand with the hydrocarbon attached
and then to attach the elongated ligand to the support
matrix. This procedure will minimize the possibility of
hydrophobic interactions of the space interfering with the
intended affinity chromatography.
Example 5.4
Figure 5.20 (37) shows the effect of hydrocarbon
spacers on the reversible binding of glycogen phosphorylase b
to alkyl agarose supports. The columns were equilibrated at
22°C with a buffer of 50 mM sodium ,&glycerophosphate, 50
mM 2-mercaptoethanol and 1mM EDTA at pH 7.0 before
applying the protein sample to the column. Nonadsorbed
protein was washed off with the same buffer. Elution,
initiated at the arrow in the figure, was carried out with a
Affinity Chromatography 439
FRACTION NUMBER
5.3.3 Ligands
The ligand is usually a small molecule, covalently
attached to the solid support, that displays a special and
unique affinity for the molecule to be purified. This small
molecule must possess chemical groups that can be modified
for linkage to the solid support without destroying or
seriously altering interactions with the designated protein.
When the target protein has a high molecular weight, the
ligand groups which interact with that protein must be
sufficiently distant from the solid matrix to minimize steric
interference. The use of “spacers” to create this distance
has just been discussed.
Glyoxylase Glutathione PH
3 B - ffydroxysteroid pi
dehydrogenase
Derivative of
Type of Ligand Sepharose Comments
A 4.1 2.3
B 2.5 1.5
C 1.5 1.0
D 0.5 0.3
I I I I I I I I
0 1 2 3 4 5 6 7 0
Time of heating (hours)
Equilibration buffers
Molarlty 5 - loo mM
PH 5.5 - 9.0
Netill Ions Na, K, Mg, Ca, SC, Bo, Zm, Cu, Co, Ni,
Mn, Fe, Al, Cc.
ligands. The two techniques used most often are pH and salt
elution. The ionic strength of the eluant may be increased
in steps, pulses or gradients (54).
Nonspecific
Molarity 0.025 - 6 M
Specific elutants
Molarity 0.001 - 25 mM
~Q~~~N(&
((0 2s so
RETENTIONVOLUME (cc)
FILLER TUBE
LACK STOP
ING
OLLECT RING
IXED SEAL
ADJUSTABLE
SEAL
SUPPORT RIM
;fJL;STABLE
COLUMN TUBE
BED SUPPORT
FIXED CELL
;;JA;STABLE
FOOT UNIT
\ ,
(A) Mixing/adsorption /a) Washing
/
0’
0
0
0
/’
k l’
Wong and Charm (60) have taken the porous fabric bag
approach and converted it into a continuous belt process,
shown in Figure 5.28. The porous belt containing the
affinity particles forms a continuous loop which moves
through a series of tanks so that affinity adsorption,
washing, elution and regeneration occur sequentially. This
process requires that the ligate have a very high affinity for
the sorbent material.
A
Wash Derorbing
buffer agent
FURTHER
PROCESSING
IB
I Fraction collector I
rair 1
-irELn
_------TV--
---I
1
r---------
+&YE. R sz ’ s3
“0
81L-4l-l
“C
Pl
Rl R3 R4
iI
’ t I
’ ’ I
I I I
I I I
I I I
IL5
D
’
P .__? r___+----
I
M,
n-l I
J
Iii
i
c’ ,-*
--4 C __
1
i
n
t--* 11
I ’ f I
I; ' I i
11;
II ! !
t ’
, LB_ ___-_~~~~_~___~__-__J I
L--------_----_-___- __________ ~~~~_.__~~-~~~~~J
Oistributor
Anqulor
Position
- - --- -
o**.o”. **o
08
0
l o* O
.
PORTS
AFFINITY OR
FLOW
PATHS
-
INERT SUPPORT
ADSORBING DESORBING
BEAD RECYCLE
ADDITIONAL PROTEIN
RECOVERY
B)
ADSORUENT
SUSPENSION
SEPARATION
MEMBRANE
0
0 02 004 006 0.8
TIME (DIMENSIONLESS)
Luciferase Flavin
Concanavalin A (I - Antitrypsin
Herpes-specific membrane glycoproteins
Horseradish peroxidase
Human alkaline phosphatase
Interferon
Immunoglobulin A
Receptors for insulin
Receptors for epidermal growth factor
Porcine enteropeptidase
Rat brain glycopeptides
Thyrotropin
Ricinus communis (I- Fetoprotein
Wheat germ agglutin Erythropoietin
Glycophorin A
Receptors for insulin
Receptors for somatomedin C
Retinal glycoproteins
Concen*rat!on Pellicon
cassetfe system to 500 ml
I
Pasteurize with addition of
cilratc lo 0.5 M pH 7.55. 10 h 6O’C
+
Desalt on Sephadcx C-SO
2.5 liter column
I
750 ml solution
I
Sterile filter
I
Lyophilize
20% PEG
supernatant 115 0.655 75,325 70.2
DEAE unadsorbed
+ wash 215 0.321 69,015 64.4
0.8
0.2
-7 -6 -5
LOG ( TOTAL PEPTIDE) (14
Homogenize 1 kg E. cofi
+
Treat homogenate wilh 30% (NH.&Q
+
Treat supernatant with 65% (NH&SO4
+
Dissolve and dialyze pellet
1
Load on 17 ml column of anti-interferon-agar
1
Wash column; elute interferon at pH 2.5
1
Adjust interferon fractions to pH 4.5 (IM Tris)
and load on to CM-cellulose
c
Elute interferon with 0.5 M ammonium acetate
Fc IgG Urea
Binding Protein
Protein Eluent
a- Fetoprotein Urea
Choriogonadotropin Mg Cl2
Thyrotropin Guanidine
Urokinase Glycine - II Cl
After Chromatography
Nonadherent to column 10.9 z 1.2
5.8 REFERENCES
10. Nishikawa, A.H., Bailon, P., Ramel, A.H., Adv Expl Med
m, 42:33 (1974)
14. Yang, C.M., Tsao, G.T., Adv Biochem Enq, 25:l (1982)
15. Arnold, F.H., Blanch, H.W., Wilke, C.R., The Chem Enq
.I, 30:B9 (1985)
30. Ohlson, S., Hansson, L., Larsson, P.O., et al., FEBS Lett,
93:5 (1978)
39. Mosbach, K., Adv Enzvmol Relat Areas Mol Biol, 46:205
(1978)
40. Lis, H., Lotan, R., Sharon, N., Ann N Y Acad Sci,
234:232 (1974)
47. Lowe, C.R., Hans, M., Spibey, N., et al., Anal Biochem,
104:23 (1980)