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Determination of the structure of agarose gels by gel chromatography
B I O C H I M I C A ET B I O P H Y S I C A ACTA
199
Bt~A 25733 D E T E R M I N A T I O N OF T H E STRUCTURE OF AGAROSE GELS BY GEL CHROMATOGRAPHY* T ( ) I < V A I ~ D C. I . A U R E X T
I @ar/m~n! ((f 3ledica! Chemistry, University of Uppsala, Uppsala (Swe,~ten) (1,~ccci\xd A u g u s t 2 2 n d , 1960)
SUMMARY
Well-characterized fractions of the synthetic polysaccharide Ficoll have been chronlatographed on columns of pearl-condensed agarose gels of concentrations between 2 and 8 %. The results agree with the theory that the Ficoll is sterically excluded from the gel, which is made up of a random three-dimensional network of long fibers. The agarose fibers, which were estimated to be approx. 5° A in diameter, appear to contain 35-5o °o water.
INTRODUCTION
It has been proposed that the main selection nlechanisnl in gel chromatography (gel filtration) is a steric exclusion of solutes fronl the gel grains that constitute the solid phase in the chromatographic column ~-a. In an earlier paper of this series, LAURENT AND KILLANDER2 showed that the exclusion of proteins from dextran gels could be regarded as the exclusion of spheres from a three-dimensional network of randomly distributed rigid rods. A later report showed that data obtained from chromatography of proteins on a hyaluronic acid gel could be explained by the same nlechanism 4. The present communication describes a sinlilar investigation with agarose gels, which have also proved useful for preparative gel chromatography'5,6. The results provide an estimate of the size of the agarose fibers. M ~TERIAL
The Ficoll fractions were described in the preceding paperL Pearl-condensed agarose gels for gel chromatography were kindly supplied by Dr. B. GELOTTE, Pharmacia, Uppsala. The agarose concentrations of the gels and their lot numbers were: 2 °..o, 13°. 4 O~/o,Io7, I26 and I 5 I °' j6, .o, IO2 ; and 8 °"o, IoI. Human serum albumin was kindly supplied by Mr. H. BJORLING, AB KABI, Stockholm. METHODS
The agarose columns were packed in closed tubes with diameters of 2 cm and lengths of 20 to 32 cm. The construction of the chromatographic tubes was similar * T h i s p a p e r is N o . 14 in t h e s e r i e s I n t e r a c t i o n molecules.
between polysaccharides
and other macro-
t3iochzm. Biophys. ~4cta, 130 (1967) i 9 9 - 2 o §
200
T.C.
LAURENT
to that described by PORATH AND BENNICH8. The columns were eluted with upward flow at a rate of 4 ml/h using a peristaltic pump. The Ficoll samples (about 5 mg each) were applied in a volume of i ml. The effluent was collected in 2-ml fractions, as determined by weight, and the fractions were analyzed by the anthrone reaction 7,'~. Albumin was applied in samples of applox. 40 mg and was detected in the eluate by spectrophotometly at 280 mff. The total volumes of the columns were determined by chromatographing all20, which was located in the effluent with a Packard Tri-Carb liquid scintillation spectrometer. Corrections were made for the volume occupied by agarose itself and for the tritium exchange between the polysaccharide and water 1°. The void volumes of columns with agarose gels from 4 to 8 % concentrations were determined with a high-molecular-weight dextran (FDR 922, Pharmacia, mol. wt. 2.1o 6) or with the highest-molecular-weight fraction (F-IA) obtained from Ficoll F-I + 2 (7) by gel filtration on 2 % agarose. The void volumes of columns packed with 2 % agarose gels were estimated from runs with high-molecular-weight hyaluronie acid n or DNA F-1A c 0.7 Q
0.6
F-4+12 F-5
f_
~o.5 o
~
-6
F-16
0.4
8 0.3 g
~
0.2
/
2~ <
0.1
2;
'f
30
Vo
50
F-1A
70 T
Vt n f s o m e F i c o l l f r a c t i o n s o n a c o l u m n of a g a r o s e of S °Io c o n c e n t r a t i o n .
F-16
~ "+ 1"
Oot
r-'-+
o ~o t
60
Elution vol.(ml)
F i g . I. C h r o m a t o g r a p h y
*~ 0"3I
40
4'0
Vo F i g . 2. C h r o m a t o g r a p h y
¢.
_ F-1
so
~o Elution
vol.(ml)
7~0
J
J 8O
Vt
of s o m e F i c o l l f r a c t i o n s o n at c o l u m n of a g a r o s e of 0 ° o c o n c e n t r a t i o n .
B i o c h i m . t~iophys. A c t a , 1 3 6 (1967) I 9 9 2 0 5
STRUCTURE
OF AGAROSE GELS
201
(kindly supplied by Dr. O. ZETTERQUIST).The estimates are, however, uncertain (see below). Hyaluronie acid was analyzed in the eluate by the carbazol reaction n and D N A was determined by speetrophotometry at 260 raft. ttESULTS
Representative chromatograms of the various Ficoll fractions on agarose gels are shown in Figs. 1- 4. The partition coefficients, Kay, between the gel phase and the liquid phase for the various fractions were calculated with the relationship2: /~'av -
Ve-
~o
(i)
V t - Vo
Vo and Vt are the void volume and the total volume of the column and Ve the elution volume of the solute. The results are given in Table I.
~0.6
F-5 /~
~o,5
F-16 F-6
O,4 .c_ 0.3 c 0.2
\\\
0.1
o
bo
~o
Vo
do
60
70
80
1' vt
Elution vol.(ml)
l:ig. 3. C h r o m a t o g r a p h y
of s o m e F i c o l l f r a c t i o n s o n a c o l u m n o f a g a r o s e of 4 °o c o n c e n t r a t i o n .
C .o
"C< 0.3
oz
Pa F-5
EE o.2 oO
DNA
.~_~ ~L C
0.1
2o
< I
30 ~ 40
Vo Fig. 4. C h r o m a t o g r a p h y tration.
50
60
i
l
70
Elution vol. (ml)
L
L
801
t
90
i
100
Vt
of s o m e F i c o l l f r a c t i o n s a n d D N A
o n a c o l u m n o f a g a r o s e of 2 % c o n c e n -
Biochim. Biophys. Acta, 136 (1967) 199-2o.5
202
T. C. L A U R E N T
TABLE
[
PARTITION COJSIgFICIENTS, I~,~v, OBTAINED FOR THF FICOLL FRACTIONS AND SERUM ALBUMIN l N GILL CHROMATOGRAPIIY ON VARIOUS A1;AROSE GILLS
Ficoll fraction
Radicls of equiz,ale~t sDJ1cv# (d)
F - 4 + 12 F 13 F- 5 F- r 4 F-6 F-15 F-7 F- I 0 l ¢- I 7 ScrtlDl albumin
50.0 49.o 46.8 43-2 38.4 34.5 30.5 2(>.4 1S.8
Kay 2o'; Agarose lo! 13o
4°o Agarose lot lO 7
4 °o Agarosc lol 126
4 '~, Agarose lot z5r
6 '!i, Agaros¢ lot [()2
0.,%5
0.03 od~5 0.00 °-7° 0.7 I °.78 o.8 l o.83 O,85
0.57
0.01
o.35
o.3c
o.43 o.45 0.51
o.37 o.4o o.44 o.47 O.5 I O.57 O.Go o.7I
o.87 0.94 0.93 0.93
3 5 . 5 ( r e f . 2)
o.72
o.73
().~O
0.50 O.02 0.67
d 0,', Agaro,~c
l()t lOl
o.48
DISCUSSION
A theoretical model was proposed in an earlier paper to explain the gel chromatography process 2. The gel was treated as a three-dimensional network of rigid fibers, randomly distributed and infinitely long, and the partition of a substance between the gel and a solution was assumed to be determined by the space available to the molecules in the network. If the molecules are spherical, the partition coefficient can be calculated from an equation given by OGSTONla and written here in a modified form : Kay ~ e x p / - - ~ . L ( r s
b
rr)2/
(')
The partition coefficient, Kay, is equal to that fraction of the gel which is available to the nlolecules, rs is tile molecular radius of the substance. The rs values used in this paper were Stokes radii calculated from diffusion constants 14. rr is the radius of the fiber. L is the concentration of the fiber in the gel expressed as cm fiber/cm a. The model accounted very well for the chromatographic behavior of proteins oi1 various dextran gels" and on a hyaluronic acid gel 4, but the results indicated that the fiber diameters in the two types of gels were differenl. Therefore, gel chromatography was proposed as a convenient method for the characterization of gels, i.e. the determination of fiber diameters 4. The purposes of the present study were: first, to test the validity of our model for gel c h l o m a t o g r a p h y in still another s y s t e m ; and second, to determine the structure of agarose gels, which must be v e r y different from the polysaccharide gels studied earlier. To obtain homogeneous test substances for the chromatographic studies, the polysaccharide Ficoll was separated into sharp fractionsL Their molecular radii w e l e determined on a dextran geF and are tabulated in Table I. To confirm that a Ficoll polymer behaved in gel chromatography like a protein with the same radius, serum albumin was also chromatographed on the 4 and 8 °o agarose gels (see Table I). Biochim. t3iopkys. _4cta, 136 (1967) 1 9 9 - 2 o 5
S T R U C T U R E OF A G A R O S E G E L S
20~
The partition coefficients obtained for albumin are very close to those which would be expected for a Ficoll fraction with a radius of 35.5 •To calculate partition coefficients from gel chromatography data, it is necessary to have accurate values for the void volumes and total volumes of the columns. The / agarose should be correct, but there void volumes determined for columns of 4-8 o,,o is no criterion for judging the correctness of the values obtained for the 2 °o columns. In the absence of any rigorous method for the determination of the void volume in such cases, we have arbitrarily used the front of a DNA peak or a hyaluronic acid peak, as shown in Fig. 4. Therefore, the results obtained with 2 o/o agarose gels cannot be given much weight in the calculations. Differences in properties among agarose gels of the same concentrations have been reported* Three different batches of agarose 4 o,/J• were tested, and the data in Table I indicate that variations do occur**. All of the values used in the calculations below were obtained with lot xo7. To test whether the data given in Table I conform to Eqn. 2, the method of plotting proposed by SIEGEL AN[) MONTY15 was adopted. Eqn. 2 can be rearranged to : •
(-- In Kay)ite = (:r.L)Z/2 (l"s q- rr)
(3)
( In Kav)~/'i should thus be linearly related to the molecular radius, rs, of the Ficoll fractions. The plots shown in Fig. 5 indicate that this is the case for gels of 4, 6, and 8 % agarose. Probably the relationship holds also for 2 % agarose, but uncertainties in the data prevent drawing any firm conclusion. Apparently, the theoretical
8%
,
0.8
0.4
J
j•
J J 0.2
2 °/o
o.~
J J
J
-20
-10
0
10
20
30
40
50
Moleculor roctius(r s )(X) Fig. 5. P l o t t o t e s t if e x p e r i m e n t a l d a t a c o n f o r m t o E q n . 2. F o r d e t a i l s see t e x t . * B . ~ ) B E R G A N D L . PttlLIPSON, p e r s o n a l c o m m u n i c a t i o n . ** T h e b a t c h e s w e r e p r e p a r e d d i f f e r e n t l y a c c o r d i n g t o t h e m a n u f a c t u r e r .
Biochim. Biophys..4eta, 136 ( i 9 0 7 ) 1 9 9 - 2 o 5
204
T.C. LAURENT
model proposed earlier for dextran and hyaluronic acid gels accounts satisfactorily for gel filtration behavior on agarose gels as well. Fig. 5 m a y be used for the calculation of fiber parameters in the agarose (Table II). The intersection of the lines on the abscissa gives the negative value of the fiber radius. The radii are about 25 A, considerably larger than those of dextran (7 A) TABLE 1 1 PARAMI6TERS
CALCULATED
FOR
1;iber radi~*s (cm "~ ro s)
TIlE
FIBERS
IN
7"ola/ !eJ*gth <{/ fibem (1.) (CHg/CIll 3
Agarose, Agarose, Agarosc, Agarosc
8 c~'o 6% 4 °o 2 o,o
THE
2o.o 5.53 24.o 5.o*~ 25.6 2.30 Not possible to determine
10
II)
AGAROSI"
GELS
Co*~cem~'atio*~ orgat~ic material i*z fiber (g,,ml)
o.O5 o.<~5 o.S2
and hyaluronic acid (3.5 A) fibers. The parameter L (cm fiber/cm a gel) can be calculated from the slope of the line. The values are about five tinles lower than the corresponding values for dextran gels of the same concentrations. F r o m the known concentration of agarose in the gel and the intersection on the ordinate it is also possible to calculate the concentration of organic material in the fiber. The value o.65 to o.82 g/ml seems reasonable, as it indicates t h a t the h y d r a t e d agarose fibers contain 35-5o % water. The value of 1.6 used for the density of agarose in these calculations was estimated from the partial specific volume of polysaceharides similar to agarose. The difference between the value (o.82) obtained for the 4 % gel and t h a t (o.65) found for the 6 % and 8 % gels cannot be regarded as significant because the ordinate intercepts are based on rather long extrapolations and are squared in the calculations. The results described herein indicate that the exclusion properties of agarose gels can be explained in terms of a model in which the gel is considered to be composed of r a n d o m l y oriented and moderately h y d r a t e d fibers with dianleters of approx. 5o A. ACKNOWLEDGEMENTS This work was supported b y the Swedish Medical Research Council (Grant no. I3x-4), the Swedish Cancer Society and Gustaf V : s 8o-~rsfond. REFERENCES I J . PORATtt, Pz*re Appl. Chem., 0 (t903) 233.
2 3 4 5 0 7 8 9
T. PT. S. S. T. J. T.
C. LAURENT aXD J. KILLANm~;R, J. Chromatog., 14 (tq64) 317 . G. SQUIRE, Arch. Biochem. I~iophys., lo 7 (1904) 17I. C. LAURENT, Biochem. J., 93 (1904) 1o(~. HJERT~N, Yliochim. Biophys. Acta, 79 (I904) 393. BENGTSSON AND ],. PHILIPSON, Biochim. Biophys. Acta, 79 (t964) 399C. LAUREN'r AND K. A. GRAXAT~, Biochim. Biophys. Acta, 136 (t907) 19I. PORaTH .aND H. BENNICtt, Arch. Biochem. Biophys. Suppl., I (~96") I52. A. SCOTT JR. :XND IE. H. MELVIN, A~zal. Chem., 25 (I953) 1650.
Biochim. Bioph),s..4cla, 130 (i9(>7) I99--2o5
STRUCTURE OF AGAROSE GELS
205
N. V. B. MARSDEN, Ann. N . Y . Acad. Scz., 125 (1965) 428. T. C. LAURENT AND J. GERGELY, .J. Biol. Chem., 212 (1955) 325. Z. DlSCHE, J. Biol. Chem., 167 (1947) 189. A. G. OGSTON, Trans. Faraday Soe., 54 (1958) 1754. E. J. COHN AND J. T. EDSALL, Proteins, Amino Acids and Peptides, Reinhold, New Y o r k , 1943, p. 4o2. 15 L. M. SIEGEL AND I£. J. MONTY, Biochim. Biophys. dcta, I I 2 (1966) 346.
IO II 12 a3 14
Biochim. Biophys. Acta, 136 (1967) 199-2o 5
199
Bt~A 25733 D E T E R M I N A T I O N OF T H E STRUCTURE OF AGAROSE GELS BY GEL CHROMATOGRAPHY* T ( ) I < V A I ~ D C. I . A U R E X T
I @ar/m~n! ((f 3ledica! Chemistry, University of Uppsala, Uppsala (Swe,~ten) (1,~ccci\xd A u g u s t 2 2 n d , 1960)
SUMMARY
Well-characterized fractions of the synthetic polysaccharide Ficoll have been chronlatographed on columns of pearl-condensed agarose gels of concentrations between 2 and 8 %. The results agree with the theory that the Ficoll is sterically excluded from the gel, which is made up of a random three-dimensional network of long fibers. The agarose fibers, which were estimated to be approx. 5° A in diameter, appear to contain 35-5o °o water.
INTRODUCTION
It has been proposed that the main selection nlechanisnl in gel chromatography (gel filtration) is a steric exclusion of solutes fronl the gel grains that constitute the solid phase in the chromatographic column ~-a. In an earlier paper of this series, LAURENT AND KILLANDER2 showed that the exclusion of proteins from dextran gels could be regarded as the exclusion of spheres from a three-dimensional network of randomly distributed rigid rods. A later report showed that data obtained from chromatography of proteins on a hyaluronic acid gel could be explained by the same nlechanism 4. The present communication describes a sinlilar investigation with agarose gels, which have also proved useful for preparative gel chromatography'5,6. The results provide an estimate of the size of the agarose fibers. M ~TERIAL
The Ficoll fractions were described in the preceding paperL Pearl-condensed agarose gels for gel chromatography were kindly supplied by Dr. B. GELOTTE, Pharmacia, Uppsala. The agarose concentrations of the gels and their lot numbers were: 2 °..o, 13°. 4 O~/o,Io7, I26 and I 5 I °' j6, .o, IO2 ; and 8 °"o, IoI. Human serum albumin was kindly supplied by Mr. H. BJORLING, AB KABI, Stockholm. METHODS
The agarose columns were packed in closed tubes with diameters of 2 cm and lengths of 20 to 32 cm. The construction of the chromatographic tubes was similar * T h i s p a p e r is N o . 14 in t h e s e r i e s I n t e r a c t i o n molecules.
between polysaccharides
and other macro-
t3iochzm. Biophys. ~4cta, 130 (1967) i 9 9 - 2 o §
200
T.C.
LAURENT
to that described by PORATH AND BENNICH8. The columns were eluted with upward flow at a rate of 4 ml/h using a peristaltic pump. The Ficoll samples (about 5 mg each) were applied in a volume of i ml. The effluent was collected in 2-ml fractions, as determined by weight, and the fractions were analyzed by the anthrone reaction 7,'~. Albumin was applied in samples of applox. 40 mg and was detected in the eluate by spectrophotometly at 280 mff. The total volumes of the columns were determined by chromatographing all20, which was located in the effluent with a Packard Tri-Carb liquid scintillation spectrometer. Corrections were made for the volume occupied by agarose itself and for the tritium exchange between the polysaccharide and water 1°. The void volumes of columns with agarose gels from 4 to 8 % concentrations were determined with a high-molecular-weight dextran (FDR 922, Pharmacia, mol. wt. 2.1o 6) or with the highest-molecular-weight fraction (F-IA) obtained from Ficoll F-I + 2 (7) by gel filtration on 2 % agarose. The void volumes of columns packed with 2 % agarose gels were estimated from runs with high-molecular-weight hyaluronie acid n or DNA F-1A c 0.7 Q
0.6
F-4+12 F-5
f_
~o.5 o
~
-6
F-16
0.4
8 0.3 g
~
0.2
/
2~ <
0.1
2;
'f
30
Vo
50
F-1A
70 T
Vt n f s o m e F i c o l l f r a c t i o n s o n a c o l u m n of a g a r o s e of S °Io c o n c e n t r a t i o n .
F-16
~ "+ 1"
Oot
r-'-+
o ~o t
60
Elution vol.(ml)
F i g . I. C h r o m a t o g r a p h y
*~ 0"3I
40
4'0
Vo F i g . 2. C h r o m a t o g r a p h y
¢.
_ F-1
so
~o Elution
vol.(ml)
7~0
J
J 8O
Vt
of s o m e F i c o l l f r a c t i o n s o n at c o l u m n of a g a r o s e of 0 ° o c o n c e n t r a t i o n .
B i o c h i m . t~iophys. A c t a , 1 3 6 (1967) I 9 9 2 0 5
STRUCTURE
OF AGAROSE GELS
201
(kindly supplied by Dr. O. ZETTERQUIST).The estimates are, however, uncertain (see below). Hyaluronie acid was analyzed in the eluate by the carbazol reaction n and D N A was determined by speetrophotometry at 260 raft. ttESULTS
Representative chromatograms of the various Ficoll fractions on agarose gels are shown in Figs. 1- 4. The partition coefficients, Kay, between the gel phase and the liquid phase for the various fractions were calculated with the relationship2: /~'av -
Ve-
~o
(i)
V t - Vo
Vo and Vt are the void volume and the total volume of the column and Ve the elution volume of the solute. The results are given in Table I.
~0.6
F-5 /~
~o,5
F-16 F-6
O,4 .c_ 0.3 c 0.2
\\\
0.1
o
bo
~o
Vo
do
60
70
80
1' vt
Elution vol.(ml)
l:ig. 3. C h r o m a t o g r a p h y
of s o m e F i c o l l f r a c t i o n s o n a c o l u m n o f a g a r o s e of 4 °o c o n c e n t r a t i o n .
C .o
"C< 0.3
oz
Pa F-5
EE o.2 oO
DNA
.~_~ ~L C
0.1
2o
< I
30 ~ 40
Vo Fig. 4. C h r o m a t o g r a p h y tration.
50
60
i
l
70
Elution vol. (ml)
L
L
801
t
90
i
100
Vt
of s o m e F i c o l l f r a c t i o n s a n d D N A
o n a c o l u m n o f a g a r o s e of 2 % c o n c e n -
Biochim. Biophys. Acta, 136 (1967) 199-2o.5
202
T. C. L A U R E N T
TABLE
[
PARTITION COJSIgFICIENTS, I~,~v, OBTAINED FOR THF FICOLL FRACTIONS AND SERUM ALBUMIN l N GILL CHROMATOGRAPIIY ON VARIOUS A1;AROSE GILLS
Ficoll fraction
Radicls of equiz,ale~t sDJ1cv# (d)
F - 4 + 12 F 13 F- 5 F- r 4 F-6 F-15 F-7 F- I 0 l ¢- I 7 ScrtlDl albumin
50.0 49.o 46.8 43-2 38.4 34.5 30.5 2(>.4 1S.8
Kay 2o'; Agarose lo! 13o
4°o Agarose lot lO 7
4 °o Agarosc lol 126
4 '~, Agarose lot z5r
6 '!i, Agaros¢ lot [()2
0.,%5
0.03 od~5 0.00 °-7° 0.7 I °.78 o.8 l o.83 O,85
0.57
0.01
o.35
o.3c
o.43 o.45 0.51
o.37 o.4o o.44 o.47 O.5 I O.57 O.Go o.7I
o.87 0.94 0.93 0.93
3 5 . 5 ( r e f . 2)
o.72
o.73
().~O
0.50 O.02 0.67
d 0,', Agaro,~c
l()t lOl
o.48
DISCUSSION
A theoretical model was proposed in an earlier paper to explain the gel chromatography process 2. The gel was treated as a three-dimensional network of rigid fibers, randomly distributed and infinitely long, and the partition of a substance between the gel and a solution was assumed to be determined by the space available to the molecules in the network. If the molecules are spherical, the partition coefficient can be calculated from an equation given by OGSTONla and written here in a modified form : Kay ~ e x p / - - ~ . L ( r s
b
rr)2/
(')
The partition coefficient, Kay, is equal to that fraction of the gel which is available to the nlolecules, rs is tile molecular radius of the substance. The rs values used in this paper were Stokes radii calculated from diffusion constants 14. rr is the radius of the fiber. L is the concentration of the fiber in the gel expressed as cm fiber/cm a. The model accounted very well for the chromatographic behavior of proteins oi1 various dextran gels" and on a hyaluronic acid gel 4, but the results indicated that the fiber diameters in the two types of gels were differenl. Therefore, gel chromatography was proposed as a convenient method for the characterization of gels, i.e. the determination of fiber diameters 4. The purposes of the present study were: first, to test the validity of our model for gel c h l o m a t o g r a p h y in still another s y s t e m ; and second, to determine the structure of agarose gels, which must be v e r y different from the polysaccharide gels studied earlier. To obtain homogeneous test substances for the chromatographic studies, the polysaccharide Ficoll was separated into sharp fractionsL Their molecular radii w e l e determined on a dextran geF and are tabulated in Table I. To confirm that a Ficoll polymer behaved in gel chromatography like a protein with the same radius, serum albumin was also chromatographed on the 4 and 8 °o agarose gels (see Table I). Biochim. t3iopkys. _4cta, 136 (1967) 1 9 9 - 2 o 5
S T R U C T U R E OF A G A R O S E G E L S
20~
The partition coefficients obtained for albumin are very close to those which would be expected for a Ficoll fraction with a radius of 35.5 •To calculate partition coefficients from gel chromatography data, it is necessary to have accurate values for the void volumes and total volumes of the columns. The / agarose should be correct, but there void volumes determined for columns of 4-8 o,,o is no criterion for judging the correctness of the values obtained for the 2 °o columns. In the absence of any rigorous method for the determination of the void volume in such cases, we have arbitrarily used the front of a DNA peak or a hyaluronic acid peak, as shown in Fig. 4. Therefore, the results obtained with 2 o/o agarose gels cannot be given much weight in the calculations. Differences in properties among agarose gels of the same concentrations have been reported* Three different batches of agarose 4 o,/J• were tested, and the data in Table I indicate that variations do occur**. All of the values used in the calculations below were obtained with lot xo7. To test whether the data given in Table I conform to Eqn. 2, the method of plotting proposed by SIEGEL AN[) MONTY15 was adopted. Eqn. 2 can be rearranged to : •
(-- In Kay)ite = (:r.L)Z/2 (l"s q- rr)
(3)
( In Kav)~/'i should thus be linearly related to the molecular radius, rs, of the Ficoll fractions. The plots shown in Fig. 5 indicate that this is the case for gels of 4, 6, and 8 % agarose. Probably the relationship holds also for 2 % agarose, but uncertainties in the data prevent drawing any firm conclusion. Apparently, the theoretical
8%
,
0.8
0.4
J
j•
J J 0.2
2 °/o
o.~
J J
J
-20
-10
0
10
20
30
40
50
Moleculor roctius(r s )(X) Fig. 5. P l o t t o t e s t if e x p e r i m e n t a l d a t a c o n f o r m t o E q n . 2. F o r d e t a i l s see t e x t . * B . ~ ) B E R G A N D L . PttlLIPSON, p e r s o n a l c o m m u n i c a t i o n . ** T h e b a t c h e s w e r e p r e p a r e d d i f f e r e n t l y a c c o r d i n g t o t h e m a n u f a c t u r e r .
Biochim. Biophys..4eta, 136 ( i 9 0 7 ) 1 9 9 - 2 o 5
204
T.C. LAURENT
model proposed earlier for dextran and hyaluronic acid gels accounts satisfactorily for gel filtration behavior on agarose gels as well. Fig. 5 m a y be used for the calculation of fiber parameters in the agarose (Table II). The intersection of the lines on the abscissa gives the negative value of the fiber radius. The radii are about 25 A, considerably larger than those of dextran (7 A) TABLE 1 1 PARAMI6TERS
CALCULATED
FOR
1;iber radi~*s (cm "~ ro s)
TIlE
FIBERS
IN
7"ola/ !eJ*gth <{/ fibem (1.) (CHg/CIll 3
Agarose, Agarose, Agarosc, Agarosc
8 c~'o 6% 4 °o 2 o,o
THE
2o.o 5.53 24.o 5.o*~ 25.6 2.30 Not possible to determine
10
II)
AGAROSI"
GELS
Co*~cem~'atio*~ orgat~ic material i*z fiber (g,,ml)
o.O5 o.<~5 o.S2
and hyaluronic acid (3.5 A) fibers. The parameter L (cm fiber/cm a gel) can be calculated from the slope of the line. The values are about five tinles lower than the corresponding values for dextran gels of the same concentrations. F r o m the known concentration of agarose in the gel and the intersection on the ordinate it is also possible to calculate the concentration of organic material in the fiber. The value o.65 to o.82 g/ml seems reasonable, as it indicates t h a t the h y d r a t e d agarose fibers contain 35-5o % water. The value of 1.6 used for the density of agarose in these calculations was estimated from the partial specific volume of polysaceharides similar to agarose. The difference between the value (o.82) obtained for the 4 % gel and t h a t (o.65) found for the 6 % and 8 % gels cannot be regarded as significant because the ordinate intercepts are based on rather long extrapolations and are squared in the calculations. The results described herein indicate that the exclusion properties of agarose gels can be explained in terms of a model in which the gel is considered to be composed of r a n d o m l y oriented and moderately h y d r a t e d fibers with dianleters of approx. 5o A. ACKNOWLEDGEMENTS This work was supported b y the Swedish Medical Research Council (Grant no. I3x-4), the Swedish Cancer Society and Gustaf V : s 8o-~rsfond. REFERENCES I J . PORATtt, Pz*re Appl. Chem., 0 (t903) 233.
2 3 4 5 0 7 8 9
T. PT. S. S. T. J. T.
C. LAURENT aXD J. KILLANm~;R, J. Chromatog., 14 (tq64) 317 . G. SQUIRE, Arch. Biochem. I~iophys., lo 7 (1904) 17I. C. LAURENT, Biochem. J., 93 (1904) 1o(~. HJERT~N, Yliochim. Biophys. Acta, 79 (I904) 393. BENGTSSON AND ],. PHILIPSON, Biochim. Biophys. Acta, 79 (t964) 399C. LAUREN'r AND K. A. GRAXAT~, Biochim. Biophys. Acta, 136 (t907) 19I. PORaTH .aND H. BENNICtt, Arch. Biochem. Biophys. Suppl., I (~96") I52. A. SCOTT JR. :XND IE. H. MELVIN, A~zal. Chem., 25 (I953) 1650.
Biochim. Bioph),s..4cla, 130 (i9(>7) I99--2o5
STRUCTURE OF AGAROSE GELS
205
N. V. B. MARSDEN, Ann. N . Y . Acad. Scz., 125 (1965) 428. T. C. LAURENT AND J. GERGELY, .J. Biol. Chem., 212 (1955) 325. Z. DlSCHE, J. Biol. Chem., 167 (1947) 189. A. G. OGSTON, Trans. Faraday Soe., 54 (1958) 1754. E. J. COHN AND J. T. EDSALL, Proteins, Amino Acids and Peptides, Reinhold, New Y o r k , 1943, p. 4o2. 15 L. M. SIEGEL AND I£. J. MONTY, Biochim. Biophys. dcta, I I 2 (1966) 346.
IO II 12 a3 14
Biochim. Biophys. Acta, 136 (1967) 199-2o 5