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Molecular weight determination by polyacrylamide gradient gel electrophoresis
Blood Transfusion and Immunohaematology Tome XXVII. - - N ° 6. - - 1984
691
Molecular weight determination by polyacrylamide gradient gel electrophoresis by P. Lambin Laboratoire d'Immunochimie des Prot6ines, Centre National de Transfusion Sanguine, PARIS.
U
LTRACENTRIFUGATIONand gel filtration h a v e b e e n u s e d f o r molecular weight d e t e r m i n a t i o n of proteins and p o l y p e p t i d e chains. More recently, it a p p e a r e d t h a t electrophoretic techniques using p o l y a c r y l a m i d e gels as s u p p p o r t i n g m e d i u m can be utilized for this p u r p o s e w i t h a c c u r a c y a n d rapidness. As a m a t t e r of fact, p o l y a c r y l a m i d e gel is a good s u p p o r t i n g med i u m for electrophoresis not only by p r c v e n t i n g diffusion and convection b u t also b y i n t e r a c t i n g with the m i g r a t i n g particles. This i n t e r a c t i o n depends on the particule size. I t has b e e n t e r m e d " m o lecular sieving effect". Thus in p o l y a c r y l a m i d e gels, proteins are s e p a r a t e d according both to charge and m o l e c u l a r size. The sieving effect being related to gel density, it is necessary to recall briefly w h a t is the s t r u c t u r e of p o l y a c r y l a m i d e gels.
3 Structure of p o l y a c r y I a m i d e gels Polyacrylamide gels are the p r o d u c t of the p o l y m e r i z a t i o n and cross-linking of the m o n o m e r of a c r y l a m i d e and a crosslinker: N, N'-methylene-bis-acrylamide. Gel density is defined by two values T and C.
692 T c o r r e s p o n d s to the total p e r c e n t a g e a c r y l a m i d e and bis-acrylamide.
L A M B I N P.
concentration
of b o t h
C to the p e r c e n t a g e c o n c e n t r a t i o n of c r o s s l i n k e r relative to the total c o n c e n t r a t i o n T. Schematically two kinds of gels can be u s e d f o r p r o t e i n s separation: gels of c o n s t a n t c o n c e n t r a t i o n a n d p o l y a c r y l a m i d e gradients. Gels of c o n s t a n t c o n c e n t r a t i o n in c o n j o n c t i o n w i t h a discontinuous b u f f e r s y s t e m w e r e i n t r o d u c e d b y ORSTEIN [17] a n d DAVIS [2]. Several y e a r s later, the use of p o l y a c r y l a m i d e gradients was d e s c r i b e d b y MARGOLIS a n d KENRICK [14]. R e p r o d u c i b l e a n d precise profils of gradients (concave or linear) can b e n o w o b t a i n e d b y a u t o m a t i c gradients m a k e r s , peristaltic p u m p and a vertical slab gel system. I n o r d e r to o b t a i n o p t i m u m conditions of polymerization, time, t e m p e r a t u r e , p H a n d c o n c e n t r a t i o n of reagents m u s t b e carefully precised [9, 12]. Practically, T can v a r y f r o m 2 to 30% a n d C f r o m 3 to 8.5%. As well as the use of density g r a d i e n t u l t r a c e n t r i f u g a t i o n h a d o p e n e d u p a new d i m e n s i o n for the s e p a r a t i o n of proteins, polyacrylamide gradients allowed a b e t t e r resolution t h a n gels of c o n s t a n t concentration. The gradients can b e u s e d for the s e p a r a t i o n of m u l t i c o m p o n e n t s m i x t u r e s in one d i m e n t i o n a l [14] or in two dimentional techniques [16].
General principles of molecular weight determination of proteins by electrophoresis T r e a t m e n t of proteins b y an ionic d e t e r g e n t such as s o d i u m dodecyl sulfate (SDS) increases c o n s i d e r a b l y their e l e c t r o p h o r e t i c mobility and provides a relative u n i f o r m i t y of t h e i r charge density in such a m a n n e r t h a t their m i g r a t i o n distance in a p o l y a c r y l a m i d e gel can be directly related to their m o l e c u l a r weight (MW) [18, 8, 26, 15]. I n the absence of ionic detergents, e l e c t r o p h o r e t i c mobility depends b o t h on charge a n d m o l e c u l a r size and t h e r e f o r e only an indirect m e a s u r e m e n t of the MW is possible. The m e t h o d s u s e d can be classified into f o u r m a j o r categories according to their p e r f o r m a n c e in the p r e s e n c e or in the absence of SDS a n d the use of c o n s t a n t c o n c e n t r a t i o n or linear gradient of polyacrylamide. 1. MW d e t e r m i n a t i o n in gel of c o n s t a n t c o n c e n t r a t i o n w i t h proteins previously t r e a t e d b y SDS was i n t r o d u c e d b y SHAPIRO et al. [25]. In this technique, a linear relationship is o b t a i n e d b y plotting log
MOLECULAR WEIGHT DETERMINATION
693
(MW) against relative mobility. However, the extent of this relationship is limited for a given gel concentration: 10% gels for MW between 11,000 to 70,000: 5% gels for MW between 25,000 and 200,000 [3]. In addition, this relationship cannot be applied with a good accuracy to glycoproteins [20, 1, 6, 5, 27]. 2. MW d e t e r m i n a t i o n in gels of a constant concentration and in the absence of SDS has been described b y several authors and in p a r t i c u l a r by HEDRICK and SMITH [7] and by RODBAgD et al. [21, 22]. In these methods, several electrophoreses are p e r f o r m e d in gels of different densities. A r e t a r d a t i o n coefficient is obtained for a given protein. This coefficient is itself related to the MW. The m a j o r inconvenient of these techniques is the repetition of electrophoreses in gels of different concentrations. 3. MW d e t e r m i n a t i o n in gradient gels c o m b i n e d with SDS was i n t r o d u c e d by LAMBIN et al. [12, 9]. This m e t h o d provides a good accuracy for a wider range of MW than conventional gels. 4. Several methods have been described for MW d e t e r m i n a t i o n in gradient gels and in the absence of SDS [4, 13, 24]. We will r e p o r t here the m e t h o d developped in our lab [10, 11]. Molecular weight determination in the presence of SDS
After electrophoresis in a gradient of polyacrylamide, a linear relationship was empirically established [12] between the log (MW) of a protein and the log of the polyacrylamide concentration (T) reached by this protein. This relationship can be written as follows log (MW) = a log (T) + b (1) where a and b are the slope and intercept of the linear regression respectively. This relationship proved to be suitable for proteins having MWs between 104 to 106 with 3 to 30% linear gradients of polyacrylamide. It can be also applied to polypeptide chains of proteins after reduction of disulfide bonds by reducing agents such as 2- mercaptoethanol. The preliminary results obtained with 10 proteins were confirmed with m o r e than 40 proteins and polypeptide chains [9, 10]. This n u m b e r of proteins allowed a statistical analysis of the results concerning the extend of the relationship and the accuracy of the d e t e r m i n a t i o n of the MW of an u n k n o w n protein. Fig. l a and l b show the results of two experiments p e r f o r m e d in 3 to 30% linear gradients with b o t h reduced and u n r e d u c e d proteins. Best linear fit and 95% confidence limits, for a single observation, were calculated. Fig. 2a and 2b, show the correlation between expected and observed MWs.
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MOLECULAR WEIGHT DETERMINATION
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LAMBIN P.
S i m i l a r e x p e r i m e n t s w e r e p e r f o r m e d w i t h d i f f e r e n t p r o f i l s of g r a d i e n t s a n d r e l a t i v e p e r c e n t a g e of b i s - a c r y l a m i d e , (see table, I). F r o m t h e s e e x p e r i m e n t s , it w a s p o s s i b l e to d r a w t h e f o l l o w i n g conclusion: TABLE I Data obtained with three different structures of gel and two di[[erent protein incubation conditions. GEL STRUCTURE
T C T C T C T C T C
= = = = = = = = = =
3-30% 8.4% 3-30% 8.4% 3-30% 3.8% 3-30% 3.8% 2-10% 3.8%
MEAN
PROTEIN TREATMENT
N
r
SDS
34
0.998
5.9
SDS + 2-Met
31
0.997
4.9
SDS
33
0.995
7.3
SDS + 2-Mer
31
0.991
7.8
SDS
8
0.996
5.5
DEVIATION
(%)
a) a s i m i l a r a c c u r a c y w a s o b t a i n e d i n t h e a b s e n c e o r i n t h e p r e s e n c e of r e d u c i n g a g e n t : i n f a c t c o r r e l a t i o n c o e f f i c i e n t s w e r e r e s p e c t i v e l y 0.998 a n d 0.997 a n d m e a n d e v i a t i o n s , -- 5.9% a n d +- 4.9% (C = 8.4%). b) T h e i n c r e a s e i n c r o s s l i n k e r f r o m 3.8% to 8.4% i m p r o v e s s l i g h t l y t h e a c c u r a c y f o r t h e e v a l u a t i o n of a MW: m e a n d e v i a t i o n s w e r e r e s p e c t i v e l y -+ 7.3% a n d +--5.9% i n SDS. c) G l y c o p r o t e i n s a p p e a r to b e h a v e i n t h e s a m e w a y as do o t h e r p r o t e i n s . Of t h e 34 p r o t e i n s a n a l y z e d , 15 w e r e g l y c o p r o t e i n s w i t h c a r b o h y d r a t e c o n t e n t r a n g i n g f r o m 4 to 45%; slopes, i n t e r c e p t s , c o r r e l a t i o n c o e f f i c i e n t s a n d m e a n d e v i a t i o n s o b t a i n e d w i t h glycoproteins a n d proteins w i t h o u t c a r b o h y d r a t e s are given in Table H. Differences b e t w e e n the two regression lines were small a n d n o t s t a t i s t i c a l l y s i g n i f i c a n t (t: 0.45 < 1.96; p < 0.05). I t s h o u l d b e n o t e d t h a t w h e n p r o t e i n s a r e s u b j e c t e d to e l e c t r o p h o r e s i s i n t h e a b s e n c e of SDS i n c u b a t i o n , n o l i n e a r r e g r e s s i o n c a n b e e s t a b l i s h e d . I n c o n t r a s t , a l i n e a r r e g r e s s i o n w a s o b t a i n e d i n t h e case of p r o t e i n s m o n o m e r s a n d their homologous oligomers which have the same charge density p e r m o l e c u l e (see Fig. 3).
MOLECULAR WEIGHT DETERMINATION
697
TABLE II
Results obtained by 3 to 30% gradient gel electrophoresis (C = 8.4%) for proteins w i t h or w i t h o u t carbohydrates.
PROTEIN
N
SLOPE
INTERCEPT
W i t h c a r b o h y d r a t e s .. Without carbohydrates All p r o t e i n s . . . . . . . . . .
15 19 34
- - 3.421 3.426 - - 3.430
8.68 8.68 8.69
-
-
MEAN DEVIATION
(%)
0.998 0.996 0.998
MW 10
6
-
IgA
10 5
Tf
'
'
'
'
'
'
I
1
I
FIc. 3 . - L i n e a r r e g r e s s i o n o b t a i n e d w i t h t h r e e p r o t e i n m o n o m e r s a n d t h e i r h o m o l o g o u s o l i g o m e r s a f t e r elect r o p h o r e s i s i n a 3 to 30% g r a d i e n t gel in t h e a b s e n c e of SDS (Alb: a l b u m i n , Tf: t r a n s f e r r i n , a n d IgA: i m m u n o g l o b u l i n A).
6.6 5.3 5.9
698
LAMBIN P.
Validity of relation (1) was c o n f i r m e d b y e x p e r i m e n t a l a n d comp u t e r s t i m u l a t i o n studies of PODUSLO a n d RODBAI~D [19]. SDS t r e a t m e n t of p r o t e i n s a n d linear p o l y a c r y l a m i d e g r a d i e n t gel e l e c t r o p h o r e s i s p e r m i t the e s t i m a t i o n of the MW of a p r o t e i n in the r a n g e of 10 4 to 10 6 in a single gel w i t h an e r r o r of -+ 6% w h a t e v e r the c a r b o h y d r a t e content. Molecular w e i g h t d e t e r m i n a t i o n in the a b s e n c e of S D S SDS being a dissociating agent, the MW of a native p r o t e i n m a y differ f r o m the MW f o u n d in the p r e s e n c e of SDS w h e n this p r o t e i n is c o m p o s e d of several subunits held t o g e t h e r b y non-covalent bonds. As previously m e n t i o n e d , in the absence of ionic detergents, the e ] e c t r o p h o r e t i c mobility of a p r o t e i n in a p o l y a c r y l a m i d e gel, depends b o t h on charge a n d size. I n these conditions, only an indirect e s t i m a t i o n of MW is possible. As s h o w n b y LAMBIN a n d FINE [ 11] linear gradient of polyacrylam i d e can b e succesfully u s e d f o r this estimation. I n this m e t h o d , isolated p r o t e i n s or p r o t e i n s m i x t u r e s are s u b j e c t e d to e l e c t r o p h o r e s i s in the s a m e slab gel a n d at c o n s t a n t voltage during several periods of t i m e b e t w e e n 1 to 8 hours, After 8 hours, the gels are stained and the p r o t e i n s position is m e a s u r e d . A relationship was first established b e t w e e n the distance of m i g r a t i o n a n d t i m e of electrophoresis. I n fact, it w a s f o u n d t h a t the b e s t c o r r e l a t i o n coefficient b e t w e e n t i m e a n d distance for all p r o t e i n s u n d e r s t u d y (of v e r y different free e l e c t r o p h o r e t i c mobilities a n d shape) w a s o b t a i n e d w h e n plotting the s q u a r e r o o t of the t i m e of e l e c t r o p h o r e s i s versus m i g r a t i o n distances according to the equation. ta~ - a D + b (2) t being the t i m e of e l e c t r o p h o r e s i s D the distance m i g r a t e d b y a p r o t e i n a a n d b Mope and i n t e r c e p t of the regression lines. W h e n volt × h o u r p r o d u c t s are b e t w e e n 40 a n d 800, c o r r e l a t i o n coefficients are b e t t e r t h a n 0.99 for all the p r o t e i n s s u b j e c t e d to electrophoresis. This e q u a t i o n w a s validated over an extended r a n g e of conditions such as p H ( f r o m 7 to 10) a n d b u f f e r s c o m p o s i t i o n ( s o d i u m - p h o s p h a t e , Tris-borate, Veronal-Tris). H o w e v e r , for p r a c t i c a l reasons, these b u f f e r s m u s t b e selected in o r d e r to give a sufficient e l e c t r o p h o r e t i c mobility to the p r o t e i n s u n d e r study. Fig. 4 illustrates the results o b t a i n e d in one e x p e r i m e n t .
699
MOLECULAR H/EIGHT DETERMINATION
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60
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Distance (ram)
FIG. 4. - - Plots of square root of time of electrophoresis (in Veronal-Tris-Glycine buffer pH 9.8) against distance migrated by 10 non-denatured proteins. a: b: c: d:
6~2 macroglobulin, thyroglobulin, IgA (dimer), catalase,
e: f: g: h:
IgA (monomer), ceruloplasmin, albumin, streptokinase,
i: ovalbumin, j : soybean inhibitor.
The l i n e a r r e g r e s s i o n s w e r e u t i l i z e d in a s e c o n d step f o r MW d e t e r m i n a t i o n . As s h o w n in figure 5 w h e n log (MWs) a r e p l o t t e d a g a i n s t log of slopes (a) of the r e g r e s s i o n lines o b t a i n e d as above, t h e r e is a l i n e a r r e l a t i o n s h i p b e t w e e n t h e two values. Figure 5 also shows the 95% c o n f i d e n c e l i m i t s o b t a i n e d for the e s t i m a t i o n of the MW of an u n k n o w p r o t e i n . Deviations between expected and o b s e r v e d MW a r e m o r e i m p o r t a n t t h a n in t h e p r e s e n c e of SDS b u t i n f e r i o r to 20%. This a c c u r a c y is of p r o b a b i l i t y t h a t MW f o u n d by the c o n s i s t s of one or bonds.
q u i t e s u f f i c i e n t to c o n c l u d e w i t h a high d e g r e e the MW c o r r e s p o n d s once, twice or m o r e the SDS m e t h o d , a n d t h e r e f o r e t h a t t h e m o l e c u l e several s u b u n i t s h e l d t o g e t h e r by non-covalent
700
L A M B I N P.
MW 106
,,':e
i/
105
, /.j,/ / 104 •01
FI~. 5.
.02
-
-
.
.
.04 .
.
. .06 . .
.08 .1 .
.2
.4
SLope
Plots of log MWs of 10 proteins versus log of slopes (see fig. 4). (--.) best linear fit (. . . . ) 95% confidence limits.
W h e n volt × h o u r p r o d u c t is a b o v e 1,000, equation (2) is no m o r e verified a n d as p r e d i c t e d b y RODBARD et al. [23] log of t i m e of electrop h o r e s i s b e c o m e s p r o p o r t i o n a l to the distance of migration. This was checked w i t h p r o t e i n s of MWs b e t w e e n 6 × 104 a n d 106; c o r r e l a t i o n coefficients being a b o v e 0.998 (LAMBIN et al. u n p u b l i s h e d data): These results c o n f i r m t h a t t h e r e is no d e a d stop for a p r o t e i n in a gradient; given enough time, it can r e a c h a n y finite distance. I t should b e n o t e d t h a t slopes of regression lines can b e u s e d as previously d e c r i b e d to d e t e r m i n e MWs of proteins. Linear gradients of p o l y a c r y l a m i d e p r o v i d e at the p r e s e n t time, the b e s t a c c u r a c y for the e s t i m a t i o n o f the MW of a p r o t e i n or a glycoprotein. I t s e e m s p r o b a b l e t h a t in these gels, the m o l e c u l a r sieving effect could p r e d o m i n a t e a n d minimize the differences in charge a n d shape of the p r o t e i n s w h i c h influence the results o b s e r v e d b y e l e c t r o p h o r e s i s in conventional p o l y a c r y l a m i d e gels.
MOLECULAR WEIGHT DETERMINATION
701
I n s u m m a r y , we suggest the following strategy for MW estimation of an u n k n o w n protein by electrophoresis in linear gradients of polyacrylamide. 1. MW estimation of the protein in SDS. 2. MW estimation of its polypeptide chains in SDS and a reducing agent. 3. MW estimation of the native protein, and c o m p a r i s o n of this result with that obtained in SDS in o r d e r to determine the n u m b e r of its subunits, the MW obtained in SDS being taken as a reference because of the best a c c u r a c y given by this method. Request reprints from: P. LAMBIN, C.N.T.S., Laboratoire d'Immunochimie des Prot6ines, 6, rue A.-Cabanel, 75739 PARIS Cedex 15.
REFERENCES [1] BANKERG.A. and COTMANC.W. - - Measurement of free electrophoretic mobility and retardation coefficient of protein-sodium dodecyl sulfate complexed by gel electrophoresis. J. Biol. Chem., 247, 5856, 1972. [2] DAvis B.J. - - Disc electrophoresis: methods and application to h u m a n serum proteins. Annals N e w York Acad. Sci., 121, 404, 1964. [3] DUNKERA.K. and RUECKERTR.R. - - Observations on molecular weight determination on polyacrylamide gel. J. Biol. Chem., 244, 5074, 1969. [4] FELGENHAUERN. - - Evaluation of molecular size by gel electrophoresis techniques. Hoppe-Seyler's Z. Physiol. Chem., 355, 1281, 1974. [5] FRANK R.N. and RODBARDD. - - Precision of sodium dodecyl sulfatepolyacrylamide-gel electrophoresis for the molecular weight estimation of a membrane glycoprotein: studies on bovine rhodopsin. Arch. Biochem. Biophys., 171, 1, 1975. [6] GREFRATH S.P. and REYNOLDS J.A. - - The molecular weight of the major glycoprotein from the human erythrocyte membrane. Prec. Nat. Acad. Sci. U.S.A., 71, 3913, 1974. [7] HEDRICKff.I. and SMITH A.ff. - - Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys., 126, 155, 1968.
LAMBIN P.
702
[8] KATZ S., SHAW M.E., CHILLAGS. and MILLER J. - - Protein-ionic d e t e r g e n t interaction. Volume a n d e l e c t r o p h o r e t i c charges p r o d u c e d by s o d i u m dodecyl sulfate r e a c t i o n w i t h bovine s e r u m albumin. Y. Biol. Chem., 247, 5228, 1972. [9] LAMBIN P . - Reliability of m o l e c u l a r weight d e t e r m i n a t i o n of p r o t e i n s by p o l y a c r y l a m i d e g r a d i e n t gel e l e c t r o p h o r e s i s in the presence of s o d i u m dodecyl sulfate. Anal. Biochem., 85, 114, 1978. [10] LAMmN P. - - Linear p o l y a c r y l a m i d e g r a d i e n t gels for m o l e c u l a r weight e s t i m a t i o n of p r o t e i n s in ,~ p r o c e e d i n g s of the I n t e r n a t i o n a l conference on E l e c t r o p h o r e s i s ~>, (B.J. R a d o l a ed.), de Gruyter, Berlin, pp. 227 (1980). [11] LAMBINP. and FINE J.M. - - Molecular weight e s t i m a t i o n of p r o t e i n s b y electrophoresis in linear p o l y a c r y l a m i d e g r a d i e n t gels in the absence of d e n a t u r i n g agents. Anal. Biochem., 98, 160, 1979. [12] LAMBINP., ROCHU D. and FINE J.M. - - A new m e t h o d for d e t e r m i n a t i o n of m o l e c u l a r weights of p r o t e i n s b y e l e c t r o p h o r e s i s across a s o d i u m dodecyl sulfate (SDS) p o l y a c r y l a m i d e g r a d i e n t gel. Anal. Biochem., 74, 567, 1976. [13] MANWELL C. - - A s i m p l i f i e d e l e c t r o p h o r e t i c system for d e t e r m i n i n g m o l e c u l a r weights of proteins. Biochem. J., 165, 487, 1977. [14] MARGOLIS J. and KENRICK K.G. - - P o l y a c r y l a m i d e gel electrophoresis across a m o l e c u l a r sieve gradient. Nature, 214, 1334, 1967. [15] MATTICE W.L., RISER J.M. and CLARK D.S. Conformational properties of the complexes f o r m e d b y p r o t e i n s and s o d i u m dodecyl sulfate. Biochemistry, 15, 4264, 1976. [16] O'FARELL P.H. - - High r e s o l u t i o n two-dimensional e l e c t r o p h o r e s i s of proteins. J. Biol. Chem., 250, 4007, 1975. -
-
[17] ORSTEIN L. - - Disc-electrophoresis: b a c k g r o u n d a n d theory. Annals New York Acad. Sci., 121, 321, 1964. [18] PITT RIVERS R. a n d IMPIOMBATOF.S.A. - - The binding of s o d i u m dodecyl sulfate to various proteins. Biochem. J., 109, 825, 1968. [19] PODUSEO J.F. a n d RODBARD D. - - Molecular weight e s t i m a t i o n using s o d i u m dodecyl sulfate-pore g r a d i e n t electrophoresis. Anal. Biochem., 101, 394, 1980. [20] REYNOLDS J. a n d TANFORD C. The gross c o n f o r m a t i o n of proteins o d i u m dodecyl sulfate complexes. J. Biol. Chem., 245, 5161, 1970. -
-
[21] ROOBARDD. - - E s t i m a t i o n of m o l e c u l a r weight b y gel f i l t r a t i o n a n d gel electrophoresis. In ,, Methods of proteins separation ,, (Catsimpoolas, N. ed.), vol. 2, pp. 145, Plenum Press, New York, 1976. [22] RODBARDD. a n d CPtRAMB¢tCHA. E s t i m a t i o n of m o l e c u l a r radius, free m o b i l i t y a n d valence using p o l y a c r y l a m i d e gel electrophoresis. Anal. Biochem., 40, 95, 1971. -
-
[23] RODBARD D., KAPADIA G. a n d CHRAMBACH A. - -
Pore g r a d i e n t electrophoresis. Anal. Biochem., 40, 135, 1971. [24] ROTHE G.M. and PURKHANBABA.- - D e t e r m i n a t i o n of m o l e c u l a r weights a n d stokes' radii of n o n - d e n a t u r e d p r o t e i n s by p o l y a c r y l a m i d e gradient gel electrophoresis. Electrophoresis, 3, 33, 1982.
MOLECULAR WEIGHT DETERMINATION
703
[25] SHAPIRO A.L., VINUELA E. and MAIZEL J.U. Molecular weight estimation of p o l y p e p t i d e chains b y electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Research Commun., 28, 815, 1967. [26] SHIRHAMA K., TsuJII K. and TAKAGI T. - - F r e e - b o u n d a r y electrophoresis of s o d i u m dodecyl sulfate-protein p o l y p e p t i d e complexes with special reference to SDS-polyacrylamide gel electrophoresis. J. Biochem. (Tokyo), 75, 309, 1974. [27] WEBER K. a n d OSBORN M. - - Proteins a n d s o d i u m dodecyl sulfate m o l e c u l a r weight d e t e r m i n a t i o n on p o l y a c r y l a m i d e gels and related procedures. I n ,, The proteins ,, (3rd ed.) (H. NEURATH a n d R.L. HILL, eds). Vol. 1, pp. 179, New York, 1975. -
-
691
Molecular weight determination by polyacrylamide gradient gel electrophoresis by P. Lambin Laboratoire d'Immunochimie des Prot6ines, Centre National de Transfusion Sanguine, PARIS.
U
LTRACENTRIFUGATIONand gel filtration h a v e b e e n u s e d f o r molecular weight d e t e r m i n a t i o n of proteins and p o l y p e p t i d e chains. More recently, it a p p e a r e d t h a t electrophoretic techniques using p o l y a c r y l a m i d e gels as s u p p p o r t i n g m e d i u m can be utilized for this p u r p o s e w i t h a c c u r a c y a n d rapidness. As a m a t t e r of fact, p o l y a c r y l a m i d e gel is a good s u p p o r t i n g med i u m for electrophoresis not only by p r c v e n t i n g diffusion and convection b u t also b y i n t e r a c t i n g with the m i g r a t i n g particles. This i n t e r a c t i o n depends on the particule size. I t has b e e n t e r m e d " m o lecular sieving effect". Thus in p o l y a c r y l a m i d e gels, proteins are s e p a r a t e d according both to charge and m o l e c u l a r size. The sieving effect being related to gel density, it is necessary to recall briefly w h a t is the s t r u c t u r e of p o l y a c r y l a m i d e gels.
3 Structure of p o l y a c r y I a m i d e gels Polyacrylamide gels are the p r o d u c t of the p o l y m e r i z a t i o n and cross-linking of the m o n o m e r of a c r y l a m i d e and a crosslinker: N, N'-methylene-bis-acrylamide. Gel density is defined by two values T and C.
692 T c o r r e s p o n d s to the total p e r c e n t a g e a c r y l a m i d e and bis-acrylamide.
L A M B I N P.
concentration
of b o t h
C to the p e r c e n t a g e c o n c e n t r a t i o n of c r o s s l i n k e r relative to the total c o n c e n t r a t i o n T. Schematically two kinds of gels can be u s e d f o r p r o t e i n s separation: gels of c o n s t a n t c o n c e n t r a t i o n a n d p o l y a c r y l a m i d e gradients. Gels of c o n s t a n t c o n c e n t r a t i o n in c o n j o n c t i o n w i t h a discontinuous b u f f e r s y s t e m w e r e i n t r o d u c e d b y ORSTEIN [17] a n d DAVIS [2]. Several y e a r s later, the use of p o l y a c r y l a m i d e gradients was d e s c r i b e d b y MARGOLIS a n d KENRICK [14]. R e p r o d u c i b l e a n d precise profils of gradients (concave or linear) can b e n o w o b t a i n e d b y a u t o m a t i c gradients m a k e r s , peristaltic p u m p and a vertical slab gel system. I n o r d e r to o b t a i n o p t i m u m conditions of polymerization, time, t e m p e r a t u r e , p H a n d c o n c e n t r a t i o n of reagents m u s t b e carefully precised [9, 12]. Practically, T can v a r y f r o m 2 to 30% a n d C f r o m 3 to 8.5%. As well as the use of density g r a d i e n t u l t r a c e n t r i f u g a t i o n h a d o p e n e d u p a new d i m e n s i o n for the s e p a r a t i o n of proteins, polyacrylamide gradients allowed a b e t t e r resolution t h a n gels of c o n s t a n t concentration. The gradients can b e u s e d for the s e p a r a t i o n of m u l t i c o m p o n e n t s m i x t u r e s in one d i m e n t i o n a l [14] or in two dimentional techniques [16].
General principles of molecular weight determination of proteins by electrophoresis T r e a t m e n t of proteins b y an ionic d e t e r g e n t such as s o d i u m dodecyl sulfate (SDS) increases c o n s i d e r a b l y their e l e c t r o p h o r e t i c mobility and provides a relative u n i f o r m i t y of t h e i r charge density in such a m a n n e r t h a t their m i g r a t i o n distance in a p o l y a c r y l a m i d e gel can be directly related to their m o l e c u l a r weight (MW) [18, 8, 26, 15]. I n the absence of ionic detergents, e l e c t r o p h o r e t i c mobility depends b o t h on charge a n d m o l e c u l a r size and t h e r e f o r e only an indirect m e a s u r e m e n t of the MW is possible. The m e t h o d s u s e d can be classified into f o u r m a j o r categories according to their p e r f o r m a n c e in the p r e s e n c e or in the absence of SDS a n d the use of c o n s t a n t c o n c e n t r a t i o n or linear gradient of polyacrylamide. 1. MW d e t e r m i n a t i o n in gel of c o n s t a n t c o n c e n t r a t i o n w i t h proteins previously t r e a t e d b y SDS was i n t r o d u c e d b y SHAPIRO et al. [25]. In this technique, a linear relationship is o b t a i n e d b y plotting log
MOLECULAR WEIGHT DETERMINATION
693
(MW) against relative mobility. However, the extent of this relationship is limited for a given gel concentration: 10% gels for MW between 11,000 to 70,000: 5% gels for MW between 25,000 and 200,000 [3]. In addition, this relationship cannot be applied with a good accuracy to glycoproteins [20, 1, 6, 5, 27]. 2. MW d e t e r m i n a t i o n in gels of a constant concentration and in the absence of SDS has been described b y several authors and in p a r t i c u l a r by HEDRICK and SMITH [7] and by RODBAgD et al. [21, 22]. In these methods, several electrophoreses are p e r f o r m e d in gels of different densities. A r e t a r d a t i o n coefficient is obtained for a given protein. This coefficient is itself related to the MW. The m a j o r inconvenient of these techniques is the repetition of electrophoreses in gels of different concentrations. 3. MW d e t e r m i n a t i o n in gradient gels c o m b i n e d with SDS was i n t r o d u c e d by LAMBIN et al. [12, 9]. This m e t h o d provides a good accuracy for a wider range of MW than conventional gels. 4. Several methods have been described for MW d e t e r m i n a t i o n in gradient gels and in the absence of SDS [4, 13, 24]. We will r e p o r t here the m e t h o d developped in our lab [10, 11]. Molecular weight determination in the presence of SDS
After electrophoresis in a gradient of polyacrylamide, a linear relationship was empirically established [12] between the log (MW) of a protein and the log of the polyacrylamide concentration (T) reached by this protein. This relationship can be written as follows log (MW) = a log (T) + b (1) where a and b are the slope and intercept of the linear regression respectively. This relationship proved to be suitable for proteins having MWs between 104 to 106 with 3 to 30% linear gradients of polyacrylamide. It can be also applied to polypeptide chains of proteins after reduction of disulfide bonds by reducing agents such as 2- mercaptoethanol. The preliminary results obtained with 10 proteins were confirmed with m o r e than 40 proteins and polypeptide chains [9, 10]. This n u m b e r of proteins allowed a statistical analysis of the results concerning the extend of the relationship and the accuracy of the d e t e r m i n a t i o n of the MW of an u n k n o w n protein. Fig. l a and l b show the results of two experiments p e r f o r m e d in 3 to 30% linear gradients with b o t h reduced and u n r e d u c e d proteins. Best linear fit and 95% confidence limits, for a single observation, were calculated. Fig. 2a and 2b, show the correlation between expected and observed MWs.
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MOLECULAR WEIGHT DETERMINATION
695
0
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LAMBIN P.
S i m i l a r e x p e r i m e n t s w e r e p e r f o r m e d w i t h d i f f e r e n t p r o f i l s of g r a d i e n t s a n d r e l a t i v e p e r c e n t a g e of b i s - a c r y l a m i d e , (see table, I). F r o m t h e s e e x p e r i m e n t s , it w a s p o s s i b l e to d r a w t h e f o l l o w i n g conclusion: TABLE I Data obtained with three different structures of gel and two di[[erent protein incubation conditions. GEL STRUCTURE
T C T C T C T C T C
= = = = = = = = = =
3-30% 8.4% 3-30% 8.4% 3-30% 3.8% 3-30% 3.8% 2-10% 3.8%
MEAN
PROTEIN TREATMENT
N
r
SDS
34
0.998
5.9
SDS + 2-Met
31
0.997
4.9
SDS
33
0.995
7.3
SDS + 2-Mer
31
0.991
7.8
SDS
8
0.996
5.5
DEVIATION
(%)
a) a s i m i l a r a c c u r a c y w a s o b t a i n e d i n t h e a b s e n c e o r i n t h e p r e s e n c e of r e d u c i n g a g e n t : i n f a c t c o r r e l a t i o n c o e f f i c i e n t s w e r e r e s p e c t i v e l y 0.998 a n d 0.997 a n d m e a n d e v i a t i o n s , -- 5.9% a n d +- 4.9% (C = 8.4%). b) T h e i n c r e a s e i n c r o s s l i n k e r f r o m 3.8% to 8.4% i m p r o v e s s l i g h t l y t h e a c c u r a c y f o r t h e e v a l u a t i o n of a MW: m e a n d e v i a t i o n s w e r e r e s p e c t i v e l y -+ 7.3% a n d +--5.9% i n SDS. c) G l y c o p r o t e i n s a p p e a r to b e h a v e i n t h e s a m e w a y as do o t h e r p r o t e i n s . Of t h e 34 p r o t e i n s a n a l y z e d , 15 w e r e g l y c o p r o t e i n s w i t h c a r b o h y d r a t e c o n t e n t r a n g i n g f r o m 4 to 45%; slopes, i n t e r c e p t s , c o r r e l a t i o n c o e f f i c i e n t s a n d m e a n d e v i a t i o n s o b t a i n e d w i t h glycoproteins a n d proteins w i t h o u t c a r b o h y d r a t e s are given in Table H. Differences b e t w e e n the two regression lines were small a n d n o t s t a t i s t i c a l l y s i g n i f i c a n t (t: 0.45 < 1.96; p < 0.05). I t s h o u l d b e n o t e d t h a t w h e n p r o t e i n s a r e s u b j e c t e d to e l e c t r o p h o r e s i s i n t h e a b s e n c e of SDS i n c u b a t i o n , n o l i n e a r r e g r e s s i o n c a n b e e s t a b l i s h e d . I n c o n t r a s t , a l i n e a r r e g r e s s i o n w a s o b t a i n e d i n t h e case of p r o t e i n s m o n o m e r s a n d their homologous oligomers which have the same charge density p e r m o l e c u l e (see Fig. 3).
MOLECULAR WEIGHT DETERMINATION
697
TABLE II
Results obtained by 3 to 30% gradient gel electrophoresis (C = 8.4%) for proteins w i t h or w i t h o u t carbohydrates.
PROTEIN
N
SLOPE
INTERCEPT
W i t h c a r b o h y d r a t e s .. Without carbohydrates All p r o t e i n s . . . . . . . . . .
15 19 34
- - 3.421 3.426 - - 3.430
8.68 8.68 8.69
-
-
MEAN DEVIATION
(%)
0.998 0.996 0.998
MW 10
6
-
IgA
10 5
Tf
'
'
'
'
'
'
I
1
I
FIc. 3 . - L i n e a r r e g r e s s i o n o b t a i n e d w i t h t h r e e p r o t e i n m o n o m e r s a n d t h e i r h o m o l o g o u s o l i g o m e r s a f t e r elect r o p h o r e s i s i n a 3 to 30% g r a d i e n t gel in t h e a b s e n c e of SDS (Alb: a l b u m i n , Tf: t r a n s f e r r i n , a n d IgA: i m m u n o g l o b u l i n A).
6.6 5.3 5.9
698
LAMBIN P.
Validity of relation (1) was c o n f i r m e d b y e x p e r i m e n t a l a n d comp u t e r s t i m u l a t i o n studies of PODUSLO a n d RODBAI~D [19]. SDS t r e a t m e n t of p r o t e i n s a n d linear p o l y a c r y l a m i d e g r a d i e n t gel e l e c t r o p h o r e s i s p e r m i t the e s t i m a t i o n of the MW of a p r o t e i n in the r a n g e of 10 4 to 10 6 in a single gel w i t h an e r r o r of -+ 6% w h a t e v e r the c a r b o h y d r a t e content. Molecular w e i g h t d e t e r m i n a t i o n in the a b s e n c e of S D S SDS being a dissociating agent, the MW of a native p r o t e i n m a y differ f r o m the MW f o u n d in the p r e s e n c e of SDS w h e n this p r o t e i n is c o m p o s e d of several subunits held t o g e t h e r b y non-covalent bonds. As previously m e n t i o n e d , in the absence of ionic detergents, the e ] e c t r o p h o r e t i c mobility of a p r o t e i n in a p o l y a c r y l a m i d e gel, depends b o t h on charge a n d size. I n these conditions, only an indirect e s t i m a t i o n of MW is possible. As s h o w n b y LAMBIN a n d FINE [ 11] linear gradient of polyacrylam i d e can b e succesfully u s e d f o r this estimation. I n this m e t h o d , isolated p r o t e i n s or p r o t e i n s m i x t u r e s are s u b j e c t e d to e l e c t r o p h o r e s i s in the s a m e slab gel a n d at c o n s t a n t voltage during several periods of t i m e b e t w e e n 1 to 8 hours, After 8 hours, the gels are stained and the p r o t e i n s position is m e a s u r e d . A relationship was first established b e t w e e n the distance of m i g r a t i o n a n d t i m e of electrophoresis. I n fact, it w a s f o u n d t h a t the b e s t c o r r e l a t i o n coefficient b e t w e e n t i m e a n d distance for all p r o t e i n s u n d e r s t u d y (of v e r y different free e l e c t r o p h o r e t i c mobilities a n d shape) w a s o b t a i n e d w h e n plotting the s q u a r e r o o t of the t i m e of e l e c t r o p h o r e s i s versus m i g r a t i o n distances according to the equation. ta~ - a D + b (2) t being the t i m e of e l e c t r o p h o r e s i s D the distance m i g r a t e d b y a p r o t e i n a a n d b Mope and i n t e r c e p t of the regression lines. W h e n volt × h o u r p r o d u c t s are b e t w e e n 40 a n d 800, c o r r e l a t i o n coefficients are b e t t e r t h a n 0.99 for all the p r o t e i n s s u b j e c t e d to electrophoresis. This e q u a t i o n w a s validated over an extended r a n g e of conditions such as p H ( f r o m 7 to 10) a n d b u f f e r s c o m p o s i t i o n ( s o d i u m - p h o s p h a t e , Tris-borate, Veronal-Tris). H o w e v e r , for p r a c t i c a l reasons, these b u f f e r s m u s t b e selected in o r d e r to give a sufficient e l e c t r o p h o r e t i c mobility to the p r o t e i n s u n d e r study. Fig. 4 illustrates the results o b t a i n e d in one e x p e r i m e n t .
699
MOLECULAR H/EIGHT DETERMINATION
Time ( hoV-Eg~urs) •
f
o
7-
6-
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54 3 2
J
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I
20
40
-"
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l
Distance (ram)
FIG. 4. - - Plots of square root of time of electrophoresis (in Veronal-Tris-Glycine buffer pH 9.8) against distance migrated by 10 non-denatured proteins. a: b: c: d:
6~2 macroglobulin, thyroglobulin, IgA (dimer), catalase,
e: f: g: h:
IgA (monomer), ceruloplasmin, albumin, streptokinase,
i: ovalbumin, j : soybean inhibitor.
The l i n e a r r e g r e s s i o n s w e r e u t i l i z e d in a s e c o n d step f o r MW d e t e r m i n a t i o n . As s h o w n in figure 5 w h e n log (MWs) a r e p l o t t e d a g a i n s t log of slopes (a) of the r e g r e s s i o n lines o b t a i n e d as above, t h e r e is a l i n e a r r e l a t i o n s h i p b e t w e e n t h e two values. Figure 5 also shows the 95% c o n f i d e n c e l i m i t s o b t a i n e d for the e s t i m a t i o n of the MW of an u n k n o w p r o t e i n . Deviations between expected and o b s e r v e d MW a r e m o r e i m p o r t a n t t h a n in t h e p r e s e n c e of SDS b u t i n f e r i o r to 20%. This a c c u r a c y is of p r o b a b i l i t y t h a t MW f o u n d by the c o n s i s t s of one or bonds.
q u i t e s u f f i c i e n t to c o n c l u d e w i t h a high d e g r e e the MW c o r r e s p o n d s once, twice or m o r e the SDS m e t h o d , a n d t h e r e f o r e t h a t t h e m o l e c u l e several s u b u n i t s h e l d t o g e t h e r by non-covalent
700
L A M B I N P.
MW 106
,,':e
i/
105
, /.j,/ / 104 •01
FI~. 5.
.02
-
-
.
.
.04 .
.
. .06 . .
.08 .1 .
.2
.4
SLope
Plots of log MWs of 10 proteins versus log of slopes (see fig. 4). (--.) best linear fit (. . . . ) 95% confidence limits.
W h e n volt × h o u r p r o d u c t is a b o v e 1,000, equation (2) is no m o r e verified a n d as p r e d i c t e d b y RODBARD et al. [23] log of t i m e of electrop h o r e s i s b e c o m e s p r o p o r t i o n a l to the distance of migration. This was checked w i t h p r o t e i n s of MWs b e t w e e n 6 × 104 a n d 106; c o r r e l a t i o n coefficients being a b o v e 0.998 (LAMBIN et al. u n p u b l i s h e d data): These results c o n f i r m t h a t t h e r e is no d e a d stop for a p r o t e i n in a gradient; given enough time, it can r e a c h a n y finite distance. I t should b e n o t e d t h a t slopes of regression lines can b e u s e d as previously d e c r i b e d to d e t e r m i n e MWs of proteins. Linear gradients of p o l y a c r y l a m i d e p r o v i d e at the p r e s e n t time, the b e s t a c c u r a c y for the e s t i m a t i o n o f the MW of a p r o t e i n or a glycoprotein. I t s e e m s p r o b a b l e t h a t in these gels, the m o l e c u l a r sieving effect could p r e d o m i n a t e a n d minimize the differences in charge a n d shape of the p r o t e i n s w h i c h influence the results o b s e r v e d b y e l e c t r o p h o r e s i s in conventional p o l y a c r y l a m i d e gels.
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I n s u m m a r y , we suggest the following strategy for MW estimation of an u n k n o w n protein by electrophoresis in linear gradients of polyacrylamide. 1. MW estimation of the protein in SDS. 2. MW estimation of its polypeptide chains in SDS and a reducing agent. 3. MW estimation of the native protein, and c o m p a r i s o n of this result with that obtained in SDS in o r d e r to determine the n u m b e r of its subunits, the MW obtained in SDS being taken as a reference because of the best a c c u r a c y given by this method. Request reprints from: P. LAMBIN, C.N.T.S., Laboratoire d'Immunochimie des Prot6ines, 6, rue A.-Cabanel, 75739 PARIS Cedex 15.
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