- Email: [email protected]
[22] Recovery of functional proteins in sodium dodecyl sulfate gels
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN S D S GELS
263
A disadvantage in using the ion-retardation resin is its cost; this can be minimized by preparing it from Dowex 1. Although the adsorption of most ions is reversible and regeneration of the resin can be achieved by simply washing with water, SDS appears to be bound very strongly to the resin, since only a 9% recovery of bound [a~S]SDS could be achieved by elution with a 1.0 M NaCl solution. 9 Thus, the resin cannot be reutilized conveniently. The principal advantages of the method are the following: it is a rapid, one-step procedure that avoids excessive loss of proteins and peptides through adsorption on the resin and results in an effectively complete removal of SDS under appropriate conditions.
[22] R e c o v e r y o f F u n c t i o n a l P r o t e i n s in S o d i u m D o d e c y l Sulfate Gels By AD SPANOS and ULRICH HOBSCHER
The use of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) has greatly facilitated the detection, purification, and characterization of proteins from complex mixtures. 1,2 With this technique, proteins are dissociated into their constituent polypeptides and separated according to their molecular weight. After native gel electrophoresis, in which proteins are separated according to both size and charge, it has been possible to detect the enzymic activity of proteins? However, enzymic activities can also be detected following SDSpolyacrylamide gel electrophoresis (PAGE) by removal of the SDS, elution of the protein from the gel, and renaturing it. 4 Alternatively, the renaturation and enzyme assay steps can be performed in the intact gel. This is especially suitable for enzymes that rely on high-molecular-weight substrates or cofactors, such as DNA, RNA, or proteins, since these can be polymerized into the gel) "6 We describe here a procedure to recover the catalytic activity of enzymes after SDS-PAGE within the intact gel. The method was originally I K. 2 U. a O. 4 K. 5 A.
Weber, T. R. Pringle, and M. Osborn, this series, Vol. 26, p. 3. K. L a e m m l i , Nature (London) 227, 680 (1970). Gabriel, this series, Vol. 22, p. 578. Weber and J. Kuter, J. Biol. Chem. 246, 4504 (1971). Spanos, S. G. Sedgwick, G. T. Yarranton, U. Hiibscher, and G. R. Banks, Nucl. Acids Res. 9, 1825 (1981). 6 A. L. Rosenthal and S. A. Lacks, Anal. Biochem. 80, 76 (1977).
METHODS IN ENZYMOLOGY,VOL. 91
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181991-4
264
CHAIN SEPARATION
[22]
developed for enzymes involved in D N A transactions 7 (e.g., D N A polymerases and their associated nucleases), but can also be applied to others, and it has the advantage that either homogeneous, enriched, or even crude enzyme fractions can be analyzed. Questions of isoenzyme or precursor structure and proteolytic degradation can be a t t a c k e d / since the enzymically active bands can be correlated with Coomassie Blue stain (for details, see below). Identification of mutant or cloned enzymes is also possible .5,7 H o w e v e r , the detection o f some enzymes may be hampered by the absence of a sensitive or appropriate assay or by the fact that some enzymes require at least two different polypeptide chains for enzymic activity. Finally, the introduction of two-dimensional electrophoretic systems in connection with e n z y m e renaturation may extend this analytical technique.a Experimental Procedures Preparation o f Gels and Electrophoresis Reagents. Acrylamide (>99% pure), N,N'-methylbisacrylamide, and N , N , N ' , N ' - t e t r a m e t h y l e t h y l e n e d i a m i n e ( T E M E D ) were from Bio-Rad; SDS, "especially p u r e " product No. 30176, ammonium persulfate and glycine were from BDH; and Trizma base (rids), 5-bromo-4-chloro-3indolyl-/3-o-galactoside (X-Gal), naphthol-AS-MX-phosphate, Fast Blue RR salt, and isopropyl/3-o-thiogalactopyranoside were from Sigma. Calf thymus D N A was purchased from Worthington. Strains. Cultures o f wild-type Ustilago maydis and of Escherichia coli wild-type and polA mutants were grown as described, s Cultures ofE. coli strain J G l l 3 were grown overnight in phosphate-free medium to induce alkaline phosphatase synthesis, a Synthesis of/3-galactosidase was induced in E. coli CGSC 4515 b y overnight growth in L broth containing 10 -4 M isopropyl-/3-D-thiogalactopyranoside. TM Control cultures of E. coli 4515-625, which are unable to synthesize/3-galactosidase, were grown under similar conditions. 11 Enzymes. Escherichia coli D N A polymerase I was purified up to the phosphocellulose stage from extracts of heat-induced cells containing the u. Hiibscher, A. Spanos, W. Albert, F. Grummt, and G. R. Banks, Proc. Natl. Acad. Sci. U.S.A. 78, 6771 (1981). a G. Scheele, J. Pash, and W. Bieger, Anal. Biochem. 112, 304 (1981). 9 A. Torriani, in "Procedures in Nucleic Acid Research" (G. L. Cantoni and D. R. Davies, eds.), p. 224. Harper & Row, New York, 1966. 10j. Miller, ed., "Experiments in Molecular Genetics." Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1971. 11 W. A. Newton, J. R. Beckwith, D. Zipser, and S. Brenner, J. Mol. Biol. 14, 290 (1965).
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN S D S GELS
265
ypolA + C 1857 lysogen, tz,la Alkaline phosphatase from E. coli and DNase I were from P-L Biochemicals and Worthington, respectively. Escherichia coil/3-galactosidase was purchased from Sigma. DNA Templates. Calf thymus DNA was dissolved in 20 mM Tris-HC1, pH 7.5, 20 mM NaC1 overnight at 4° and adjusted to a concentration of 3 mg/ml. It could then be denatured by heating the solution at 95 ° for 15 min. To make gapped DNA, MgCh (to 5 mM) and DNase I (to 0.1 ixg/ml) were added to the native DNA solution (3 mg/ml), which was prewarmed at 37° for 15 min. After incubation for 6 min at 37°, proteins were extracted by an equal volume of phenol and chloroform, the aqueous phase was made 200 mM in NaC1, and the DNA was precipitated with ethanol. The DNA pellet was dissolved in 20 mM Tris-HC1, pH 7.5,200 mM NaCI and the precipitation was repeated three times. The DNA was finally dissolved in 20 mM Tris-HC1, pH 7.5, 20 mM NaC1 at 2 mg/ml and stored at - 2 0 °. Agarose gel electrophoresis showed that it possessed a molecular weight of (0.5-5.0) x 106. Gapped DNA was labeled by nick translation 14 or its 3'-termini were labeled using [a-32p]dTTP and the Klenow fragment of DNA polymerase I. 5"15The DNA (0.5 mg) was incubated in a 3-ml reaction mixture containing 50 mM Tris-HCl, pH 7.5, 7 mM MgCI2 1 mM 2-mercaptoethanol, 40 nmol each of dGTP, dCTP, dATP, 50/xCi of [aa2p]dTTP, and 50 units of the E. coil DNA polymerase I Klenow fragment for 30 min at 37°. The DNA was purified, resuspended, and stored as above. If rdquired, the labeled DNA may be denatured before use in the gel.
Solutions and Electrophoresis Stock solutions: see tabulation on p. 266. Electrophoresis buffer: 6 g of Trizma-base, 28.8 g of glycine per liter containing 0.1% SDS and 2 mM EDTA Sample buffer A: 0.04 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 10 mM 2-mercaptoethanol S ample buffer B: 0.025 M Tris-HCl, pH 6.8, 2 mM EDTA, 30% glycerol, 0.6 M 2-mercaptoethanol, and 5% (w/v) SDS Sample buffer C: 0.065 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 0.12 M 2-mercaptoethanol and 1% SDS The SDS-gel electrophoresis is carried out essentially as described by Laemmli. 2 The source of SDS is particularly important, as impurities in 12 W. S. Kelly and K. H. Stump, J. Biol. Chem. 254, 3206 (1979). ~a C, C. Richardson, C. L. Schildkraut, H. V. Aposhian, and A. Kornberg, J. Biol. Chem. 239, 222 (1964). 14 p. W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg, J. Mol. Biol. 113, 237 (1977). ~5 H. Klenow and I. Henningsen, Proc. Natl. Acad. Sei. U.S.A. 65, 168 (1970).
266
CHAIN SEPARATION
[22] Final concentration in
Acrylamide- bisacrylamide, 30%/0.8% Tris-HCl, 1 M, pH 6.8 Tris-HCl, 1 M, pH 8.8 EDTA, 0.2 M, pH 8.0 SDS 10% w/v (freshly made) Substrate (DNA or RNA) Ammonium persulfate TEMED
Separating gel
Stackinggel
5-20% -0.375 M 0.002 M 0.1% As indicated in text 0.05% 0.05%
5% 0.065 M -0.002 M 0.1% -0.1% 0.1%
particular preparations prevent renaturation of enzymes after electrophoresis. 5 It has been found that especially pure SDS from B D H is satisfactory. Depending on the molecular weight of the enzyme under investigation, the acrylamide concentration in the separation gel may be varied from 5 to 20%, and gradient gels may also be used. When highmolecular-weight substrates are included in the gel, they are added prior to T E M E D and ammonium persulfate. The gel is poured without degassing into a slab (0.15 × 17 × 18 cm). A 5% stacking gel (4 cm) containing 0.065 M Tris-HC1 (pH 6.8) is layered on top of the separating gel, and a comb allowing up to 100/.tl of sample loading capacity is used. After loading the samples, electrophoresis is performed at room temperature (20-25 °) at 50-70 V for 15 hr or at 150 V for 4 hr. Sample Preparation. Unless otherwise stated, all operations were carried out at or near 0 °. The use of protease inhibitors is recommended during the preparation and purification of all samples (see Hiibscher et al.r). Freshly prepared or frozen cells (1 g) are resuspended in 3 ml of buffer A and disrupted by using a French pressure cell for unicellular organisms or a Dounce homogenizer for mammalian cells or tissues. Crude e n z y m e preparations (10-20 mg of protein per milliliter) are obtained by centrifugation at 25,000 g for 30 min. Cells and aliquots of samples are stored at - 8 0 ° or in liquid nitrogen, until further use. Crude extracts or homogeneous enzymes (10-100/zl) are thawed at 0° diluted 4 : 1 in freshly made buffer B, and immediately heated for 3 min at 37 ° or at 100° (see below). Buffer C can also be used with the same efficiency. To enhance r e c o v e r y of activity in samples with low protein concentrations, bovine serum albumin (BSA) (10 mg/ml, preheated at 100° for 15 min) is added to the samples at a final concentration of 1 mg/ml. This " c a r r i e r "
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN S D S GELS
267
can also be included in the gel at a concentration of 10/xg/ml. However, this is not recommended when nucleases and DNA polymerases are determined, because BSA preparations may contain nucleases. TM Renaturation of Enzymes. After electrophoresis it is essential to remove the SDS immediately from the gel and to allow the polypeptides to renature. As mentioned, the source of the SDS and also other factors may be important in optimizing the recovery of a particular enzyme (see below). After completion of electrophoresis, the gel is rinsed in renaturation buffer (50 mM Tris-HC1, pH 7.5, 1 mM EDTA, and, if required, 5 mM 2-mercaptoethanol, and then shaken in 1 liter of this buffer at room temperature with changes after 30 and 60 min. Although some enzymes can be assayed immediately after this initial washing step, it is found that others renature only if the gel is kept for a further 3-24 hr at 4°. Prolonged standing may result in loss of enzyme from the gel, especially with small polypeptides. However, this can be tested by comparing identical sampies, one of which is stained with Coomassie Blue immediately after electrophoresis and the other after completion of the enzyme assay. To measure the renaturation kinetics, single-gel tracks can be sliced out and assayed after different times of renaturation. Unless otherwise stated, gels are washed for 1 hr and then left in renaturation buffer for 24 hr at 0° with several changes. Staining and Autoradiography of Gels. After most enzyme assays, the proteins in the gel can be stained with 0.25% (w/v) Coomassie Blue, 50% methanol, 10% acetic acid for 90 min, then destained, first in 50% methanol, 10% acetic acid for 90 min, and finally, in 5% methanol, 7% acetic acid for 24 hr. Staining of gels before autoradiography may result in some loss of radioactivity (determined for DNA polymerasesS). In such cases, gels can first be directly autoradiographed and then stained. After autoradiography, the dried gel can be swelled in distilled water for 60 min, stained, and then destained as described above. Rehydration of gels containing 10% acrylamide may lead to cracking of the gel. Often the enzyme assay is more sensitive than protein staining and can measure enzymes into the picogram range. 17 Where the assay involves the use of a radioactive isotope, such as azp, the gel may be dried onto Whatman 3 MM paper and then autoradiographed. It is possible in certain cases to obtain an autoradiogram of the undried gel by placing it on a glass plate, covering it with cling film and then the X-ray film. This is specially applicable to assays using 32p and for monitoring nuclease activity (see section on nucleases below). ~6 A. Spanos, unpublished results, 1979. ~7 A. Spanos, unpublished results, 1978.
268
CHAIN SEPARATION
[22l
Results and Discussion
Detection of Enzymes in Gels General Remarks. Existing procedures to assay enzymes following electrophoresis in native gels or in other separating systems 8 can be used or adapted to detect enzymes in SDS-polyacrylamide gels. Polypeptides with known enzymic activity can be located by incubating the gel in a specific assay mixture. Visualization can be effected by the formation or removal of an insoluble colored, fluorescent, or radioactive compound at the position of the enzyme band. High-molecular-weight molecules, such as DNA, RNA, or proteins, can be polymerized into the gel mixtures and then serve as substrates or cofactors for enzymes, such as nucleases, DNA and RNA polymerases, DNA methylases, DNA and RNA binding proteins and proteases. The presence of these substrates in the gels does not alter the mobility of polypeptides. The assays of five different classes of enzymes and proteins are now described in more detail to give insight into the potential and the limitations of this technique. Nucleases. A general outline of nuclease renaturation and assay is shown in Fig. 1. Natural or synthetic, single or double-stranded DNA or RNA and D N A - R N A hybrids can be polymerized into the gel and serve as substrates for deoxyribonuclease (DNase), ribonuclease (RNase), or RNase H enzymes. The enzymic hydrolysis and subsequent localized removal of nucleic acid in the gel is detected as a dark band after staining with ethidium bromide (Fig. 2A and C). When radioactively labeled DNA or RNA is polymerized into the gel, autoradiography or fluorography TM techniques detect nuclease activity as a clear band on the black autoradiogram (Fig. 2B and D). The latter approach is more sensitive; very small quantities of natural or synthetic templates can be used, and this allows the detection of either weak or highly specific nucleases (compare Fig. 2A and B or 2C and D). Finally, the protein bands in the gel can be stained with Coomassie Blue before autoradiography. DNases. Both single strand- and double strand-specific DNA endonucleases and exonucleases can be identified by using the appropriate radioactive substrate (Fig. 1). After electrophoresis and renaturation, the gel is incubated in 0.05 M Tris-HC1, pH 7.5, 5 mM MgCI~, and 1 mM CaCI2 at 37°, for times that should be determined experimentally. The DNA in the gel is stained with ethidium bromide (1-5/~g/ml) for 30 rain, rinsed, and photographed using a far-UV light box. This staining does not interfere with the nuclease activity, and further incubations of the gel are still possible. 5,6 Active nucleases show activity within 24 hr of incubation, 18 R. A. Laskey and A. D. Mills, Eur. J. Biochem. 54, 335 (1975).
[22]
RECOVERYOF FUNCTIONALPROTEINSIN SDS GELS DNA p o l y m e r a s e
DNA substrate in gel:
Nueiease 32
IIIIIIIIIII
IIII111111111111
lJlllJlllllllJllllllll
Assay:
269
IIHHllIIIIIIII
I111111111111P
32p
~IlIII[IHIIHIIIIIII
a~p i HI I I I I l i l l i l i l ( [ l i l l l l
IIIIllllilHlll
32
lilt lilt ItJilililiilit
P IIIIIlilllltllllllllilLI
g a p p e d DNA
a2P-labeled DNA
[~-32P]dTTP, dATP, dGTP, dCTP, MgCI2, MSH, buffer
MgCI2, MSH, buffer
32 IIIll111111111ll p
Illllllllllllllllll
32]~) IIIIIIIIIIIIIIIIIIIIIIII
a2 p
llIIHl]lilllllli
IIIlllllllllllll
I 32~:) I III] [ l l l l l IIII II IIIIIII
tllliltllllllLltllll
DNA synthesis
HIHHIIINI~ IIHHIIIIILIIIILIIH
D N A degradation
r
__J wash with TCA, sodium pyrophosphate
stain
dry
|
d a r k band in clear background ~
I autoradiograph
~
clear band in dark background
FIG. 1. Detection of catalytic DNA polymerase and associated exonuclease activities following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Details are outlined in the text.
while weaker nuclease bands are detected after several days of incubation only. In this case, it is advisable to include an inhibitor of bacterial growth (0.01% sodium azide). When nucleases in crude extracts are investigated, long renaturation times (> 14 hr) at 4 ° should be carried out in the presence of 2 mM EDTA to prevent premature degradation of substrate by very active nucleases that can subsequently obscure weaker activities. Assay conditions such as D N A source or concentration, temperature, or pH in the gel can be adjusted to optimal detection of specific nucleases. The assay can be made more sensitive if radioactively labeled DNA, such as bacterial, phage, or plasmid D N A is used in the gel. Gapped calf thymus DNA labeled in v i t r o with a2p at the 3' or 5' termini serves as good templates for endonucleases and 5' ~ 3'- or 3' --~ 5'-exonucleases (see also Spanos e t al. s for details). This has the advantage that the removal of only the 3' or 5' termini, without degradation of the rest of the DNA, by very specific or weak nucleases can be detected by autoradiography. Most of the D N A at the position of such nuclease activity remains intact
270
CHAIN SEPARATION
B
A 1
25K
234
[22]
1
2
34
25K
Fio. 2. Deoxyribonuclease activity detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels (10% acrylamide) containing per milliliter 5/~g of gapped DNA labeled at the 3' termini with asp (10,000 cpm//~g) were prepared as described in the text. After electrophoresis and renaturation, the gels were incubated for 36 hr in assay mixture (see text), stained with ethidium bromide (5 tzg/ml) for 30 min, and then photographed under UV illumination. They were then dried and subjected to autoradiography at room temperature. Figure 2A and C are the ethidium bromide-stained gels, and Fig. 2B and D are the corresponding autoradiograms. Gel A: Wells contained 200 tzg of the following Escherichia coli extracts: 1, Escherichia coli JGII2 (polAl); 2, E. cord BR15 (polA-); 3, E. coli JGll3 (polA+); and 4, E. coli Hfr KLI6 recC22. Gel 2C: Wells contained 200 ~g of freshly prepared Ustilago maydis (wild-type) crude extract; 2, the same as 1, except that the extract was stored at -80 ° for 1 month. a n d is s t a i n e d b y e t h i d i u m b r o m i d e (Fig. 2). I f sufficient l a b e l e d D N A is u s e d ( 5 - 1 0 / z g / m l , 10,000 cpm//.~g), its d e g r a d a t i o n c a n be identified b y b o t h s t a i n i n g a n d a u t o r a d i o g r a p h y . I n i t i a l D N a s e a n a l y s i s should b e d o n e u s i n g a large g a p p e d calf t h y m u s D N A ( M W > 0.5 × 106) l a b e l e d to a high specific a c t i v i t y ( > 10,000 cpm//zg). T h e u s e o f 3'- or Y - l a b e l e d t e r m i n i in D N A facilitates the specific a s s a y i n g o f 3' ---> 5'- or 5' ---> 3 ' - e x o n u c l e a s e s . 5
[22]
RECOVERY OF FUNCTIONALPROTEINSIN SDS GELS
C
271
D
21
21
i
FIG. 2 (continued) To follow the kinetics of e n z y m e activity during the assay, the gel m a y be layered on a glass plate and autoradiographed (see Methods). Other radioisotopes, such as 3H or 14C, m a y be used as well, and the gels can be autoradiographed using a fluorographic method. TM R N a s e s . High-molecular-weight ribosomal R N A from E. coli can be included as a substrate in the gel. Total R N A from yeast is not suitable because, owing to its smaller molecular weight, it is lost f r o m the gel to a great extent during renaturation and incubation. Radioactive synthetic ribopolynucleotides or in vivo labeled ribosomal R N A are polymerized at 1 tzg/ml or less into the gel, and hydrolysis of templates is then detected as for D N a s e s . The specificity of R N a s e s can be investigated with substrates, such as a2P-labeled single-stranded synthetic R N A homopolymers, or with a double-stranded synthetic D N A - R N A hybrid, such as poly(rA) • oligo(dT) for R N a s e H. TM 19j. Huet, A. Sentenac, and P. Fromageot, FEBS Lett. 94, 28 (1978).
272
CHAIN SEPARATION
[22]
DNA Polymerases. These enzymes are detected with the technique described above by including a template DNA that has been nicked or gapped 5,~ (Fig. 1). After electrophoresis, removal of the SDS, and renaturation, the gel is incubated in a reaction mixture specific for the DNA polymerase investigated. As an example, for E. coli DNA polymerase I, the gel is incubated in 2-3 gel volumes of 50 mM Tris-HC1 (pH 7.5), 7 mM MgCI2, 3 mM 2-mercaptoethanol, 12/.tM each of dGTP, dCTP, and dATP, and 1/zCi of [t~-a2P]dTTP per milliliter (>2000 cplrdpmol) at 37° for 16-24 hr. It is then washed for 60 min with two liter changes of 5% trichloroacetic acid (TCA) containing 1% sodium pyrophosphate, left for 40 hr at 4° with at least three changes of TCA solution (removal of [a-a2p]dTTP can be monitored b y a Geiger counter). The gel is dried onto Whatman 3 MM filter paper and autoradiographed, enzyme activity being detected as a dark band where the 3zP-labeled deoxyribonucleoside monophosphate is incorporated into the acid-insoluble DNA template. Exposure time can vary, depending on the activity, and should be determined empirically. The autoradiograms may be scanned by a densitometer to determine quantitatively the intensity of a polymerase activity bandy Alternatively, the activity band can be excised from a gel and counted in a scintillation counter. An example of E. coil DNA polymerase I is illustrated in Fig. 3, which shows that a proteolytic fragment, called the Klenow fragment, 15 is created during the last step of purification. Although this fragment and intermediate proteolytic fragments are present in the preceding purification step, they are not detectable by staining, and this emphasizes the sensitivity of the assay. A few facts should be mentioned at this stage. 1. Very high specific activity, and therefore low concentration, of the labeled dNTP is essential. 5 2. Removal of vital cofactors during electrophoresis and the presence of nucleases that overlap the DNA polymerase band may prevent detection of polymerase activity, a problem likely in crude enzyme fractions. 3. It is possible that a radioactive band resulting from the polymerase assay may be due to binding of the radioactive triphosphate to a protein in the gel. To test this, the gel is incubated with DNase I (10/zg/ml, under the conditions mentioned for DNases) following a DNA polymerase assay. If the radioactive band is still present, it may be due to triphosphate binding. However, removal of the label by DNase I does not exclude the possibility of DNA-dependent binding of the triphosphate by a protein. To investigate this, the radioactive band may be cut out, the DNA extracted, and protein removed by phenol extraction. The radioactivity present in the aqueous phase should then be acid-insoluble but become soluble after DNase I treatment.
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN SDS GELS
A 1
23
273
B 1
23 ii
i liill ? i ¸
i ¸ iii~!i ¸
iiiiii,i(i i: iii~!ii!ili~i~:i~ ~:~iiii' !~~ij:iii ~
109K
76K
\
FIG. 3. Escherichia coli DNA polymerase I activity detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel (7.5% acrylamide) containing 80 t~g of gapped calf thymus DNA per milliliter was run and assayed as described in the text. After drying, it was stained with Coomassie Blue (B) and autoradiographed (A) (2 hr of exposure), Wells contained: 1, hydroxyapatite fraction (for final purification step, see Richardson et alAS), 20 units; 2 and 3, 50 and 20 units, respectively, of the preceding purification step of phosphocellulose chromatography.
4. I f D N A is p r e s e n t in c r u d e e n z y m e f r a c t i o n s , it will e n t e r t h e gel d u r i n g e l e c t r o p h o r e s i s , a n d it is t h e r e f o r e p o s s i b l e t h a t D N A p o l y m e r a s e a c t i v i t i e s a r e d e t e c t e d w i t h o u t a t e m p l a t e in t h e gel. D N A and R N A Binding Proteins. C e r t a i n p r o t e i n s , s u c h as h i s t o n e s , c a n b e d e t e c t e d a f t e r S D S - P A G E b e c a u s e t h e y b i n d to D N A a n d p r e v e n t it f r o m s t a i n i n g w i t h e t h i d i u m b r o m i d e . 2° T h i s m a y in f a c t b e m i s t a k e n f o r n u c l e a s e a c t i v i t y , a n d it is i m p o r t a n t to t e s t s i m u l t a n e o u s l y w i t h a nuclease assay using a radioactively labeled DNA substrate. In the case of a z0 A. L. Rosenthal and S. A. Lacks, J. Biol. Chem. 253, 8674 (1978).
274
CHAIN SEPARATION
[22]
DNA binding protein, the latter assay is negative. Such binding, furthermore, does not usually require divalent cations, and there is no timedependent increase of the band intensity. If low concentrations of radioactive DNA or RNA are used to detect nucleases, dark bands as well as "holes" due to nuclease degradation may appear on the autoradiogram (Fig. 2C and D). These bands have been interpreted as being localized concentrations of the template caused by nucleic acid binding proteins. TM This is because some of the DNA or RNA is normally lost uniformly from the gel during renaturation and incubation, owing to diffusion, and this loss is assumed to be prevented at the position of DNA or RNA binding proteins, thus resulting in a darker band on the autoradiogram. However, nonspecific trapping of the template by some polypeptides is another possible explanation for this phenomenon. Another method has been introduced that transfers the protein after renaturation to nitrocellulose paper. 21 The filter containing the proteins is soaked in a radioactive DNA solution and upon binding to the protein the radioactive bands are detected by autoradiography at places of DNAdependent protein binding. This method is more quantitative and allows the use of a wide range of specific DNA templates or smaller fragments. [3-Galactosidase. An example of an electropherogram of enzyme recovery using E. coli fl-galactosidases is shown in Fig. 4. Crude wild-type extracts and a pure commercially obtained enzyme were electrophoresed and then renatured for 3 hr. The gel was incubated in 0.1 M sodium phosphate (pH 7.0), 0.01 M KC1, 0.001 M MgSO4, 0.005 M 2-mercaptoethanol containing 40 /zg of 5-bromo-4-chloro-3-indolyl-flo-galactoside per milliliter (freshly prepared). After 1-2 hr, a blue band appears in the 130,000-MW region in the gel if the sample is not boiled before loading. When the gel is then further incubated overnight, weaker bands of higher molecular weight appear, suggesting dimers and trimers of the enzyme (Fig. 4A). If, however, the enzyme is boiled before loading, more than 90% of the monomer is lost and the higher-molecularweight bands disappear. In addition, no fl-galactosidase activity can be detected in the mutant strain lacking the fl-galactosidase activity. Alkaline Phosphatase. Sample preparation, electrophoresis, and renaturation are as previously described, except that the gel is washed in renaturation buffer for 30 min and renatured for 1 hr at room temperature. The enzymic activity is then assayed in 0.08 M Tris-HCl (pH 8.0) containing per milliliter 1.6 mg of Fast Blue RR salt and 1 mg of naphthol-ASMX-phosphate. The formation of the azo pigment can be seen after 10 min if a purified enzyme is tested (Fig. 4C). The reaction is completed after 3 hr. As mentioned for fl-galactosidases, heating also removes aggregates. Unlike the crude extract for/3-galactosidase, alkaline phosphatase 21 B. Bowen, J. Steinberg, U. K. Laernmli, and H. Weintraub, Nucl. Acids Res. 8, 1 (1980).
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN SDS GELS
A 2
B 3
275
C
1
1
2
3
4
!! iii i~i!/iiiii? ~¸¸
130K-60K i~ i ii!~ ~ ~ ~
~i ~ ~,~ ~L~
ilij~!!/ i i!!j!:
!;i;~¸:~!J jii! iii~
~ ki!~LII! ~;~iii~ ~iii i!ii !!i!i!iiii! ~
;i';i i!i/~ii !JiJ~i i i'~i~i ~i ~i,i ~i d~!!i!!ii
~
u
i¸
d?
FIG. 4. Assay of/3-galactosidase (A) and alkaline phosphatase (C) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (A) In 6.5% acrylamide; the samples were prepared and incubated at 37° for 3 min (no boiling) before electrophoresis. The gel was assayed for fl-galactosidase as described in the text and photographed. Wells contained: 1,200/xg of crude extract from induced cells; 2 and 3, 20 units and 40 units of commercially obtained /3-galactosidase. (B) Commercial/3-galactosidase was electrophoresed on the same gel but incubated in assay buffer in the absence of substrate and then stained with Coomassie Blue. (C) In 10% acrylamide; the wells contained: 1, commercial alkaline phosphatase (5 units) boiled for 3 min before loading onto the gel; 2 as in 1, but treated for 3 min at 37°; 3, induced cell crude extract (200/zg) treated as in lane 1; 4, induced cell extract (200/xg) treated as in lane 2.
a c t i v i t y in c r u d e e x t r a c t s o f i n d u c e d vated by boiling.
E. coli c e l l s 9 is n o t i r r e v e r s i b l y i n a c t i -
Transfer of Enzymes from Gels to DEAE Paper P r o t e i n a n d e n z y m e c a n b e t r a n s f e r r e d t o D E A E p a p e r f r o m S D S gels. A f t e r e l e c t r o p h o r e s i s , t h e g e l is w a s h e d in r e n a t u r a t i o n b u f f e r f o r 1 0 - 2 0 h r t o r e m o v e S D S . T h e t r a n s f e r is p e r f o r m e d b y a b l o t t i n g t e c h n i q u e u s e d
276
CHAINSEPARATION
[22]
for DNA in agarose gels. z2 Several enzymes (DNA polymerases, /3galactosidase, and alkaline phosphatase) have been detected directly by assaying the DEAE paper as described for the gels. In the case of DNA polymerase, the template DNA (100/zg/ml) has to be added to the reaction mix. The enzyme transfer, however, is less efficient, as described for the nitrocellulose method, 2~ although the enzyme recovery is estimated to be higher than 10%. ~3
Problems Encountered in Assaying Enzymes If a sensitive assay exists for a particular enzyme following SDSPAGE, there may still be problems in its detection. 1. The fully functional enzyme may be oligomeric. Such enzymes, if they possess identical subunits may be detected on the gel by varying the conditions of electrophoresis, such as lowering the SDS concentration in the gel, the absence of 2-mercaptoethanol in the sample, renaturation buffer, and omitting boiling the sample prior to loading onto the gel. Under these conditions the enzyme activity may be seen at the position of the monomer or dimer, etc. Overloading the gel with sample may also result in detectable amounts of renaturable enzyme. If enzymic activity requires two or more polypeptides of different molecular weights, then detection is not possible unless the subunits are eluted from the gel and mixed. However, overlapping of polypeptides of similar size on the gel may be sufficient to detect activity. 2. Essential cofactors like small molecules or metals could be separated during electrophoresis or eluted during renaturation. 3. The protein itself may be eluted from the gel during renaturation or incubation. This may be overcome in certain cases by eluting the polypeptide onto paper (e.g., DEAE paper or nitrocellulose) before renaturation and assay. 4. The inability to detect enzymes may be due to nonoptimal renaturation or assay conditions. 5. Proteolysis of the enzyme may occur during purification of preparation of the sample, and in some cases this happens even in the presence of SDS. 1 Therefore the use of effective proteolysis inhibitors may be important to analyze enzymes at different stages of extraction and purification. Conclusions We predict that the basic principles involved in the assay of the enzymes described above can be applied to the detection of many more ~ F. Winberg and M. L. HammarskjSld, Nucl. Acids Res. 8, 253 (1980). ~s A. Spanos, unpublished results, 1981.
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN S D S GELS
277
APPLICATIONS OF FUNCTIONAL ENZYME RECOVERIES AFTER SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS
Enzyme
Principle of detection
DNA polymerase" Exonucleasesa Endonucleases~
Radioactive labeling of gapped DNA in the gel Loss of Y-OH or Y-OH labeled ends from DNA in the gel Loss of unlabeled or labeled (nick translated) DNA in the gel DNA methylaseb Methylation of unmethylated DNA in the gel using [3sS]adenosylmethionine label RNA polymerasesc Synthesis of RNA using double-stranded DNA in the gel Alkaline phosphataseb Formation of azo pigment in the presence of naphthol AS-MX phosphatase and Fast Blue RR salt /3-Galactosidaseb Formation of 5-bromo-4-chloro-indigo dye from X-Gal Single-stranded binding proteinb Binding of single-stranded DNA in the gel Ap4A-binding protein~ ZH-labeled Ap4A binds to protein in the gel a Spanos et al. 5 b A. Spanos, unpublished results, 1981. U. Hiibscher, unpublished results, 1981. e n z y m e s after t h e i r s e p a r a t i o n on S D S - p o l y a c r y l a m i d e gels. E n z y m e s c a n t h u s b e a s s a y e d in t h e gel o r e l u t e d a n d t h e n a s s a y e d b y c o n v e n t i o n a l t e c h n i q u e s . T h e t a b l e d o c u m e n t s a f e w e x a m p l e s o f s u c c e s s f u l approaches. Both prokaryotic and eukaryotic, monomeric and multimeric D N A p o l y m e r a s e s , for i n s t a n c e , h a v e b e e n d e t e c t e d in gels. D N A p o l y m e r a s e s I, I I , a n d I I I o f E . coli a n d D N A p o l y m e r a s e s a , /3, a n d 7 f r o m s e v e r a l e u k a r y o t i c t i s s u e s h a v e b e e n t e s t e d . ~,7"24 T h u s this t e c h nique has permitted identification of the catalytic subunits of enzymes, e v o l u t i o n a r y c o m p a r i s o n s , a n d a s s e s s m e n t o f t h e influence o f e n d o g e n o u s p r o t e o l y t i c c l e a v a g e on t h e i r s t r u c t u r e . 7 F u t u r e a p p l i c a t i o n s m a y i n c l u d e t h e r a p i d i d e n t i f i c a t i o n o f m u t a n t s d e f i n e d in a p a r t i c u l a r e n z y m e a c t i v i t y a n d , in a s u i t a b l e h o s t , o f t h e a c t i v i t y c o d e d b y c l o n e d g e n e s . It s e e m s p o s s i b l e to c o m b i n e this a p p r o a c h w i t h t h e b e t t e r r e s o l u t i o n o b t a i n e d b y t w o - d i m e n s i o n a l s e p a r a t i o n s , a n d this p r o m i s e s a n e v e n g r e a t e r utility o f this m e t h o d . 2~ Acknowledgments The authors thank Drs. S. G. Sedgwick and G. R. Banks for many useful suggestions, for expert assistance in growing and selecting mutant strains (S. G. S.), and for critical reading the manuscript. Part of this work was supported by the Swiss National Science Foundation, Grant 3.006-0.81. 24 A. Spanos and U. H~bscher, unpublished results, 1981. 25 A. Spanos, unpublished results, 1981.
RECOVERY OF FUNCTIONAL PROTEINS IN S D S GELS
263
A disadvantage in using the ion-retardation resin is its cost; this can be minimized by preparing it from Dowex 1. Although the adsorption of most ions is reversible and regeneration of the resin can be achieved by simply washing with water, SDS appears to be bound very strongly to the resin, since only a 9% recovery of bound [a~S]SDS could be achieved by elution with a 1.0 M NaCl solution. 9 Thus, the resin cannot be reutilized conveniently. The principal advantages of the method are the following: it is a rapid, one-step procedure that avoids excessive loss of proteins and peptides through adsorption on the resin and results in an effectively complete removal of SDS under appropriate conditions.
[22] R e c o v e r y o f F u n c t i o n a l P r o t e i n s in S o d i u m D o d e c y l Sulfate Gels By AD SPANOS and ULRICH HOBSCHER
The use of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) has greatly facilitated the detection, purification, and characterization of proteins from complex mixtures. 1,2 With this technique, proteins are dissociated into their constituent polypeptides and separated according to their molecular weight. After native gel electrophoresis, in which proteins are separated according to both size and charge, it has been possible to detect the enzymic activity of proteins? However, enzymic activities can also be detected following SDSpolyacrylamide gel electrophoresis (PAGE) by removal of the SDS, elution of the protein from the gel, and renaturing it. 4 Alternatively, the renaturation and enzyme assay steps can be performed in the intact gel. This is especially suitable for enzymes that rely on high-molecular-weight substrates or cofactors, such as DNA, RNA, or proteins, since these can be polymerized into the gel) "6 We describe here a procedure to recover the catalytic activity of enzymes after SDS-PAGE within the intact gel. The method was originally I K. 2 U. a O. 4 K. 5 A.
Weber, T. R. Pringle, and M. Osborn, this series, Vol. 26, p. 3. K. L a e m m l i , Nature (London) 227, 680 (1970). Gabriel, this series, Vol. 22, p. 578. Weber and J. Kuter, J. Biol. Chem. 246, 4504 (1971). Spanos, S. G. Sedgwick, G. T. Yarranton, U. Hiibscher, and G. R. Banks, Nucl. Acids Res. 9, 1825 (1981). 6 A. L. Rosenthal and S. A. Lacks, Anal. Biochem. 80, 76 (1977).
METHODS IN ENZYMOLOGY,VOL. 91
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181991-4
264
CHAIN SEPARATION
[22]
developed for enzymes involved in D N A transactions 7 (e.g., D N A polymerases and their associated nucleases), but can also be applied to others, and it has the advantage that either homogeneous, enriched, or even crude enzyme fractions can be analyzed. Questions of isoenzyme or precursor structure and proteolytic degradation can be a t t a c k e d / since the enzymically active bands can be correlated with Coomassie Blue stain (for details, see below). Identification of mutant or cloned enzymes is also possible .5,7 H o w e v e r , the detection o f some enzymes may be hampered by the absence of a sensitive or appropriate assay or by the fact that some enzymes require at least two different polypeptide chains for enzymic activity. Finally, the introduction of two-dimensional electrophoretic systems in connection with e n z y m e renaturation may extend this analytical technique.a Experimental Procedures Preparation o f Gels and Electrophoresis Reagents. Acrylamide (>99% pure), N,N'-methylbisacrylamide, and N , N , N ' , N ' - t e t r a m e t h y l e t h y l e n e d i a m i n e ( T E M E D ) were from Bio-Rad; SDS, "especially p u r e " product No. 30176, ammonium persulfate and glycine were from BDH; and Trizma base (rids), 5-bromo-4-chloro-3indolyl-/3-o-galactoside (X-Gal), naphthol-AS-MX-phosphate, Fast Blue RR salt, and isopropyl/3-o-thiogalactopyranoside were from Sigma. Calf thymus D N A was purchased from Worthington. Strains. Cultures o f wild-type Ustilago maydis and of Escherichia coli wild-type and polA mutants were grown as described, s Cultures ofE. coli strain J G l l 3 were grown overnight in phosphate-free medium to induce alkaline phosphatase synthesis, a Synthesis of/3-galactosidase was induced in E. coli CGSC 4515 b y overnight growth in L broth containing 10 -4 M isopropyl-/3-D-thiogalactopyranoside. TM Control cultures of E. coli 4515-625, which are unable to synthesize/3-galactosidase, were grown under similar conditions. 11 Enzymes. Escherichia coli D N A polymerase I was purified up to the phosphocellulose stage from extracts of heat-induced cells containing the u. Hiibscher, A. Spanos, W. Albert, F. Grummt, and G. R. Banks, Proc. Natl. Acad. Sci. U.S.A. 78, 6771 (1981). a G. Scheele, J. Pash, and W. Bieger, Anal. Biochem. 112, 304 (1981). 9 A. Torriani, in "Procedures in Nucleic Acid Research" (G. L. Cantoni and D. R. Davies, eds.), p. 224. Harper & Row, New York, 1966. 10j. Miller, ed., "Experiments in Molecular Genetics." Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1971. 11 W. A. Newton, J. R. Beckwith, D. Zipser, and S. Brenner, J. Mol. Biol. 14, 290 (1965).
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN S D S GELS
265
ypolA + C 1857 lysogen, tz,la Alkaline phosphatase from E. coli and DNase I were from P-L Biochemicals and Worthington, respectively. Escherichia coil/3-galactosidase was purchased from Sigma. DNA Templates. Calf thymus DNA was dissolved in 20 mM Tris-HC1, pH 7.5, 20 mM NaC1 overnight at 4° and adjusted to a concentration of 3 mg/ml. It could then be denatured by heating the solution at 95 ° for 15 min. To make gapped DNA, MgCh (to 5 mM) and DNase I (to 0.1 ixg/ml) were added to the native DNA solution (3 mg/ml), which was prewarmed at 37° for 15 min. After incubation for 6 min at 37°, proteins were extracted by an equal volume of phenol and chloroform, the aqueous phase was made 200 mM in NaC1, and the DNA was precipitated with ethanol. The DNA pellet was dissolved in 20 mM Tris-HC1, pH 7.5,200 mM NaCI and the precipitation was repeated three times. The DNA was finally dissolved in 20 mM Tris-HC1, pH 7.5, 20 mM NaC1 at 2 mg/ml and stored at - 2 0 °. Agarose gel electrophoresis showed that it possessed a molecular weight of (0.5-5.0) x 106. Gapped DNA was labeled by nick translation 14 or its 3'-termini were labeled using [a-32p]dTTP and the Klenow fragment of DNA polymerase I. 5"15The DNA (0.5 mg) was incubated in a 3-ml reaction mixture containing 50 mM Tris-HCl, pH 7.5, 7 mM MgCI2 1 mM 2-mercaptoethanol, 40 nmol each of dGTP, dCTP, dATP, 50/xCi of [aa2p]dTTP, and 50 units of the E. coil DNA polymerase I Klenow fragment for 30 min at 37°. The DNA was purified, resuspended, and stored as above. If rdquired, the labeled DNA may be denatured before use in the gel.
Solutions and Electrophoresis Stock solutions: see tabulation on p. 266. Electrophoresis buffer: 6 g of Trizma-base, 28.8 g of glycine per liter containing 0.1% SDS and 2 mM EDTA Sample buffer A: 0.04 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 10 mM 2-mercaptoethanol S ample buffer B: 0.025 M Tris-HCl, pH 6.8, 2 mM EDTA, 30% glycerol, 0.6 M 2-mercaptoethanol, and 5% (w/v) SDS Sample buffer C: 0.065 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 0.12 M 2-mercaptoethanol and 1% SDS The SDS-gel electrophoresis is carried out essentially as described by Laemmli. 2 The source of SDS is particularly important, as impurities in 12 W. S. Kelly and K. H. Stump, J. Biol. Chem. 254, 3206 (1979). ~a C, C. Richardson, C. L. Schildkraut, H. V. Aposhian, and A. Kornberg, J. Biol. Chem. 239, 222 (1964). 14 p. W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg, J. Mol. Biol. 113, 237 (1977). ~5 H. Klenow and I. Henningsen, Proc. Natl. Acad. Sei. U.S.A. 65, 168 (1970).
266
CHAIN SEPARATION
[22] Final concentration in
Acrylamide- bisacrylamide, 30%/0.8% Tris-HCl, 1 M, pH 6.8 Tris-HCl, 1 M, pH 8.8 EDTA, 0.2 M, pH 8.0 SDS 10% w/v (freshly made) Substrate (DNA or RNA) Ammonium persulfate TEMED
Separating gel
Stackinggel
5-20% -0.375 M 0.002 M 0.1% As indicated in text 0.05% 0.05%
5% 0.065 M -0.002 M 0.1% -0.1% 0.1%
particular preparations prevent renaturation of enzymes after electrophoresis. 5 It has been found that especially pure SDS from B D H is satisfactory. Depending on the molecular weight of the enzyme under investigation, the acrylamide concentration in the separation gel may be varied from 5 to 20%, and gradient gels may also be used. When highmolecular-weight substrates are included in the gel, they are added prior to T E M E D and ammonium persulfate. The gel is poured without degassing into a slab (0.15 × 17 × 18 cm). A 5% stacking gel (4 cm) containing 0.065 M Tris-HC1 (pH 6.8) is layered on top of the separating gel, and a comb allowing up to 100/.tl of sample loading capacity is used. After loading the samples, electrophoresis is performed at room temperature (20-25 °) at 50-70 V for 15 hr or at 150 V for 4 hr. Sample Preparation. Unless otherwise stated, all operations were carried out at or near 0 °. The use of protease inhibitors is recommended during the preparation and purification of all samples (see Hiibscher et al.r). Freshly prepared or frozen cells (1 g) are resuspended in 3 ml of buffer A and disrupted by using a French pressure cell for unicellular organisms or a Dounce homogenizer for mammalian cells or tissues. Crude e n z y m e preparations (10-20 mg of protein per milliliter) are obtained by centrifugation at 25,000 g for 30 min. Cells and aliquots of samples are stored at - 8 0 ° or in liquid nitrogen, until further use. Crude extracts or homogeneous enzymes (10-100/zl) are thawed at 0° diluted 4 : 1 in freshly made buffer B, and immediately heated for 3 min at 37 ° or at 100° (see below). Buffer C can also be used with the same efficiency. To enhance r e c o v e r y of activity in samples with low protein concentrations, bovine serum albumin (BSA) (10 mg/ml, preheated at 100° for 15 min) is added to the samples at a final concentration of 1 mg/ml. This " c a r r i e r "
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN S D S GELS
267
can also be included in the gel at a concentration of 10/xg/ml. However, this is not recommended when nucleases and DNA polymerases are determined, because BSA preparations may contain nucleases. TM Renaturation of Enzymes. After electrophoresis it is essential to remove the SDS immediately from the gel and to allow the polypeptides to renature. As mentioned, the source of the SDS and also other factors may be important in optimizing the recovery of a particular enzyme (see below). After completion of electrophoresis, the gel is rinsed in renaturation buffer (50 mM Tris-HC1, pH 7.5, 1 mM EDTA, and, if required, 5 mM 2-mercaptoethanol, and then shaken in 1 liter of this buffer at room temperature with changes after 30 and 60 min. Although some enzymes can be assayed immediately after this initial washing step, it is found that others renature only if the gel is kept for a further 3-24 hr at 4°. Prolonged standing may result in loss of enzyme from the gel, especially with small polypeptides. However, this can be tested by comparing identical sampies, one of which is stained with Coomassie Blue immediately after electrophoresis and the other after completion of the enzyme assay. To measure the renaturation kinetics, single-gel tracks can be sliced out and assayed after different times of renaturation. Unless otherwise stated, gels are washed for 1 hr and then left in renaturation buffer for 24 hr at 0° with several changes. Staining and Autoradiography of Gels. After most enzyme assays, the proteins in the gel can be stained with 0.25% (w/v) Coomassie Blue, 50% methanol, 10% acetic acid for 90 min, then destained, first in 50% methanol, 10% acetic acid for 90 min, and finally, in 5% methanol, 7% acetic acid for 24 hr. Staining of gels before autoradiography may result in some loss of radioactivity (determined for DNA polymerasesS). In such cases, gels can first be directly autoradiographed and then stained. After autoradiography, the dried gel can be swelled in distilled water for 60 min, stained, and then destained as described above. Rehydration of gels containing 10% acrylamide may lead to cracking of the gel. Often the enzyme assay is more sensitive than protein staining and can measure enzymes into the picogram range. 17 Where the assay involves the use of a radioactive isotope, such as azp, the gel may be dried onto Whatman 3 MM paper and then autoradiographed. It is possible in certain cases to obtain an autoradiogram of the undried gel by placing it on a glass plate, covering it with cling film and then the X-ray film. This is specially applicable to assays using 32p and for monitoring nuclease activity (see section on nucleases below). ~6 A. Spanos, unpublished results, 1979. ~7 A. Spanos, unpublished results, 1978.
268
CHAIN SEPARATION
[22l
Results and Discussion
Detection of Enzymes in Gels General Remarks. Existing procedures to assay enzymes following electrophoresis in native gels or in other separating systems 8 can be used or adapted to detect enzymes in SDS-polyacrylamide gels. Polypeptides with known enzymic activity can be located by incubating the gel in a specific assay mixture. Visualization can be effected by the formation or removal of an insoluble colored, fluorescent, or radioactive compound at the position of the enzyme band. High-molecular-weight molecules, such as DNA, RNA, or proteins, can be polymerized into the gel mixtures and then serve as substrates or cofactors for enzymes, such as nucleases, DNA and RNA polymerases, DNA methylases, DNA and RNA binding proteins and proteases. The presence of these substrates in the gels does not alter the mobility of polypeptides. The assays of five different classes of enzymes and proteins are now described in more detail to give insight into the potential and the limitations of this technique. Nucleases. A general outline of nuclease renaturation and assay is shown in Fig. 1. Natural or synthetic, single or double-stranded DNA or RNA and D N A - R N A hybrids can be polymerized into the gel and serve as substrates for deoxyribonuclease (DNase), ribonuclease (RNase), or RNase H enzymes. The enzymic hydrolysis and subsequent localized removal of nucleic acid in the gel is detected as a dark band after staining with ethidium bromide (Fig. 2A and C). When radioactively labeled DNA or RNA is polymerized into the gel, autoradiography or fluorography TM techniques detect nuclease activity as a clear band on the black autoradiogram (Fig. 2B and D). The latter approach is more sensitive; very small quantities of natural or synthetic templates can be used, and this allows the detection of either weak or highly specific nucleases (compare Fig. 2A and B or 2C and D). Finally, the protein bands in the gel can be stained with Coomassie Blue before autoradiography. DNases. Both single strand- and double strand-specific DNA endonucleases and exonucleases can be identified by using the appropriate radioactive substrate (Fig. 1). After electrophoresis and renaturation, the gel is incubated in 0.05 M Tris-HC1, pH 7.5, 5 mM MgCI~, and 1 mM CaCI2 at 37°, for times that should be determined experimentally. The DNA in the gel is stained with ethidium bromide (1-5/~g/ml) for 30 rain, rinsed, and photographed using a far-UV light box. This staining does not interfere with the nuclease activity, and further incubations of the gel are still possible. 5,6 Active nucleases show activity within 24 hr of incubation, 18 R. A. Laskey and A. D. Mills, Eur. J. Biochem. 54, 335 (1975).
[22]
RECOVERYOF FUNCTIONALPROTEINSIN SDS GELS DNA p o l y m e r a s e
DNA substrate in gel:
Nueiease 32
IIIIIIIIIII
IIII111111111111
lJlllJlllllllJllllllll
Assay:
269
IIHHllIIIIIIII
I111111111111P
32p
~IlIII[IHIIHIIIIIII
a~p i HI I I I I l i l l i l i l ( [ l i l l l l
IIIIllllilHlll
32
lilt lilt ItJilililiilit
P IIIIIlilllltllllllllilLI
g a p p e d DNA
a2P-labeled DNA
[~-32P]dTTP, dATP, dGTP, dCTP, MgCI2, MSH, buffer
MgCI2, MSH, buffer
32 IIIll111111111ll p
Illllllllllllllllll
32]~) IIIIIIIIIIIIIIIIIIIIIIII
a2 p
llIIHl]lilllllli
IIIlllllllllllll
I 32~:) I III] [ l l l l l IIII II IIIIIII
tllliltllllllLltllll
DNA synthesis
HIHHIIINI~ IIHHIIIIILIIIILIIH
D N A degradation
r
__J wash with TCA, sodium pyrophosphate
stain
dry
|
d a r k band in clear background ~
I autoradiograph
~
clear band in dark background
FIG. 1. Detection of catalytic DNA polymerase and associated exonuclease activities following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Details are outlined in the text.
while weaker nuclease bands are detected after several days of incubation only. In this case, it is advisable to include an inhibitor of bacterial growth (0.01% sodium azide). When nucleases in crude extracts are investigated, long renaturation times (> 14 hr) at 4 ° should be carried out in the presence of 2 mM EDTA to prevent premature degradation of substrate by very active nucleases that can subsequently obscure weaker activities. Assay conditions such as D N A source or concentration, temperature, or pH in the gel can be adjusted to optimal detection of specific nucleases. The assay can be made more sensitive if radioactively labeled DNA, such as bacterial, phage, or plasmid D N A is used in the gel. Gapped calf thymus DNA labeled in v i t r o with a2p at the 3' or 5' termini serves as good templates for endonucleases and 5' ~ 3'- or 3' --~ 5'-exonucleases (see also Spanos e t al. s for details). This has the advantage that the removal of only the 3' or 5' termini, without degradation of the rest of the DNA, by very specific or weak nucleases can be detected by autoradiography. Most of the D N A at the position of such nuclease activity remains intact
270
CHAIN SEPARATION
B
A 1
25K
234
[22]
1
2
34
25K
Fio. 2. Deoxyribonuclease activity detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels (10% acrylamide) containing per milliliter 5/~g of gapped DNA labeled at the 3' termini with asp (10,000 cpm//~g) were prepared as described in the text. After electrophoresis and renaturation, the gels were incubated for 36 hr in assay mixture (see text), stained with ethidium bromide (5 tzg/ml) for 30 min, and then photographed under UV illumination. They were then dried and subjected to autoradiography at room temperature. Figure 2A and C are the ethidium bromide-stained gels, and Fig. 2B and D are the corresponding autoradiograms. Gel A: Wells contained 200 tzg of the following Escherichia coli extracts: 1, Escherichia coli JGII2 (polAl); 2, E. cord BR15 (polA-); 3, E. coli JGll3 (polA+); and 4, E. coli Hfr KLI6 recC22. Gel 2C: Wells contained 200 ~g of freshly prepared Ustilago maydis (wild-type) crude extract; 2, the same as 1, except that the extract was stored at -80 ° for 1 month. a n d is s t a i n e d b y e t h i d i u m b r o m i d e (Fig. 2). I f sufficient l a b e l e d D N A is u s e d ( 5 - 1 0 / z g / m l , 10,000 cpm//.~g), its d e g r a d a t i o n c a n be identified b y b o t h s t a i n i n g a n d a u t o r a d i o g r a p h y . I n i t i a l D N a s e a n a l y s i s should b e d o n e u s i n g a large g a p p e d calf t h y m u s D N A ( M W > 0.5 × 106) l a b e l e d to a high specific a c t i v i t y ( > 10,000 cpm//zg). T h e u s e o f 3'- or Y - l a b e l e d t e r m i n i in D N A facilitates the specific a s s a y i n g o f 3' ---> 5'- or 5' ---> 3 ' - e x o n u c l e a s e s . 5
[22]
RECOVERY OF FUNCTIONALPROTEINSIN SDS GELS
C
271
D
21
21
i
FIG. 2 (continued) To follow the kinetics of e n z y m e activity during the assay, the gel m a y be layered on a glass plate and autoradiographed (see Methods). Other radioisotopes, such as 3H or 14C, m a y be used as well, and the gels can be autoradiographed using a fluorographic method. TM R N a s e s . High-molecular-weight ribosomal R N A from E. coli can be included as a substrate in the gel. Total R N A from yeast is not suitable because, owing to its smaller molecular weight, it is lost f r o m the gel to a great extent during renaturation and incubation. Radioactive synthetic ribopolynucleotides or in vivo labeled ribosomal R N A are polymerized at 1 tzg/ml or less into the gel, and hydrolysis of templates is then detected as for D N a s e s . The specificity of R N a s e s can be investigated with substrates, such as a2P-labeled single-stranded synthetic R N A homopolymers, or with a double-stranded synthetic D N A - R N A hybrid, such as poly(rA) • oligo(dT) for R N a s e H. TM 19j. Huet, A. Sentenac, and P. Fromageot, FEBS Lett. 94, 28 (1978).
272
CHAIN SEPARATION
[22]
DNA Polymerases. These enzymes are detected with the technique described above by including a template DNA that has been nicked or gapped 5,~ (Fig. 1). After electrophoresis, removal of the SDS, and renaturation, the gel is incubated in a reaction mixture specific for the DNA polymerase investigated. As an example, for E. coli DNA polymerase I, the gel is incubated in 2-3 gel volumes of 50 mM Tris-HC1 (pH 7.5), 7 mM MgCI2, 3 mM 2-mercaptoethanol, 12/.tM each of dGTP, dCTP, and dATP, and 1/zCi of [t~-a2P]dTTP per milliliter (>2000 cplrdpmol) at 37° for 16-24 hr. It is then washed for 60 min with two liter changes of 5% trichloroacetic acid (TCA) containing 1% sodium pyrophosphate, left for 40 hr at 4° with at least three changes of TCA solution (removal of [a-a2p]dTTP can be monitored b y a Geiger counter). The gel is dried onto Whatman 3 MM filter paper and autoradiographed, enzyme activity being detected as a dark band where the 3zP-labeled deoxyribonucleoside monophosphate is incorporated into the acid-insoluble DNA template. Exposure time can vary, depending on the activity, and should be determined empirically. The autoradiograms may be scanned by a densitometer to determine quantitatively the intensity of a polymerase activity bandy Alternatively, the activity band can be excised from a gel and counted in a scintillation counter. An example of E. coil DNA polymerase I is illustrated in Fig. 3, which shows that a proteolytic fragment, called the Klenow fragment, 15 is created during the last step of purification. Although this fragment and intermediate proteolytic fragments are present in the preceding purification step, they are not detectable by staining, and this emphasizes the sensitivity of the assay. A few facts should be mentioned at this stage. 1. Very high specific activity, and therefore low concentration, of the labeled dNTP is essential. 5 2. Removal of vital cofactors during electrophoresis and the presence of nucleases that overlap the DNA polymerase band may prevent detection of polymerase activity, a problem likely in crude enzyme fractions. 3. It is possible that a radioactive band resulting from the polymerase assay may be due to binding of the radioactive triphosphate to a protein in the gel. To test this, the gel is incubated with DNase I (10/zg/ml, under the conditions mentioned for DNases) following a DNA polymerase assay. If the radioactive band is still present, it may be due to triphosphate binding. However, removal of the label by DNase I does not exclude the possibility of DNA-dependent binding of the triphosphate by a protein. To investigate this, the radioactive band may be cut out, the DNA extracted, and protein removed by phenol extraction. The radioactivity present in the aqueous phase should then be acid-insoluble but become soluble after DNase I treatment.
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN SDS GELS
A 1
23
273
B 1
23 ii
i liill ? i ¸
i ¸ iii~!i ¸
iiiiii,i(i i: iii~!ii!ili~i~:i~ ~:~iiii' !~~ij:iii ~
109K
76K
\
FIG. 3. Escherichia coli DNA polymerase I activity detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel (7.5% acrylamide) containing 80 t~g of gapped calf thymus DNA per milliliter was run and assayed as described in the text. After drying, it was stained with Coomassie Blue (B) and autoradiographed (A) (2 hr of exposure), Wells contained: 1, hydroxyapatite fraction (for final purification step, see Richardson et alAS), 20 units; 2 and 3, 50 and 20 units, respectively, of the preceding purification step of phosphocellulose chromatography.
4. I f D N A is p r e s e n t in c r u d e e n z y m e f r a c t i o n s , it will e n t e r t h e gel d u r i n g e l e c t r o p h o r e s i s , a n d it is t h e r e f o r e p o s s i b l e t h a t D N A p o l y m e r a s e a c t i v i t i e s a r e d e t e c t e d w i t h o u t a t e m p l a t e in t h e gel. D N A and R N A Binding Proteins. C e r t a i n p r o t e i n s , s u c h as h i s t o n e s , c a n b e d e t e c t e d a f t e r S D S - P A G E b e c a u s e t h e y b i n d to D N A a n d p r e v e n t it f r o m s t a i n i n g w i t h e t h i d i u m b r o m i d e . 2° T h i s m a y in f a c t b e m i s t a k e n f o r n u c l e a s e a c t i v i t y , a n d it is i m p o r t a n t to t e s t s i m u l t a n e o u s l y w i t h a nuclease assay using a radioactively labeled DNA substrate. In the case of a z0 A. L. Rosenthal and S. A. Lacks, J. Biol. Chem. 253, 8674 (1978).
274
CHAIN SEPARATION
[22]
DNA binding protein, the latter assay is negative. Such binding, furthermore, does not usually require divalent cations, and there is no timedependent increase of the band intensity. If low concentrations of radioactive DNA or RNA are used to detect nucleases, dark bands as well as "holes" due to nuclease degradation may appear on the autoradiogram (Fig. 2C and D). These bands have been interpreted as being localized concentrations of the template caused by nucleic acid binding proteins. TM This is because some of the DNA or RNA is normally lost uniformly from the gel during renaturation and incubation, owing to diffusion, and this loss is assumed to be prevented at the position of DNA or RNA binding proteins, thus resulting in a darker band on the autoradiogram. However, nonspecific trapping of the template by some polypeptides is another possible explanation for this phenomenon. Another method has been introduced that transfers the protein after renaturation to nitrocellulose paper. 21 The filter containing the proteins is soaked in a radioactive DNA solution and upon binding to the protein the radioactive bands are detected by autoradiography at places of DNAdependent protein binding. This method is more quantitative and allows the use of a wide range of specific DNA templates or smaller fragments. [3-Galactosidase. An example of an electropherogram of enzyme recovery using E. coli fl-galactosidases is shown in Fig. 4. Crude wild-type extracts and a pure commercially obtained enzyme were electrophoresed and then renatured for 3 hr. The gel was incubated in 0.1 M sodium phosphate (pH 7.0), 0.01 M KC1, 0.001 M MgSO4, 0.005 M 2-mercaptoethanol containing 40 /zg of 5-bromo-4-chloro-3-indolyl-flo-galactoside per milliliter (freshly prepared). After 1-2 hr, a blue band appears in the 130,000-MW region in the gel if the sample is not boiled before loading. When the gel is then further incubated overnight, weaker bands of higher molecular weight appear, suggesting dimers and trimers of the enzyme (Fig. 4A). If, however, the enzyme is boiled before loading, more than 90% of the monomer is lost and the higher-molecularweight bands disappear. In addition, no fl-galactosidase activity can be detected in the mutant strain lacking the fl-galactosidase activity. Alkaline Phosphatase. Sample preparation, electrophoresis, and renaturation are as previously described, except that the gel is washed in renaturation buffer for 30 min and renatured for 1 hr at room temperature. The enzymic activity is then assayed in 0.08 M Tris-HCl (pH 8.0) containing per milliliter 1.6 mg of Fast Blue RR salt and 1 mg of naphthol-ASMX-phosphate. The formation of the azo pigment can be seen after 10 min if a purified enzyme is tested (Fig. 4C). The reaction is completed after 3 hr. As mentioned for fl-galactosidases, heating also removes aggregates. Unlike the crude extract for/3-galactosidase, alkaline phosphatase 21 B. Bowen, J. Steinberg, U. K. Laernmli, and H. Weintraub, Nucl. Acids Res. 8, 1 (1980).
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN SDS GELS
A 2
B 3
275
C
1
1
2
3
4
!! iii i~i!/iiiii? ~¸¸
130K-60K i~ i ii!~ ~ ~ ~
~i ~ ~,~ ~L~
ilij~!!/ i i!!j!:
!;i;~¸:~!J jii! iii~
~ ki!~LII! ~;~iii~ ~iii i!ii !!i!i!iiii! ~
;i';i i!i/~ii !JiJ~i i i'~i~i ~i ~i,i ~i d~!!i!!ii
~
u
i¸
d?
FIG. 4. Assay of/3-galactosidase (A) and alkaline phosphatase (C) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (A) In 6.5% acrylamide; the samples were prepared and incubated at 37° for 3 min (no boiling) before electrophoresis. The gel was assayed for fl-galactosidase as described in the text and photographed. Wells contained: 1,200/xg of crude extract from induced cells; 2 and 3, 20 units and 40 units of commercially obtained /3-galactosidase. (B) Commercial/3-galactosidase was electrophoresed on the same gel but incubated in assay buffer in the absence of substrate and then stained with Coomassie Blue. (C) In 10% acrylamide; the wells contained: 1, commercial alkaline phosphatase (5 units) boiled for 3 min before loading onto the gel; 2 as in 1, but treated for 3 min at 37°; 3, induced cell crude extract (200/zg) treated as in lane 1; 4, induced cell extract (200/xg) treated as in lane 2.
a c t i v i t y in c r u d e e x t r a c t s o f i n d u c e d vated by boiling.
E. coli c e l l s 9 is n o t i r r e v e r s i b l y i n a c t i -
Transfer of Enzymes from Gels to DEAE Paper P r o t e i n a n d e n z y m e c a n b e t r a n s f e r r e d t o D E A E p a p e r f r o m S D S gels. A f t e r e l e c t r o p h o r e s i s , t h e g e l is w a s h e d in r e n a t u r a t i o n b u f f e r f o r 1 0 - 2 0 h r t o r e m o v e S D S . T h e t r a n s f e r is p e r f o r m e d b y a b l o t t i n g t e c h n i q u e u s e d
276
CHAINSEPARATION
[22]
for DNA in agarose gels. z2 Several enzymes (DNA polymerases, /3galactosidase, and alkaline phosphatase) have been detected directly by assaying the DEAE paper as described for the gels. In the case of DNA polymerase, the template DNA (100/zg/ml) has to be added to the reaction mix. The enzyme transfer, however, is less efficient, as described for the nitrocellulose method, 2~ although the enzyme recovery is estimated to be higher than 10%. ~3
Problems Encountered in Assaying Enzymes If a sensitive assay exists for a particular enzyme following SDSPAGE, there may still be problems in its detection. 1. The fully functional enzyme may be oligomeric. Such enzymes, if they possess identical subunits may be detected on the gel by varying the conditions of electrophoresis, such as lowering the SDS concentration in the gel, the absence of 2-mercaptoethanol in the sample, renaturation buffer, and omitting boiling the sample prior to loading onto the gel. Under these conditions the enzyme activity may be seen at the position of the monomer or dimer, etc. Overloading the gel with sample may also result in detectable amounts of renaturable enzyme. If enzymic activity requires two or more polypeptides of different molecular weights, then detection is not possible unless the subunits are eluted from the gel and mixed. However, overlapping of polypeptides of similar size on the gel may be sufficient to detect activity. 2. Essential cofactors like small molecules or metals could be separated during electrophoresis or eluted during renaturation. 3. The protein itself may be eluted from the gel during renaturation or incubation. This may be overcome in certain cases by eluting the polypeptide onto paper (e.g., DEAE paper or nitrocellulose) before renaturation and assay. 4. The inability to detect enzymes may be due to nonoptimal renaturation or assay conditions. 5. Proteolysis of the enzyme may occur during purification of preparation of the sample, and in some cases this happens even in the presence of SDS. 1 Therefore the use of effective proteolysis inhibitors may be important to analyze enzymes at different stages of extraction and purification. Conclusions We predict that the basic principles involved in the assay of the enzymes described above can be applied to the detection of many more ~ F. Winberg and M. L. HammarskjSld, Nucl. Acids Res. 8, 253 (1980). ~s A. Spanos, unpublished results, 1981.
[22]
RECOVERY OF FUNCTIONAL PROTEINS IN S D S GELS
277
APPLICATIONS OF FUNCTIONAL ENZYME RECOVERIES AFTER SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS
Enzyme
Principle of detection
DNA polymerase" Exonucleasesa Endonucleases~
Radioactive labeling of gapped DNA in the gel Loss of Y-OH or Y-OH labeled ends from DNA in the gel Loss of unlabeled or labeled (nick translated) DNA in the gel DNA methylaseb Methylation of unmethylated DNA in the gel using [3sS]adenosylmethionine label RNA polymerasesc Synthesis of RNA using double-stranded DNA in the gel Alkaline phosphataseb Formation of azo pigment in the presence of naphthol AS-MX phosphatase and Fast Blue RR salt /3-Galactosidaseb Formation of 5-bromo-4-chloro-indigo dye from X-Gal Single-stranded binding proteinb Binding of single-stranded DNA in the gel Ap4A-binding protein~ ZH-labeled Ap4A binds to protein in the gel a Spanos et al. 5 b A. Spanos, unpublished results, 1981. U. Hiibscher, unpublished results, 1981. e n z y m e s after t h e i r s e p a r a t i o n on S D S - p o l y a c r y l a m i d e gels. E n z y m e s c a n t h u s b e a s s a y e d in t h e gel o r e l u t e d a n d t h e n a s s a y e d b y c o n v e n t i o n a l t e c h n i q u e s . T h e t a b l e d o c u m e n t s a f e w e x a m p l e s o f s u c c e s s f u l approaches. Both prokaryotic and eukaryotic, monomeric and multimeric D N A p o l y m e r a s e s , for i n s t a n c e , h a v e b e e n d e t e c t e d in gels. D N A p o l y m e r a s e s I, I I , a n d I I I o f E . coli a n d D N A p o l y m e r a s e s a , /3, a n d 7 f r o m s e v e r a l e u k a r y o t i c t i s s u e s h a v e b e e n t e s t e d . ~,7"24 T h u s this t e c h nique has permitted identification of the catalytic subunits of enzymes, e v o l u t i o n a r y c o m p a r i s o n s , a n d a s s e s s m e n t o f t h e influence o f e n d o g e n o u s p r o t e o l y t i c c l e a v a g e on t h e i r s t r u c t u r e . 7 F u t u r e a p p l i c a t i o n s m a y i n c l u d e t h e r a p i d i d e n t i f i c a t i o n o f m u t a n t s d e f i n e d in a p a r t i c u l a r e n z y m e a c t i v i t y a n d , in a s u i t a b l e h o s t , o f t h e a c t i v i t y c o d e d b y c l o n e d g e n e s . It s e e m s p o s s i b l e to c o m b i n e this a p p r o a c h w i t h t h e b e t t e r r e s o l u t i o n o b t a i n e d b y t w o - d i m e n s i o n a l s e p a r a t i o n s , a n d this p r o m i s e s a n e v e n g r e a t e r utility o f this m e t h o d . 2~ Acknowledgments The authors thank Drs. S. G. Sedgwick and G. R. Banks for many useful suggestions, for expert assistance in growing and selecting mutant strains (S. G. S.), and for critical reading the manuscript. Part of this work was supported by the Swiss National Science Foundation, Grant 3.006-0.81. 24 A. Spanos and U. H~bscher, unpublished results, 1981. 25 A. Spanos, unpublished results, 1981.