Elecrrophoresis 1996, 17, 813-829

Solubilization of proteins

813

Review
Thierry Rabilloud
DBMS, CEA, Grenoble, France

Solubilization of proteins for electrophoretic analyses

Contents
1 2 2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.3 2.4 3 3.1 3.1.1 3.1.2 3.2 3.2.1 3.2.2 4 5 Introduction ........................... General principles .................... Rationale of solubilization . . . . . . . . . . . Removal of interfering substances ... Salts .................................. Lipids ................................. Nucleic acids ......................... Polysaccharides ........................ Other compounds ..................... The case of disulfide bonds . . . . . . . . . The problem of protease action . . . . . Specific solubilizations for electrophoretic separations . . . . . . . . . . . . . . . . . . Zone electrophoresis .................. Denaturing zone electrophoresis . . . . . Native zone electrophoresis . . . . . . . . . . Isoelectric focusing . . . . . . . . . . . . . . . . . . . Denaturing isoelectric focusing ....... Native isoelectric focusing ............ Concluding remarks . . . . . . . . . . . . . . . . . . References ............................ 813 813 813 815 815 815 816 817 817 817 819 820 82 1 822 823 824 824 826 827 828

Another general problem is linked to the requirements of the structural analyses methods. Although peptide mass fingerprinting and amino acid composition can be performed at a scale that is close to the one of purely analytical separations, other techniques such as microsequencing of mass analysis by electrospray will still require microgram amounts of the protein of interest. This means that even the final electrophoretic separation must be carried out on a larger scale than a purely analytical one. This in turn gives rise to new problems, such as the elimination of interfering substances, which can often be neglected in analytical separations. A section of this review will therefore be dedicated to the problem of removal of interfering substances. The aim of this review is, however, not to list the incredibly high number of solubilization processes that have been described in the literature. Such a task would be of limited help to the reader and almost impossible to carry out properly for the author. Instead I would like to concentrate on the rationale of solubilization, and then to describe the main solubilization protocols corresponding to the main electrophoretic techniques and their principal variants, with selected references to give examples of the most interesting variants in my opinion. In most cases structural analyses (e.g. microsequencing) will be performed on polypeptides, which means that all the bonds linking the polypeptide chains of the proteins must be broken prior to the analysis. This allows the use of denaturing solubilization in most cases, so that the fractionation prior to primary structural analyses will only use parameters that do not require the native threedimensional conformation, such as the isoelectric point or the polypeptide chain molecular mass. However, nondenaturing solubilizations can be required; for example, if the activity andlor conformation is needed for purification prior to the final separation steps; or if the structural analysis needs to be performed on a complete protein (e.g. mass spectrometry on a total protein). Some examples include the immunopurification with an antibody directed against a conformational epitope, or the solubilization of a multimolecular complex as a whole to take advantage of its huge but precise size to separate it from the bulk of smaller proteins [l].This explains why this review will try to cover both denaturing and nondenaturing solubilization methods.

1 Introduction
Electrophoresis is now increasingly used to resolve hundreds of proteins for analysis by amino acid composition, microsequencing, and more recently mass determination with mass spectrometry, thanks to the scaling down of these techniques. Most often, the high-resolution separation needed can only be performed on polyacrylamide gels, and sometimes by two-dimensional gel electrophoresis only. As for almost all the analytical techniques used in protein chemistry, the proteins to be analyzed must be (i) extracted from the biological sample, (ii) freed from any substances which could interfere with the analytical technique or the subsequent processes, and (iii) kept in solution during the whole separation process. These three steps represent the solubilization process, which may vary greatly from sample to sample or from one type of electrophoretic technique to another.
Correspondence: Dr. Thierry Rabilloud, DBMS, CEA-Grenoble, 17 rue des martyrs, F-38054 Grenoble Cedex 9, France (Tel: +33-7688-3212; Fax: +33-7688-5155; E-mail: thierry@sanrafael.ceng.cea.fr)
Nonstandard abbreviations: C12E8: Dodecyl octa(ethy1ene glycol); 3,4 DCI: 3,4 dichloroisocoumarin; E64: N-[N-(L-3-transcarboxyrane-2carbony1)-L-Leucyll-agmatine; NDSB: non detergent sulfobetaines; NDSB2Ol: pyridinio propane sulfonate; SB 3-14: tetradecyl dimethylammonio propane sulfonate

2 General principles
2.1 Rationale of solubilization Solubilization can be defined as a process breaking the interactions between the substances to be analyzed and interfering substances, eliminating the interfering sub0173-0835/96/0505-0813 $10.00+.25/0

Keywords: Solubilization I Two-dimensional polyacrylamide gel electrophoresis / Interfering substances

0 VCH Verlagsgesellschaft

mbH, 69451 Weinheim, 1996

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Table 1. Forces imolied in orotein cohesion and interactions with other molecules Nature of the interaction Energy of interaction (kCal/mole of bond) Disruption method(s) or agent(s)

Disulfide bond Hydrogen bond Electrostatic interactions

40 3-8 2-5

Charge-dipole Dipole-dipole Van der Waals Hvdroohobic interactions

1
0.3 0.3b ?

Reduction (facultative alkylation) Chaotropes Saltsa, charged detergents, Chaotropes Strong dipolar molecules (less efficient) Salts, dipolar molecules, chaotropes Salts, dipolar molecules, chaotropes Chaotropes, detergents

a) Salts cannot be used simply when electrophoretic separations are intended since they must be removed. b) Van der Waals bonds are very weak, but their high number in many protein-protein interactions or in the core of proteins gives them an important role

stances and preventing secondary reaggregation of the analytes during the separation process, which is electrophoresis within the scope of this paper. It is therefore mainly a process disrupting aggregative forces between the analytes (proteins in our case) and other components (proteins or nonproteinaceous compounds). Generally speaking, polypeptide chains are not linked, within the biological complexes, by covalent bonds. The only exceptions are the interchain peptide bonds made by transglutaminase [2] and the disulfide bonds. Transglutaminase leads to the formation of amide bonds, which are chemically indistinguishable from peptide bonds. Such interchain bonds cannot be disrupted by nondegradative processes [3], which are generally required if analyses other than amino acid composition are to be performed on the solubilized proteins. Oppositely, disulfide bonds are covalent bonds which are easy to break without any other degradation of the protein. However, as this is a covalent bond, breakage of disulfides will require a chemical reaction and, therefore, that the breaking reagent can enter in contact with the disulfide. This implies in turn that disulfide bonds buried in the core of a protein will not be attacked unless the protein is denatured, while disulfide bonds accessible to the solvent can be broken without protein denaturation. Disulfide bond reduction will be detailed below (Section 2.3). The other forces keeping the macromolecules together correspond to noncovalent interactions. The main interactions are listed in Table 1, with their binding energy, and some ways to disrupt them. Among these interactions, Van der Waals forces seem to play a key role in protein folding, as suggested by the hydrophobic collapse model [4]. Van der Waals forces describe the interactions between atoms, through the deformation of their electronic clouds. These interactions are, for example, responsible for the cohesion of simple hydrocarbon molecules, and drive the alkanes of more than 5C to be liquids and not gases. Because of their universal nature which applies also to very weakly polar molecules such as pure hydrocarbons, Van der Waals forces have often been called hydrophobic forces. This is not strictly true however, since they also apply to more polar atoms. The true basis for hydrophobic interactions is in fact the

presence of water. In this very peculiar, hydrogenbonded, highly polar solvent, the exposure of nonpolar groups in the solvent is thermodynamically defavored compared to the grouping of these apolar groups together. Indeed, the van der Waals forces give a contribution that is not much different between the two configurations, while the other forces (mainly hydrogen bonds) are maximized in the latter configuration and disturbed in the former (solvent destructuration), so that the energy balance is clearly in favor of the collapse of the apolar groups together [5]. This explains why hexane and water are not miscible, and also that the lateral chain of apolar amino acids (L, V, I, F, W, Y) pack together and form the hydrophobic cores of the proteins [4]. These hydrophobic interactions are also responsible for some protein-protein interactions and for the binding of lipids and other small apolar molecules to proteins. Detergents will act on such interactions by providing a stable dispersion of a hydrophobic medium in the aqueous medium, for example through the presence of micelles. Therefore, the hydrophobic molecules (e.g. lipids) do not remain collapsed in the aqueous solvent but will disaggregate into the micelles, provided that the amount of detergent is sufficient to ensure the maximal dispersion of the hydrophobic molecules. As detergents also have polar heads, which are able to contract other types of noncovalent bonds (hydrogen bonds, salt bonds for charged heads, etc.), the action of detergents will be the sum of this dispersive effect of the micelles on the hydrophobic part of the molecules and of the effect of their polar heads on the other types of bonds. This explains why various detergents show variable effects, from weak and often incomplete delipidation of membrane proteins (e.g. Tweens) to very agressive action so that exposure of the hydrophobic core in the detergentcontaining solvent is no longer energetically unfavored, with resultant denaturation (e.g. SDS). This example of detergent action points out two important facts. First, the noncovalent interactions are the basis of the three-dimensional folding of proteins and of the interaction of the proteins with other components, proteinaceous or not. Disrupting these interactions will lead to solubilization, i.e., to dissociation of the proteins

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Solubilization o f proteins

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from the other molecules with which they are able to interact, but can also lead to denaturation, i.e. disruption of the three-dimensional structure of the proteins. The ratio between both events will depend on the relative strength of the intermolecular and intramolecular interactions and of the disruptive capacity of the solubilization medium. Second, it must be kept in mind that all the noncovalent forces keeping the molecules together must be taken into account with a comparative look at the solvent. This means that the final energy of interaction depends of the interaction per se and of its effects on the solvent. If the solvent parameters are changed (dielectric constant, hydrogen bond formation, polarizability, etc.), all the resulting energies of interactions will change. This explains why chaotropes, which alter all the solvent parameters, exert such profound effects on all types of interactions. For example, by changing the hydrogen bond structure of the solvent, chaotropes decrease the energetical penalty for exposure of apolar groups and therefore favor the dispersion of hydrophobic molecules and the unfolding of the hydrophobic cores of the proteins [6]. As a conclusion, apart from the disruption of disulfide bonds, which will be described later, solubilization can be described as the way to disrupt the noncovalent forces ensuring the cohesion of supramolecular complexes, for example by the means described in Table 1, so that these complexes will be broken to the desired extent. If the three-dimensional conformation of the protein is to be kept native, there will be a difficult balance to achieve between a sufficient breaking of the undesired interactions and keeping the cohesive interactions in terms of protein three-dimensional structure. Owing to the key role of hydrophobic interactions in the threedimensional protein structure [4], this explains why the cases in which these interactions are to be broken are the most difficult, and, for example, why nondenaturing solubilization of membrane proteins is so difficult. The process will, of course, be even more difficult if multimolecular complexes are to be kept together during the solubilization and the separation process. The solubilization protocol will therefore depend on the requirements of the purification method (in our case, electrophoretic methods), on the choice of conditions (native or denaturing), and on the problems brought by the interfering non-proteinaceous substances present in the biological source of macromolecules. These interfering substances must generally be removed prior to separation of the proteins by electrophoresis, as they often interfere with the electrophoretic processes. This selective removal is a difficult part of the solubilization process; the following section of this review will therefore be devoted to this problem.

cell but proteins can be considered as an interfering substance. Some of them (e.g., coenzymes, hormones, simple sugars, nucleotides) are either so diluted that they do not give any problem or neither interact with the proteins nor with the electrophoresis (e.g. nonreducing simple sugars). Unfortunately, many other classes of compounds give rise to an interference, and the methods to eliminate them will be examined below.

2.2.1 Salts
In most cases, salts do not interfere by a strong binding to the protein, but rather by disturbing the electrophoresis process. In the cases where some proteins have a high affinity for an ion (e.g. calcium-binding proteins), the use of ion chelators or denaturation often relieves the problems. In the extreme cases where the ion is strongly coordinated (e.g. iron in the covalent heme of some cytochromes, zinc in the zinc finger structures) the strength of the coordination is so high that the molecular species which is analyzed is an ion-protein complex of defined stoichiometry, which is generally trouble-free. In general, high amounts of salts are present in halophilic organisms or in some biological fluids (e.g. urine, sweat, and, to a lesser extent, plasma and spinal fluid). The general method used to remove salts is based on dialysis, since salts have much lower molecular masses than protein. Devices which both concentrate the protein and dialyze it (vacuum dialysers, centrifugeable devices, etc.) are often used (e.g. in [7]). The major problem which may arise is loss of protein by adsorption onto the dialysis membrane, or loss by diffusion through the membrane for low molecular weight proteins. The former problem can be solved in some cases by denaturing the proteins in the presence of detergents, which will greatly reduce the adsorption to the membrane. However, it must be kept in mind that micelles are generally large enough to be retained by the membrane, so that any concentration of the sample will lead to a concomitant increase in the concentration of the detergent. Alternatively, if denaturation is tolerated, both problems can be solved by precipitation with TCA in water and resolubilization in a medium convenient for electrophoresis (e.g. in [8]). Other promising methods are based on the precipitation of proteins with dyes [9, 101. These methods are convenient substitutes of TCA precipitation for the elimination of salt. Owing to the selectivity of the protein-dye interaction, they should allow selective precipitation of proteins, and therefore an easy removal of a wide range of interfering compounds. The recent introduction of these methods has not yet permitted full evaluation of their potential.

2.2 Removal of interfering substances

Interfering substances are the compounds that interfere with the solubilization and/or the electrophoretic process, either by binding to the proteins to be analyzed, which often prevents a proper separation, or by preventing the electrophoretic process per se. If proteins are considered the valuable analytes of the sample (which is the case within the scope of this issue) everything in the

2.2.2 Lipids
Lipids give two kinds of problems depending of their supramolecular structure, i.e. as monomers or as assemblies (e.g. membranes and vesicles). As monomers, they can bind to some proteins, usually lipid carriers. Such a binding can alter the characteristics used for the electrophoretic separation (pl, molecular mass) and give rise to artifactual heterogeneity. This problem is generally a

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minor one, which is easily solved by the use of detergents in a denaturing medium for the most tenacious complexes. The problem becomes much more serious when supramolecular assemblies of lipids (membrane and derivatives thereof) are present. As a basic rule, the presence of detergents is the solution of choice to disrupt the membranes, solubilize the lipids, delipidate and solubilize the proteins bound to those membranes or vesicles. Consequently, many reviews have been written on the properties and uses of detergents and the reader is referred to some of them [ll-141, as well as to some comparative work on the efficiency of various detergents on membranes [15]. However, as stated earlier in this review, detergent acts by diluting the lipids into the micelles. A problem will therefore arise for samples with high lipid levels. In this case, there will soon be an inadequacy between the amount of lipids present in the amount of sample required to prepare the protein for the structural analyses and the amount of detergent that can be used in the desired sample volume (insufficient detergency). Two solutions may be envisioned. The first is to scale up the separation, and therefore dilute the sample, so that a correct detergency can be achieved. This is limited by the volumes which can be loaded on the electrophoretic gels. The second solution is to carry out a chemical delipidation on the sample prior to resolubilization of the proteins in the presence of detergents. Delipidation is achieved by extraction of the biological material with organic solvents [16], generally a mixture [17], and often containing chlorinated solvents [18]. Such media are often strongly denaturing, so that the subsequent resolubilization of the proteins must be made in denaturing conditions. However, more conventional and sometimes nondenaturing protein precipitation protocols, for example with ethanol or acetone, often provide a partial but useful delipidation [19, 201. Generally speaking, these media based on organic solvents remove excess lipids efficiently. However, a severe loss of proteins may be experienced, either because some proteins are soluble in organic solvents [17], or perhaps because the precipitated proteins do not resolubilize. Special attention must be paid to the final removal of the organic solvents prior to resolubilization. If the solvent is not efficiently removed, emulsion problems or precipitation by the remaining solvent may arise. If the precipitated protein pellet is dried too extensively in order to remove the solvent completely, a tight and dry pellet - impossible to resolubilize even in media of high denaturing and solubilizing power - appears, with extremely severe losses. This leads to the picture that achievement of a proper and reproducible delipidationsolubilization cycle is difficult as soon as delipidation by organic solvents is required. As a practical rule, the process becomes more and more difficult as the solvent used becomes less and less miscible with water. Consequently, partial delipidation with alcohols is often easier than with acetone, which is itself much easier than with chlorinated- or ether-based solvents.
2.2.3 Nucleic acids

and are therefore able to bind many proteins through electrostatic interactions. Second, this problem is even more severe when separation with isoelectric focusing is to be performed, as nucleic acids also bind carrier ampholytes to give complexes [21], that also bind proteins and focus to give completely artifactual results with a high amount of streaking [22]. Third, nucleic acids (especially DNA) are very long molecules that are able to increase the viscosity of the solutions considerably and also to clog the small pores of the polyacrylamide gels used to separate the proteins. When native, lowionic-strength extractions are performed, the nucleic acids stay compacted as protein-containing complexes which can easily be removed by low to medium speed centrifugation (at most 10 000 g ) . When high salt or denaturing extractions are performed, these complexes are dissociated and the nucleic acids swell in the solution, causing the problems described earlier. For these reasons, removal of nucleic acids is required, unless they are present at very low concentrations [23]. It can be effected by several methods. The first method is digestion by nucleases, initially by a mixture of RNAses and DNAses [24], followed in some occasions by removal of the digestion products (oligonucleotides) by TCA precipitation [25]. As with most of the enzymebased removal methods, the main drawbacks are linked to the parallel action of the proteases contained in the sample, thereby degrading the proteins, and the addition of extraneous proteins (the nucleases). The first drawback can be partially alleviated by digestion with S l nuclease in a urea-containing medium [26], although it has been shown that some proteases are even still active in this medium [27]. In fact, the most efficient methods use centrifugation to get rid of the excess nucleic acids. When SDS-electrophoresis is planned, DNA and large RNAs are only a problem because of their large size and viscosity, which give rise to gel clogging [28]. Ultracentrifugation has been used for years and has shown to be efficient in eliminating these large nucleic acids [29, 301. When isoelectric focusing is planned, advantage can be taken of the formation of nucleic acid-carrier ampholyte complexes to eliminate them by ultracentrifugation [25]. A last problem remains, however: some proteins still stick to nucleic acids even in the presence of high concentrations of urea [31]. These proteins can be solubilized either with competing cations such as protamine [31] or lecithins at acid pH [32], or the extraction pH can be increased so that all the proteins will behave as anions and will be repelled from the anionic nucleic acids. To avoid overswelling of the nucleic acids, which decreases the efficiency of the subsequent removal by ultracentrifugation, this increase of pH can be mediated by the addition of a basic polyamine (e.g. spermine [33]), which will precipitate the nucleic acids. Ultracentrifugation-based methods could be seen as risky methods, since large proteins could also sediment during the removal of nucleic acids, especially if this removal has to be complete and include small nucleic acids. It must, however, be kept in mind that extraction is carried out either with reagents that decrease the buoyant density of proteins (e.g. SDS), or with reagents that increase the density of the solvent (salt, urea). This will lead to decreased sedimentation of the proteins compared to

Several types of problems are encountered because of nucleic acids. First, nucleic acids behave as polyanions

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817

acids interactions can also be used (solubilization in SDS or presence of organic polycations such as spermine or oligoethyleneimine or high pH). However, severe problems can remain if low-molecular-weight, charged polysaccharides are present in the sample, especially if isoelectric focusing is used, since these polyanions will interfere with the separation process, as shown in Fig. 1. Selective precipitation of the proteins with TCA, ammonium sulfate, phenol/ammonium acetate [38], or dyes [9, lo], followed by resolubilization of the protein pellet, may be solutions, but success will strongly depend of the precise nature and size of the polysaccharide and is therefore difficult to guarantee. 2.2.5 Other compounds Apart from these major classes of interfering compounds, other interfering compounds can be found, mainly in extracts from plants. These include lignins, polyphenols, tannins, alkaloids, pigments erc. [39]. For example, polyphenols bind protein by hydrogen bonds as long as they are in the reduced state, while oxidized polyphenols bind protein by covalent bonds. The use of polyvinylpyrrolidone (PVP) to trap polyphenolic compounds is a common practice [39, 401. Otherwise, extraction with organic solvents such as acetone efficiently removes all these interfering compounds present in plant material [41, 421. The problems specifically encountered for solubilization of plant proteins and the solutions that were proposed for carrying out correct solubilization of these plant proteins were reviewed previously [43]. As a general guide, the principal methods for removal of interfering compounds, as well as their scope of application, are listed in Table 2.

Figure 1. 2-D electrophoresis of Dictyosteliurn endocytic vesicles proteins. 80 vg of endocytic vesicle proteins, purified with magnetic dextran [37], then solubilized in SDS and urea, were loaded onto the focusing gels. Focusing with carrier ampholytes, pH gradient 4-8. Second dimension: SDS-PAGE on a 10%T gel. Detection by silver staining [33]. The major streaks on the pattern are due to dextran interference.

that of the nucleic acids, which have a very high buoyant density, so that minimal protein losses due to sedimentation have been reported [29, 301. Other methods based on the selective precipitation of nucleic acids by metallic polycations such as calcium [34] or lanthanum [35] have also been described, but they are much more difficult to control and often lead to protein coprecipitation. In conclusion, removal of nucleic acids can be obtained easily by ultracentrifugation-based methods. The recent availability of miniaturized ultracentrifuges allows the use of these methods even with samples of limited size (a few hundred microliters). 2.2.4 Polysaccharides The problems due to polysaccharides are similar to those due to nucleic acids, although they are generally less severe. Uncharged polysaccharides (starch, glycogen, etc.) pose problems only because they are huge molecules, which could clog polyacrylamide gels. However, their very large size, coupled with a high buoyant density, makes them easy to remove by ultracentrifugation. The problems become much more difficult for complex polysaccharides such as mucins, hyaluronic acids, dextrans, and so on. These polysaccharides usually contain negative charges allowing them to bind proteins by electrostatic interactions. However, their generally lesser charge density, compared to nucleic acids, makes them less easy to precipitate, although precipitation has been observed in some cases [36]. The answer to the problems elicited by polysaccharides is therefore less simple than with nucleic acids. As a simple rule, ultracentrifugation will remove the high molecular weight polysaccharides, and the same methods used for preventing protein-nucleic

2.3 The case of disulfide bonds Breaking of disulfide bonds (Fig. 2) is generally achieved by an equilibrium displacement process using a large excess of free thiols. As simple alkyl thiols are very volatile (which makes their concentration difficult to control) and have a most unpleasant odor, less volatile thiols are used for biochemical purposes, such as mercaptoethanol, thioglycerol (a less awful-smelling, often neglected, substitute) or cysteamine. These chemicals are generally used at very high concentrations (0.2 M or more) to ensure a maximum displacement of the thiol-disulfide equilibrium in favor of the thiol form of the protein. Because of the intramolecular, cyclic condensation produced during oxidation of dithiothreitol (DTT) or dithioerythritol (DTE; Fig. 2), which will drive the equilibrium much more efficiently toward the oxidation of DTT and therefore toward the reduction of the protein, DTT or DTE can be used at much lower concentrations than simpler thiols (generally less than 100 mM). The use of these chemicals is, however, not without drawbacks. For example, dissolved oxygen is able to reoxidize thiols into disulfides, so that the free thiol will be consumed, upon aging of the solution, by oxygen continuously dissolving into the solution from the surrounding air. This will eventually result in the reoxidation of the protein thiols into disufides, and, in some cases, in overoxidation of free protein thiols into spurious disulfides.
This reoxidation process can also take place during electrophoresis because polyacrylamide gels are oxidizing

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Table 2. Removal of interfering compounds

Methods Detergent Ultracentrifugation Precipitation with Complex ions TCA TCA / solvent wash Ammonium sulfate Solventh Salts
-

Lipids S/Lc
+L

Nucleic acids
Sb)

Polysaccharides Vb)
VL

Pigmentsa
-

Protein recovery

S
S
-

S S
V S

V
V

V S VP) S

S
S
+dl
-

S Se
S

V
VS

SO
-

V S V

Abbreviations: S, satisfactory; V, variable; V,, variable with the size of the compound; L, limited; Lc, limited, depending on the concentration of the compound; - inefticient a) Pigments, terpenes, polypbenol and other related compounds b) Efficient only with cationic detergents (inducing in fact a precipitation), with centrifugation to remove the precipitate c) Some lipids may form an upper layer upon centrifugation; in favorable cases, this layer can be removed. d ) Salts are removed, but replaced with residual ammonium sulfate, which can be removed with 70% ethanol. e) Flotation of lipids is often induced. f) Efficient with a two-step procedure: dissociation of the proteins from nucleic acids in 0.6-0.8 M ammonium sulfate, ultracentrifugation to remove them, then quasi-saturation in ammonium sulfate to precipitate proteins. g) Variable, the solubility in concentrated ammonium sulfate not being predictable. h) Including phenol/ammonium acetate precipitation [38].

S-

+
R-SH

by a continuous influx of thiols from the electrode buffer [46]. Indeed, thiols are very weaks acids (pK, of ca. 9), so that a fraction of the molecules bear a negative charge (in the case of mercapthoethanol or DTT) and will migrate into the gel and reduce the oxidizing compounds contained in the gel, thus maintaining a reducing environment in the gel. Variants of this process use sulfonylated thiols such as mercapthoethane sulfonic acid [47], or cysteamine (a positively charged thiol) for acidic gel systems [48]. Another, more drastic, way to prevent reoxidation is to alkylate the reactive free thiols generated by protein reduction into nonreactive thioethers. Such a reaction is generally achieved by nucleophilic substitution on haloacetyl derivatives, generally iodoacetic acid or iodoacetamide [49], or by nucleophilic addition onto an activated double bond, as for substituted maleimides [50],vinylpyridine [51] or even acrylamide [52]. This alkylation process is generally compatible with post-electrophoretic primary structure analyses, such as amino acid analysis, microsequencing or mass spectrometry. In the latter case, however, care must be taken with the mass increments brought by the alkylating reagents. Moreover, the reaction specificity for thiols is often not absolute, and other protein nucleophilic groups can also react, as the &-aminogroups of lysine, with the most reactive agents, such as iodoacetic acid. This can, however, be controlled in most cases by the careful choice of the pH of the reaction [49]. When such an alkylation reaction is performed, another drawback of thiols as reducing agents is brought to light. Indeed, the free thiols that are needed to drive the thiol-disulfide equilibrium will also react with the alkylating reagent, which must therefore be present in excess with respect to the total thiols (protein plus reducing agent). This precludes the use of mercaptoethanol, and even with DTT one comes to a point where the alkylating reagent is used close to its solubility limit and where a considerable amount of byproducts (HI in

HS

S-S-R

+
R-SH

!--/OH

R-S-S-R

A O H

Figure 2. Mechanism of disulfiede (reduction) with free thiols. Lefi column: reduction with free monothiols (e.g. mercaptoethanol). Right column: reduction with cyclizable dithiols (e.g. DTT).

because of the residual persulfate, or reaction products thereof [44, 451. This process can be blocked either by using washed gels in which the spurious, oxidizing chemicals have been washed away, or, in zone electrophoresis,

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Bu~P=O

Figure 3. Mechanism of disulfide reduction with phosphines (e.g. tributylphosphine). Although the overall mechanism is known, the degree of concertation in the electron transfer process between water, the disufide and the phosphine is speculative (intermediate between brackets).

the case of iodoacetyl derivatives) are generated. The latter drawback is, however, not observed for additional reagents (NEM, vinylpyridine, acrylamide). Another additional drawback of thiols as reducing agents lies in the fact that some proteins seem resistant to reduction by mercaptoethanol or DTT. One example is the calcium-binding protein SlOOa (J. Baudier, personal communication). This is due to the fact that the thiol group of the protein is so reactive that it will either react with the first thiol reducing equivalent (see Fig. 2) but not with the second one, leading to an adduct, or even not react with the thiol reagent. Owing to these drawbacks, additional reducing reagents have been investigated, which would lead to reduction by a stoichiometric process and not by an equilibrium displacement process. Many of the agents used in organic chemistry to achieve disulfide reduction are not of practical use in biochemistry (borohydrides, sodium in acidic medium, etc.). However, trivalent phosphorus derivatives have proved to be very promising for reduction of disulfides under biochemical conditions. Of these, phosphines have been shown to be efficient, while phosphite esters are of marginal interest. Phosphines are compounds in which the phosphorus atom is linked to three simple hydrocarbon groups. The lowest members of the class (e.g. trimethylphosphine) are highly reactive and pyrophoric, while higher members (e.g. triphenylphospine) are safer to use but of very limited solubility in water. A good compromise is tributylphosphine, which was the first phosphine used for disulfide reduction in biochemistry [53]. These compounds, which react with disulfides as shown in Fig. 3, show many advantages. First, the reaction is stoichiometric, which, in turn, allows the use of a very low concentration of the reducing agent (a few mM). Second, these reagents are not as sensitive as thiols to dissolved oxygen. Third, because of the limited concentration of the agent, alkylation is much easier to perform. Phosphines interfere with alkylation with iodoacetyl derivatives. However, a typical reduction-alkylation will involve 2 mM tributylphosphine and 4 to 20 mM alkylating agent [54], instead of 50 mM DTT and 150 mM alkylating agent. Moreover, phosphines do not interfere with compounds containing double bonds, so that simple and convenient one-step protocols contacting the protein with 2 mM phosphine and 10 mM alkylating agent (final concentrations) for 30 min at room temperature and pH close to neutrality give total reactions. Some drawbacks can be found for the use of tributylphosphine. The reagent is volatile, toxic, has a rather unpleasant odor, and must be brought into water with

the help of an organic solvent. In the first uses of the reagent, propanol was used as a carrier solvent at rather high concentrations (50%) [53]. However, it was found that DMSO or DMF are suitable carrier solvents, which easily allow reducing the protein with 2 mM tributylphosphine [54]. All these drawbacks have disappeared with the introduction of a water-soluble phospine, tris(carboxyethy1)phosphine (available from Pierce), for which 1 M aqueous stock solutions can be easily prepared and stored frozen in aliquots. In addition to their superiority on thiol reagents, as soon as subsequent alkylation is performed, phosphines have the additional advantage that they reduce all protein disulfide bonds, even those resistant to DTT (as the SlOOa protein; Jacques Baudier, personal communication), and that they do not yield any adduct formation, as has been described for mercaptoethanol [45]. The use of phosphines as reducing agents should therefore be systematically envisioned as long as total reduction of all the accessible disulfides is planned, which is almost always the case when subsequent primary structure analyses are planned.

2.4 The problem of protease action

Proteases present in the sample are everything but friends for the protein biochemist, especially when structural analyses are planned. Although proteolysis is an important mechanism to mature and recycle proteins in vivo, a great amount of artifactual proteolysis can occur when the tissue or cell is broken for solubilization, owing to the breakage of the compartmentalization existing in vivo. This proteolysis should be avoided by all means, since it can lead to severe artifacts and misfacts, for example if N-terminal sequences or mass determinations are looked for, or generate artifactual polypeptides, or decrease the yield for the desired protein. Proteases may be classified according to the amino acid which effect the catalytic attack of the peptide bond. This leads to the distinction between serine, cysteine, and aspartyl proteases. Other proteases (metalloproteases) need a metal ion to effect proteolysis. Destruction of this amino acid or active site will therefore produce an efficient and irreversible means of avoiding proteolysis. In another approach to avoid proteolysis, advantage is taken of the specificities of action of proteases (e.g. pH). For example, lysosomal proteases are active at low pH and inhibited at high pH, which can be used in the solubilization process. Advantage can also be taken of the occurrence of natural or synthetic peptidic protease inhibitors, which bind to and block the active site of their substate protease(s) (e.g. trypsin inhibitors such as a1 antitrypsin, Kazal- or Kunitz-type inhibitors, etc.).

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The problem of protease action is of course most prom- quires its free thiol groups), competitive, reversible, inhiinent when native solubilization is performed, as all the bitors are often added. Many of these inhibitors are proteins, including proteases, are native and have their short peptides (e.g. pepstatin, antipain) or small molemaximal activity. In most animal cells and tissues, the cules (e.g. benzamidine), which will not interfere with main problem arises from lysosomal proteases. Since the subsequent electrophoretic analysis. However, the these proteases are most active at low to medium pH, use of large molecules (e.g. a2 macroglobulin) has also solubilization at a pH as high as possible will greatly been proposed [60], with the risk that this use will result in artifactual polypeptides arising from the addition of help in preventing proteolysis. However, this is usually not sufficient, and cocktails of inhibitors are almost the protein or of its partial degradation. Commonly used always added to the solubilization medium. In general, inhibitors, which have been used either alone [56], or, these cocktails contain both peptidic and irreversible more frequently, as various cocktails [39, 551 are listed in inhibitors [55]. The most popular irreversible inhibitor is Table 3. Comprehensive reviews of protease inhibition phenylsulfonyl fluoride (PMSF). This chemical reacts have already been published [55, 611. with the activated serine of the catalytic center of serine proteases and prevents it from playing its catalytic role, Although dramatically decreased, the problem of proso that irreversible inhibition is obtained. This chemical tease action is not abolished when denaturing solubilizahas been used by a host of workers, with variable tions are performed. Evidence of proteolysis after solubisuccess (e.g. [56, 571). Indeed, the use of PMSF is not lization in 9 M urea [57] or SDS [62] has already been dewithout drawbacks. (i) Its solubility in water is rather scribed. This is probably due to the fact that many prolow, so that it must be introduced with the help of a teases are resistant proteins, as shown by their ability to cosolvent (generally ethanol or propanol). In spite of work in dilute SDS, used for example in peptide mapthis, precipitation often occurs when PMSF is added to ping [63]. This means that their kinetics of denaturation the extraction solution, which decreases the active con- in urea- or SDS-based solutions can be slow enough to centration. This in turn leads to a severe decrease in pro- allow them to work for a nonnegligible time, while most tease inhibition [58]. (ii) PMSF is unstable and degrades of the other cellular proteins are already denatured and therefore expose a maximal number of proteolytic sites. by spontaneous hydrolysis (half-life at room temperature and pH 8 = 30 min). (iii) Some serine proteases seem to This problem of proteolysis in denaturing solutions will be quite resistant to PMSF, as human trypsin 1 [59]. of course strongly depend on the concentration of proteases in the sample, and seems to be more important in Most of the drawbacks of PMSF can be alleviated by plant tissues [57]. This work also demonstrated that addiusing other irreversible inhibitors. DFP (diisopropyl fluo- tion of protease inhibitors was of weak, if any, efficiency rophosphate) is much more potent and water soluble, to solve this problem [57]. In such difficult cases, the but is very toxic. The best compromise seems to be ami- only solution is to increase the denaturing power of the noethylbenzylsulfonyl fluoride (AEBSF), a more stable solubilization process as much as possible. For muscle and water-soluble analog of PMSF. This compound, as samples, where the degradation of the giant proteins DFP, has the drawback of introducing an electric charge nebulin and titin is a problem [62], the use of a ureato the molecule with which it reacts. This is not a pro- thiourea denaturing solutions has been proposed [46,62]. blem as far as protease inhibition is concerned. However, This solution was thought to penetrate faster into the side reactions of these inhibitors on other nucleophilic muscle, therefore blocking the action of endogenous progroups of the proteins (other serines, tyrosines or teases faster. For plant samples, which are very rich in lysines) could introduce charge artifacts that are not proteases, solubilization in boiling SDS has been procompatible with methods such as isoelectric focusing. posed [64]. In this case, the thermal denaturation synerOwing to its somewhat recent introduction, artifacts gizes the SDS denaturation and affords a faster inactivaresulting from the use of AEBSF have not been de- tion of proteases. Another solution is to homogenize the scribed yet. Other irreversible inhibitors for serine pro- sample in dilute TCA [65] or in TCA in acetone [42], teases are those based on a reactive analog of the amino which inactivates and precipitates all proteins, including acid recognized by the protease. Example of this class proteases almost instantaneously. Subsequent resolubiliare tosyl lysyl chloromethyl ketone for the inhibition of zation of the protein precipitate in an SDS buffer [65] or trypsin and tosyl phenylalanyl chloromethyl ketone for a urea-containing buffer [42], does not seem to yield any the inhibition of chymotrypsin. These inhibitors are vey reactivation of the proteases. Such procedures are probpotent but also very specific, so that they do not inhibit ably the only efficient ones for tissues very rich in proefficiently the wide range of serine proteases generally teolytic activities. present in a cell or a tissue. Irreversible inhibitors for cysteine proteinases are generally thiol-alkylating agents, such as iodoacetyl derivatives or N-ethylmaleimide or mercury compounds. Aspartyl proteases can also be irreversibly inhibited [39], but inhibition is effective only in the presence of copper ion, which precludes the use of metal chelators, which are generally added to inhibit the metalloproteases. To complement the action of irreversible inhibitors, which cannot be used under all circumstances (e.g. thiol-alkylating reagents if native solubilization of a protein re-

3 Specific solubilization for electrophoretic separations

Keeping in mind the general principles and constraints exposed above, the solubilization procedures used prior to electrophoretic separations will also depend on the constraints introduced by the separation method itself. I would therefore like to discuss the solubilization procedures which can be used for the main types of electrophoretic separations, i.e. zone electrophoresis on the one

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Table 3. Protease inhibitors

Irreversible Aspartyl proteases Cysteine proteases Inhibitors Diazonorleucine methyl ester/Cu2+ N-ethylmaleimide Iodoacetate-iodoacetamide p-Hydroxymercurybenzoate E64a) PMSF AEBSF TLCK~) TPCKb) 3-4 DCF) Soectrum General General General General General General Trypsin-like Chymotrypsin-like General Useful concentrations 2-10 mM 1-10 m M 1-10 mM 1-5 mM 0.5-10 vg/mL 0.1-1 mM 0.4-4 mM 20-100 pg/mL 70-100 pg/mL 5-200 p~

Serine proteases

Reversible inhibitors Aspartyl proteases Pepstatin Cysteine proteases Serine proteases Antipain Leupeptin Antipain Leupeptin Aprotinin

Metalloproteases

Chymostatin Benzamidine Elastinal EDTA Phosphoramidon

Pepsin, cathepsins, renin Papain-like Trypsin-like Trypsin-like Trypsin-like Trypsin, chymotrypsin, kallikreins Chymotrypsin-like Trypsin-like Elaslase-like General General

2-100 pg/mL 2-20 2-20 2-20 2-20 1-10

pg/mL pg/mL pg/mL pg/mL pg/mL

5-20 pg/mL 1-3 mM 5-50 pg/mL 0.5-5 mM 10-500 p~

a) E64: N-[N-(~-3-transcarboxyrane-2-carbonyl)-~-leucyl]-agmatine b) Tosyl-~-lysinechloromethyl ketone; inhibits also cystein, proteases of similar substrate specificity c) 3,4 DCI: 3,4 dichloro isocoumarin

hand and charge-based separations such as isoelectric focusing on the other hand. As for solubilization requirements, two-dimensional electrophoresis will behave as the first dimension, which is generally isoelectric focusing. Concerning the solubilization methods, the main difference between what is allowed for zone electrophoresis and what is allowed for isoelectric focusing comes from the fact that it will not be possible to use agents modifying the charge of the proteins in the latter case. This gives rise to a serious solubilization problem, as electrostatic repulsion beween molecules cannot be reinforced by manipulation of the charge of the molecule. Oppositely, this manipulation is often desired in zone electrophoresis to decrease the problems coming from different charge densities of the molecules and from the pH dependency of the net charge of the proteins. In view of what has been stated in the introduction, both native and denaturing solubilizations will be discussed for each main method. 3.1 Zone electrophoresis The solubility problems encountered in zone electrophoresis arise from the necessary use of discontinuous electrophoresis to achieve correct resolution. This means that a stacking process of the proteins takes place, so that the resulting concentration of the proteins within the stack is very high. Since the proteins which are separated by zone electrophoresis migrate in the same direction (the others being lost in buffers), they all have an electric charge of the same sign; electrostatic repulsion thus takes place and decreases aggregation. Unfortunately, the charge density on most proteins is not high enough to give rise to a strong electrostatic repulsion; aggregation therefore occurs at high concentrations by

the means of other binding forces (hydrogen bonds, hydrophobic interactions, etc.). This results in separations that are very sensitive to loading; this is a severe problem for structural analyses, which often require high loads of proteins. As an additional problem, proteins have widely different pls [66].This means that at a given pH, there will be anionic and cationic proteins. This may lead in turn to electrostatic interactions and therefore precipitation within the sample. In addition, zone electrophoresis will separate proteins with a given type of charge (anionic or cationic), so that proteins will be lost, because electrophoresis cannot be carried out at pH extremes where all the proteins would have the same type of charge. The best way to solve these problems is to use agents that will bind to proteins and give them an additional charge. This means in turn that separation on the basis of charge will be lost, and that separation will mainly occur according to the molecular mass. This is often not a problem, since separation on the basis of charge is much more efficiently carried out by specialized techniques such as isoelectric focusing. The use of charge modifiers, by conferring to the proteins an additional charge (generally a negative one) has several advantages for solubilization. First, it allows converting most, if not all, proteins to species of the same type of charge. By reinforcing the electrostatic repulsion, this dramatically increases the solubility of the proteins before and during the electrophoresis process. This also allows the separation of most proteins with a single type of electrophoresis (mainly by considering proteins as anions). Last but not least, charge modifiers can also dramatically increase the repulsion between proteins and interfering compounds (e.g. nucleic acids), which may considerably

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facilitate the solubilization process. In fact, these highly desirable properties have been used in most zone electrophoretic protocols, denaturing or not. 3.1.1 Denaturing zone electrophoresis In a first approximation, the world of denaturing zone electrophoresis is very simple and is contained in three letters: SDS. The somehow magic properties of SDS come in part from its ability to bind proteins at a high mass ratio (ca. 1.4 g of SDS per g of protein) basically independent of the amino acid composition and sequence of the protein [67, 681. This independence is the key of the superiority of SDS over other detergents such as decane, dodecane or tetradecane sulfonate, or decyl or tetradecyl sulfate. Indeed, the masdcharge ratio of the SDS-protein complexes will be independent of the nature of the protein, so that the resulting electrophoretic separation will be based only on the size of the initial molecule [69]. However, recent work has shown that undecyl sulfate might well even be superior to SDS, while confirming the amazing superiority of SDS over the other anionic detergents [70]. On second thought, the magic properties of SDS are those of many ionic detergents. With its long, flexible alkyl tail, SDS is able to contract hydrophobic interactions with all combinations of amino acids, which leads to massive unfolding, and thus denaturation, of proteins. However, not all detergents with C12 linear alkyl tails are so effective, dodecyl maltoside, Tween 20 and C12E8 being very mild detergents. The action of SDS is in fact largely due to its ionic head, which can break ionic interactions between proteins and, last but not least, drive an important electrostatic repulsion between SDS-protein complexes. This prevents reassociation of SDS-protein complexes, and keeps them soluble even at the very high concentrations encountered in gel electrophoresis. Other desirable properties of SDS come from the fact that its ionic head is derived from a strong ion, so that the presence of the charge is guaranteed in the pH interval 2 to 12, where most of the biochemical separations take place. To ensure maximum binding of SDS and proper denaturation of proteins, SDS has to be present in slight excess over the calculated ratio (say at least 1.5 g of SDS per g of protein), and a synergistic denaturation process has to be applied. Indeed, for some proteins for which the intramolecular cohesion is very important (and many proteases belong to this class), SDS alone may not be able to disrupt the structure of the protein, but will only coat the outside of the protein. An important negative charge, but only partial, if any, denaturation will be obtained. Healing (in SDS), as commonly described [71], induces thermal denaturation and total exposure of the protein to the solvent, so that SDS can bind to all parts of the proteins. The charge and intermolecular electrostatic repulsion induced by SDS binding generally prevents the denatured proteins from reaggregating and precipitating. However, certain membrane proteins precipitate upon heating in SDS, while they remain soluble without heating (J. L. Popot, personal communication). In these proteins, which are rich in hydrophobic residues, SDS binding to the unfolded protein might not be sufticient to overcome the host of hydrophobic interactions

between molecules, which eventually lead to reaggregation. Heating in the presence of SDS can be replaced, with almost equal efficiency, by the use of SDS and urea at room temperature [72], which avoids the protein cleavage sometimes observed by heating in SDS [72]. The efficiency and simplicity of solubilization by SDS followed by zone electrophoresis is shown by the enormous popularity of the method, as shown by the ca. 9000 citations of Laemmlis paper [71] per year. However, there are some cases for which SDS is not optimal, and for which alternative procedures have been searched. The most difficult case for SDS is represented by glycoproteins. The hydrophilic glycan moiety reduces the hydrophobic interactions between the protein and SDS, while the negative charges of the glycan (induced for example by sialic acid) induce a repulsive electrostatic interaction between SDS and the protein, both phenomena preventing the correct binding of SDS. Other difficult cases are represented by very acidic or very basic proteins (e.g. histones [73], which also exhibit an abnormal binding of SDS. These cases do not generally result in a loss of resolution, but rather in misdetermination of the molecular mass. This is usually not a problem in the planning of subsequent structural analyses, in which case electrophoresis is used as a separation method and not to determine molecular masses. However, abnormal migration can result in smears (glycoproteins) or decrease resolution (histones) or artifactual comigration. The alternatives which have been described therefore deserve attention. The limitation of SDS arises from its negative charge and maybe too short alkyl tail; the solution was to investigate the use of cationic detergent with longer alkyl tails (C14 to C16). This led to the description of cationic detergent solubilization and electrophoresis, several variants arising from the use of different detergents or gel systems [74-761. Cationic detergents indeed offer many desirable properties. They solubilize and denature protein efficiently, as long as a synergistic denaturation by urea [76] or heating [75] is carried out, but they also precipitate large polyanions, including nucleic acids and charged polysaccharides, thereby facilitating the removal of these interfering substances and also the extraction of their bound proteins [32, 761. The main problem with cationic detergents arises from the subsequent electrophoresis. High resolution electrophoresis implies the use of discontinuous electrophoresis. Discontinuous electrophoresis of cations needs to find correct leading and trailing cations and correct separation pHs, which are generally acidic [74-761. The problems thus arise from the fact that the conventional acrylamide polymerization initiators, TEMED and persulfate, are poorly efficient at low pH [77], and that persulfate precipitates many cationic detergents [78]. This led the early workers to use other polymerization systems, using either riboflavin and light [75] or a mixture of ascorbic acid, hydrogen peroxide and ferrous ion (Fentons reagent) [76] or even light and uranium [79]. The toxicity of the latter system, and the erratic polymerization allowed by the former ones, have prevented the spreading of cationic detergent electrophoresis. It must

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be emphasized here that erratic and poor polymerizations are a real problem for the subsequent analysis of proteins separated on polyacrylamide gels. Indeed, unreacted acrylamide can react with proteins [52, 80-821, inducing thiol and amine alkylation, which in turn result in N-terminus blocking, lysine modification and adducts interfering with mass spectrometry. Recent work on cationic electrophoresis, however, may bring this technique back to the front of the stage, and provide a useful alternative when SDS electrophoresis does not perform well. The first solution, which has been described, is to carry out electrophoresis at neutral pH, where standard TEMED-persulfate polymerization can take place. This is possible by the careful choice of the buffering (Tricine), leading (sodium), and trailing (arginine) ions [83]. Upon testing this protocol, I found it to give a somewhat decreased resolution compared to older ones (e.g. [75]), maybe because of the interference between persulfate and cationic detergents. .In addition, electrophoresis at neutral or slightly basic pH is not to be favored, as the side reactions between proteins and free acrylamide increase with pH [84]. In my opinion, cationic detergent electrophoresis should be reexamined in the light of the new photopolymerization system introduced by Righettis group [85]. This system has many desirable properties (reviewed in [86]), including a high polymerization efficiency over a wide range of pH down to pH 3 [77], and insensitivity to the presence of organic solvents or detergents [87, 881. This system was tested for zone electrophoresis in the cationic mode and found to give very good results [89]. Minor problems, however, before cationic detergent electrophoresis becomes as user-friendly as SDS electrophoresis are the absence of good tracking dyes or difficulties in the staining protocols; however, the compatibility of cationic detergent electrophoresis with structural analyses (e.g. microsequencing), together with desirable properties such as a high resistance to high loading, have already been nicely demonstrated [90]. 3.1.2 Native zone electrophoresis When compared to denaturing gel electrophoresis, the specific problems of native electrophoresis are mainly the result of solubility problems as soon as the protease problem is considered as solved (see introduction of Section 3.1). As an additional problem, native electrophoresis must be carried out at a pH close to neutrality. This implies in turn that many proteins will not bear a strong net charge, so that their migration in the gel is poor, or even that many proteins will bear a charge of opposite sign and will be lost. As most of the proteins have a p l below 7 [66], native electrophoresis generally separates anions (e.g. Fig. 4 in [91]). However, a system for separation of cationic proteins at neutral pH was recently described [92]. The best way to alleviate the problems inherent to charge electrophoresis is to use charge-shift electrophoresis, or derivatives thereof. The rationale of this technique is to try to recover the beneficial effects of SDS without the denaturing power by using chemicals that will bind to proteins and give them a strong neg-

Figure 4. Native gel electrophoresis. Proteins, solubilized in 0.2 M BisTris 0.1 M HC1 were loaded on a 10%T polyacrylamide gel, cast in 0.15 M Tris, 0.1 M HC1 with a 4%T stacking gel, cast in 0.2 M BisTris, 0.1 M HC1. Electrode buffer: 0.1 M Tris, 0.1 M taurine (this buffer system, previously described for 2-D electrophoresis [33], has an operative pH of 8.6). Electrophoresis was carried out overnight at 90V. The buffer system used here is more alkaline than the one described by Schagger and Von Jagow [95], but is cheaper to use and affords a better pH compromise for native electrophoresis without chargeshifting agents. From left to right: carbonic anhydrase, 200 ng; soybean trypsin inhibitor, 100 ng; Ovalbumin, 100 ng; bovine serum albumin: 100 ng; conalbumin, 100 ng. The following lanes are acidic peroxydoxin, 50 ng, under various disulfide bond reduction and alkylation conditions. This shows that this protein is present as a dimer held by disulfide bonds [91], but also by other noncovalent bonds. Protein detection by silver staining [33].

ative charge without denaturing them. Because of the resulting strong charge density, the intermolecular electrostatic repulsion is greatly increased, which dramatically improves the solubility, and therefore increases the loading capacity. Moreover, the pH dependency of the separation will be greatly decreased. Even at neutral pH, all the proteins binding the charge-shifting agent will show a strong negative charge, even those with a high PI, so that anion electrophoresis will be of almost universal use. Last but not least, differential binding of the charge shifting agent can lead to separation of proteins of similar molecular mass, by modulating the net charge of the protein complexes. The first charge shifting agents used for electrophoresis were detergents based on bile salts [93, 941. Because of their rigid polycyclic hydrophobic part, these detergents can only contract limited hydrophobic interactions, which are generally not powerful enough to lead to protein unfolding. They are thus generally considered nondenaturing detergents, although they are known to be able to inactivate some membrane proteins, maybe through subunit dissociation. However, the binding of many bile salts seems to take place on a limited number of sites on many proteins, so that they are weakly efficient as general charge shifting agents. A happy exception seems to appear in taurodeoxycholate, which seems

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to be a powerful charge shifting agent, although its use is not recommended for multiprotein complexes [95].
Another attempt was made by using sulfated alkyl oligoethylene glycol detergents [96], and these proved useful for the separation of photosynthetic proteins [97]. Here again, the versatility of these chemicals as charge shifting agents has not been sufficiently investigated. The most versatile charge shifting agent seems to be Coomassie Blue G-250, introduced by Schagger and co-workers [95,98]. Coomassie Blue binds to a very wide range of proteins (although there are still some exceptions [98]) and gives them a strong negative charge, although the binding seems to be mild enough to allow the preservation of sensitive multiprotein complexes [l, 951. This results in a good separation with fairly high protein loads, while protein recovery from the gel and subsequent analysis still appear feasible [99]. In my opinion, the main interest of this technique of native electrophoresis, as far as structural analyses are concerned, is twofold. The first case is when previous chromatographic separation of a desired activity do not yield a pure enough protein, so that the SDS pattern does not allow choosing protein bands to carry out further analyses (e.g. microsequencing). In this case, separation of the proteins under the native mode, followed by elution of the bands, detection of the activity, and reelectrophoresis of the activity containing band(s) on a denaturing gel offers a powerful separation means. The second favorable case is the one of large multiprotein complexes. Native electrophoresis offers the opportunity of a simple and powerful separation of large complexes from the bulk of low molecular weight proteins and from smaller polymeric complexes [99]. An elegant twodimensional electrophoretic separation can then be designed, with the first, native electrophoresis to separate the large complexes, and the second, denaturing, electrophoresis to separate the subunits of each complex, which can then be submitted to structural analyses [98]. A peculiar case is presented by some extremely hydrophobic proteins such as the seed storage proteins. These proteins are soluble in denaturing detergent-containing media (SDS) and in hydroorganic mixtures [loo-1021, but not in conventional aqueous buffers. Their native solubilization and analysis must therefore be performed in hydro-organic mixtures containing at least 50 O/o organic solvent. As standard polyacrylamide gelation systems are not efficient under these conditions, this has led to the design of new acrylic monomers [103-1051, which are rather hydrophobic, but which must absolutely be used with such mixed solvents, and are therefore of limited use. Another solution to the problem of carrying out electrophoresis in the presence of high amounts of organic solvents could be afforded by a new photopolymerization system, allowing polymerization of polyacrylamide gels in the presence of high proportions of organic solvents [87]. 3.2 Isoelectric focusing and two-dimensional electrophoresis
As far as solubilization is concerned, two-dimensional electrophoresis parallels isoelectric focusing, as most

high-resolution two-dimensional electrophoresis methods use isoelectric focusing (generally denaturing IEF) as the first step. Some protocols [106-1081 have used SDS-PAGE as the first dimension, which considerably decreases solubilization problems. However, difficulties are encountered in running and handling the large isoelectric focusing gels required for the second dimension. In addition, interfacing IEF gels with structural analyses is a difficult task, partly because of the interference brought about by carrier ampholytes on many occasions, and partly because of other problems such as difficulties in blotting [109-1121. These problems have prevented these variant methods from gaining widespread use. It should be also emphasized that interfacing between a restrictive SDS gel and an IEF gel is not easy [106, 1071 and cannot be guaranteed to be a trouble-free process. The solubilization problems arising for isoelectric focusing come from the fact that the electrostatic repulsion between proteins, which is widely used for zone electrophoresis, cannot be used with this technique, which requires keeping the native charge of the protein. This raises problems at three levels: (i) During the initial solubilization of the sample, there can be important interactions between proteins of widely different pZ and/ or between proteins and interfering compounds (e.g. nucleic acids). This yields poor solubilization of some components. (ii) During sample entry into the focusing gel, there is a stacking effect due to the transition between a liquid phase and a gel phase of a higher friction coefficient. This stacking increases the concentration of proteins and may result in precipitation. (iii) At, or very close to, the isoelectric point, the solubility of the proteins comes to a minimum. This can be explained by the fact that the net charge comes close to zero, with a concomitant reduction of electrostatic repulsion. This can also result in precipitation of the proteins. It should be kept in mind that, apart for point (i), the main forces responsible for protein precipitation are hydrophobic interactions and hydrogen bonds. The problem of protein solubilization for and during isoelectric focusing thus comes to a minimizing of these interactions 3.2.1 Denaturing isoelectric focusing For denaturing isoelectric focusing, the magic SDS component of zone electrophoresis is replaced by another magic component: urea. Among many effects, urea has beneficial ones for solubilization for isoelectric focusing. First, it decreases considerably the strength of hydrogen bonds. Second, as it increases the water solubility of hydrophobic compounds [6], it considerably decreases hydrophobic interactions (see Section 2.1). This considerably decreases precipitation at the solution-gel interface or close to the isoelectric point. However, the use of urea also induces denaturation, and thus exposure of the totality of the hydrophobic residues of the proteins to the solvent. This in turn increases the potential for hydrophobic interactions, so that urea alone is often not sufficient to quench completely the hydrophobic interactions. This explains why detergents, which can be viewed as specialized agents for hydrophobic interactions, are almost always included in the urea-based solubilization mixtures for isoelectric focusing. Of course, these detergents should not bear any net electrical charge, and only

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nonionic and zwitterionic detergents can be used. However, ionic detergents such as SDS can be used for the initial solubilization, prior to isoelectric focusing, in order to increase the solubilization and facilitate the removal of interfering compounds. Low amounts of SDS can be tolerated in the subsequent IEF [113], provided that high concentrations of urea [114], and of nonionic [113] or zwitterionic detergents [115], are present to ensure complete removal of the SDS from the proteins during IEF. Higher amounts of SDS must be removed prior to IEF, for example by precipitation [41]. It must therefore be kept in mind that SDS will only be useful for solubilization and for sample entry, but will not cure isoelectric precipitation problems. The use of nonionic or zwitterionic detergents in the presence of urea presents some problems due to the presence of urea itself. In concentrated urea solutions, urea is not freely dispersed in water but can form organized channels (see [116]). These channels can bind linear alkyl chains, but not branched or cyclic molecules, to form complexes of undefined stoichiometry called inclusion compounds. These complexes are much less soluble than the free solute, so that precipitation is often induced upon formation of the inclusion compounds, precipitation being stronger with increasing alkyl chain length and higher urea concentrations. Consequently, many nonionic or zwitterionic detergents with linear hydrophobic tails [117-1191, and some ionic ones [32], cannot be used in the presence of high concentrations of urea. This limits the choice of detergents to those with nonlinear alkyl tails or with short alkyl tails, which are unfortunately less efficient for quenching hydrophobic interactions Three types of detergents are therefore used for denaturing isoelectric focusing in the presence of urea, which mainly covers the field of two-dimensional electrophoresis. The first type contains a phenyl ring in the hydrophobic part, and an oligooxyethylene polar head. The main compounds for this class are Tritons (mainly Triton X-100) and Nonidet P-40 [24]. The presence of these detergents in a gel prevents subsequent blotting of the separated proteins [109]. The second type contains a linear alkyl tail and a very hydrophilic sugar-based polar head. Detergents of this type include octyl glucoside [120] and lauryl maltoside [121], and seem to be more effective solubilizers than the first type. Moreover, at least for monodimensional isoelectric focusing, these detergents give much less interference for blotting the separated proteins than Triton-type detergents [ 1101. The third type is based on sulfobetaines as polar heads. Standard sulfobetaine detergents with linear alkyl tails have received limited applications because they require low concentrations of urea. However, good results have been obtained in certain cases for membrane proteins [122-1241, although this type of protocol seems rather delicate [119]. The compatibility of sulfobetaines with urea can be greatly increased by introducing an amido group in the detergent molecule [119]. This allows the use of much higher urea concentrations (7-8 M), which has led to marked improvement of the patterns in certain applications with membrane proteins [119]. However, the lack of commercial availability of these chemicals has precluded extensive testing and use.

The last, and most used, type of sulfobetaine detergent used for isoelectric focusing is represented by CHAPS, which combines a bulky, polycyclic tail with the sulfobetaine head. This detergent has been shown to be much more efficient than Triton-type detergents [120, 1251, and has gained wide popularity in the field of two-dimensional electrophoresis. However, it seems that the 2% concentrations suggested in the early publications [ 1251 may be insufficient in some cases, and that higher concentrations (3-5%) may be required, as shown by works using two-dimensional electrophoresis [33, 1201, or onedimensional denaturing IEF and multicompartment electrophoresis [126]. Additional advantages of CHAPS include diminution of detergent interference in twodimensional electrophoresis, thereby resulting in a decreased need for equilibration between dimensions, as long as conventional, carrier ampholyte-based IEF is carried out [127]. In one-dimensional IEF, CHAPS also facilitates subsequent protein blotting (C. Cochet, personal communication). Apart from the problem of inclusion compounds, the most important problem linked to the use of urea is carbamylation. Urea in water exists in equilibrium with ammonium cyanate, the level of which increases with increasing temperature and pH [128]. Cyanate can react with amines to yield substituted ureas. In the case of proteins, this reaction takes place with the a-amino group of the N-terminus and the &-amino groups of lysines. This reaction leads to artifactual charge heterogeneity, N-terminus blocking and adduct formation detectable in mas spectrometry. Carbamylation should therefore be completely avoided. This can be easily done with some simple precautions. The use of pure urea (analytical grade) decreases the amount of cyanate present in the starting material. Avoidance of high temperatures (never heat urea-containing solutions above 37C) considerably decreases cyanate formation. In the same trend, urea-containing solutions should be stored frozen (-20C) to limit cyanate accumulation. Last but not least, a cyanate scavenger (primary amine) should be added to urea-containing solutions. In the case of isoelectric focusing, carrier ampholytes are perfectly suited for this task. Although the use of Tris has been advocated for the same purpose [129], I think that its steric hindrance and relatively weak reactivity (e.g. in other addition reactions with double bonds [SO]) make it a poor cyanate scavenger, compared to ampholytes or amino acids. If these precautions are carried out correctly proteins seem to withstand long exposures to urea without carbamylation [ 126, 1301. Because of these drawbacks of urea, substitutes have been actively searched for, with little success up to now. One direction which has been explored is that of substituted ureas [131-1331. These chemicals are much less prone to cyanate formation, and are more powerful denaturants than urea itself [6]. However, little success has been encountered, either because these chemicals interfere with acrylamide polymerization, or because these compounds are not more efficient than urea. Their increased denaturing effect comes from their increased ability to break hydrophobic interactions [6]. However, the solubilizing power of urea also comes from its ability

826

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Electrophoresis 1996, 17, 813-829

to break hydrogen bonds, and subsituted ureas seem to be less efficient than urea in this area. This explains why substituted ureas have not superseded urea as solubilizing agents for IEF. Amides have also been tried as solubilizing agents for IEF, since they are also wellknown protein denaturants [134]. Here again, little success has been encountered. Formamide, a wellknown denaturant of nucleic acids [135], could be a useful substitute, but its susceptibility t? alkaline hydrolysis with the formation of charged compounds prevents its use for IEF [136]. Sustituted amides have also been tried [131], but the general interference of these solvents with acrylamide polymerization has precluded further testing. However, the use of substituted ureas and amides could now be reinvestigated, as the use of a new photopolymerization system [85] allows one to polymerize acrylamide in the presence of high concentrations of solvents [87]. Currently, the lypical solubilization mixture prior to denaturing IEF contains urea (> 7 M), a detergent (in most cases nonionic or zwitterionic, or SDS if an additional initial solubilizing power is needed), and a buffer, usually carrier ampholytes, which avoids any interference with the subsequent IEF and protects from carbamylation. This mixture may, in some cases (e.g. membrane or nuclear proteins), not be powerful enough. This is mainly due to the fact that the pH ranges of most carrier ampholytes used are centered on a neutral or weakly acidic pH. The resulting pH of the solution is therefore only weakly acidic, so that a non-negligible amount of proteins will behave as cations. This increases proteinprotein or protein-nucleic acid complexes by electrostatic interactions, thereby decreasing the solubilization yield. In addition, proteases are still transiently active at these acidic or neutral pH ranges [27]. To alleviate these problems, the best solution is to perform the solubilization at alkaline pH. Proteases are inhibited at high pH [27], and all proteins will behave as anions, thereby increasing the electrostatic repulsion between proteins and nucleic acids or other proteins. This results in dramatically improved solubilizations, with yields very close to those obtained with SDS [137]. The alkaline pH can be obtained either by addition of a few mM of potassium carbonate to the urea-detergent-ampholytes solution [137], or by the use of alkaline ampholytes [127], or by the use of a spermine-DTT buffer, which allows better extraction of nuclear proteins [33]. In the latter buffer, spermine is used as a cyanate scavenger, and carrier ampholytes of the desired range are added after solubilization and prior to loading on the IEF gel. Practical solubilization protocols prior to denaturing I E F may be summerized as follows: (i) If no problems are encountered, use a standard urea-ampholyte-detergent mixture. Changing the detergent or mixing detergents can considerably increase the solubilization efficiency for the protein(s) of interest [121, 1251. (ii) If solubilization is still poorly efficient, use an alkaline solubilization buffer of any of the three types previously described. Do not forget that carbonate will enter into the focusing gel. This will improve the initial penetration of the proteins, but high amounts of carbonate (as in micropreparative runs with large amounts of samples) will distort the pH

gradient [138]. (iii) If problems still remain, use a ureacarrier-ampholyte-SDS buffer. Be aware that this buffer can perform nicely on analytical runs and give very poor results at the micropreparative scale due to SDS overload. Precipitation of proteins to remove the excess SDS [41, 1391 may be necessary, with the risk of losses of proteins at this step.
3.2.2 Native isoelectric focusing

All the solubilization problems described in Section 3.2 apply of course even more severely to native IEF than to denaturing IEF because many general means used in denaturing IEF - such as strong solubilizers (urea), powerful detergents or pH extremes - cannot be used. The solubilization process prior to native IEF is therefore a tedious testing process where combinations of mild detergents, weak cosolvents such as ethylene glycol [140], are tested at the analytical scale for the solubilization of the protein of interest. As a general rule, the detergents used in denaturing I E F are mild enough to be used in native IEF. The only exception are simple alkyl sulfobetaines, which denature some proteins [15] but are efficient solubilizers, so that they deserve a trial. Within this family, I would recommend resting sulfobetaines with C10 to C14 tails, either alone or in combination with nonionic detergents [141]. As nondetergent additives, polyols such as ethylene glycol, glycerol, sorbitol or nonreducing sugars are useful in some cases (e.g. [140]). The use of reducing sugars should be avoided because of the possible Amadori reaction with the amino groups of the proteins, which could induce charge heterogeneity [ 1421. Other nondetergent additives that can be very useful are dipolar, zwitterionic compounds. Amino acids have been suggested for that purpose [143], but molecules zwitterionic over a wider range of pH are required. The simplest one is carboxybetaine, which is zwitterionic, but only above pH 4, and has been used as a protein solubilizer and stabilizer [144]. More promising are the nondetergent sulfobetaines, whose use in biochemistry was recently introduced [145]. These compounds have been shown to improve solubilization in many biochemical processes, including IEF, while exhibiting no gross denaturing properties [ 145, 1461. Note that these compounds interfere weakly with acrylamide polymerization, which can be overcome by a slight increase in the initiators concentrations. The 2-D gel shown in Fig. 5 uses native IEF as the first dimension, with a cocktail of Triton X-100, a detergent sulfobetaine (SB 3-14; tetradecyl dimethylammoniopropanesulfonate) and a nondetergent sulfobetaine (NDSSB201; pyridiniopropanesulfonate) as the solubilization mixture. While the result is clearly inferior to the one obtained with denaturing IEF, correct solubility is achieved for many cellular proteins. In fact, the extraction yield with the native medium described above is 50% of the yield obtained with 9 M urea, 4% CHAPS, 40 mM DTT and 20 mM spermine. This is an important step tpwards high resolution, native 2-D electrophoresis, which may play a role as a separation tool when preservation of the protein activity is necessary for its identification prior to structural analyses.

Elertrophphoresrs 1996, 17, 813-829

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Figure 5. 2-D electrophoresis of whole cell extracts. Erythroleukemia cells are lysed either in lysis buffer N (1 M SB201, 1% SB 3-14, 0.5% Triton X-100, 0.8% Pharmalytes 3-10, 1 mM PMSF, and pepstatin, chymostatin, antipain, aprotinin, leupeptin, 10 pg/mL each) or in lysis buffer D (9 M

urea, 4% CHAPS, 20 mM spermine, 40 mM DTT). The cells are lysed in 10 volumes of lysis buffer per volume of cell pellet, for one hour at 0C (lysis in N) or room temperature (lysis in D). The extract is cleared from insolubles (mainly nucleic acids) by ultracentrifugation (200 000 g, 1 h). 2-D electrophoresis is carried out with immobilized pH gradients (pH 4-8, linear) for isoelectric focusing [33], rehydrated either in 1 M SB201, 1% SB 3-14, 0.5% Triton X 100, 0.8% Pharmalytes 3-10 (extract N) or in 9 M urea, 4% CHAPS, 0.2% Triton X-100, 0.8% Pharmalytes 3-10 and 10 mM DTT (extract D). Second dimension: 10%T SDS-PAGE. Detection by silver staining. (A) Native lysis and isoelectric focusing (buffer N). (B) Denaturing lysis and isoelectric focusing (buffer D). Arrow indicates actin.

4 Concluding remarks
As most primary structural analysis methods do not require the preservation of three-dimensional conformation, it is often advisable to switch to solubilization and separation under denaturing conditions as early as possible in the purification protocol. A few years ago, the requirements of many structural analytic methods was far beyond the capacities of electrophoretic methods. Consequently, chromatographic separations were used. These separations often require native conditions, with all the solubility and proteolysis problems described above. Thanks to the miniaturization of many structural analytic methods, electrophoresis can now be used as a micropreparative tool. This allows the use of new purification schemes, in which denaturing conditions are widely used. This in turn has many advantages in terms of solubilization yield, prevention of proteolysis and removal of interfering compounds, which are among the major pitfalls in a purification scheme. Therefore, it is often advantageous not to try to purify the desired protein to homogeneity by standard, native, chromatographic methods, but to purify it up to the point where it

can be reliably identified on a denaturing electrophoretic separation. The latter will be used, with the desired scaleup if necessary, as the final purification step. The extreme, but highly recommendable, example of this approach lies in the use of high resolution two-dimensional electrophoresis as a preparative tool. Conventional preparations that make it possible to follow biological activities sometimes result in only nanogram amounts of the purified protein, with an extremely dificult scale-up. It is therefore advisable to use these nanogram amounts, via two-dimensional gels with silver staining or radioactive detection, to carry out an identification of the protein of interest in a more crude extract (e.g. organelle preparation), whose large-scale preparation is much easier. Once this identification has been made, two-dimensional electrophoresis is used on the crude extract at the micropreparative scale to provide the microgram amounts used for structural analysis. This process using micropreparative two-dimensional electrophoresis has been greatly facilitated by the use of immobilized pH gradients, which have very high loading capacities [33, 130, 1471. Such a scheme takes full advantage of the fact that separations under denaturing conditions

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proceed with a much higher yield and reproducibility than those under native conditions. Two-dimensional electrophoresis has become the most widely used separation technique to carry out the greatest possible part of the solubilization and separation process under denaturing conditions.

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