BioSystems 6 (1975) 1 7 6 - 1 8 7 © N O R T H - H O L L A N D PUBLISHING COMPANY

SODIUM DODECYL SULFATE IN PROTEIN CHEMISTRY* T.V ~VAEHNELDT Max-Planck-Institut fiir experimentelle Medizin, Forschungsstelle Neurochemie, 3400 G6ttingen, West Germany

This review summarizes in a brief manner the main aspects of the application of sodium dodecyl sulfate (SDS) to protein chemistry. The principal problems of SDS-polyacrylamide gel electrophoresis are described, as well as the anomalous behavior o f protein-SDS complexes and the inactivation of e n z y m e s due to variable binding of SDS to the polypeptides studied. The particular value of SDS in elucidating the protein composition of biological m e m branes and in membrane-reconstitution experiments is discussed.

1. Introduction

on, in a study of the interaction between horse serum albumin and SDS, Putnam and Neurath (1945) found that two stoichiometrically mad electrophoretically discrete complexes were formed between those two components at low temperatures which they ascribed mainly to ionic forces; however, at higher temperatures additional SDS appeared to be more loosely bound. In retrospect, and quite amazingly, these two papers and related ones have largely covered the principles of today's use of SDS in protein chemistry, i.e., disaggregation of superstructures into covalently delineated constituents on one hand and stoichiometric loading of these compounds with SDS on the other. It was the advent of carrier-supported electrophoresis on polyacrylamide gels ( R a y m o n d and Weintraub, 1959; Davis, 1964), combined with the addition of SDS (Summers et al., 1965), radioactive labelling techniques (Maizel, 1966) and the empirical finding of Shapiro et al. (1967) of an inverse relation between protein molecular weight and electrophoretic migration rate which set the stage for the rapid increase of our knowledge in protein chemistry. The scope of application ranges through all biological fields, from viruses (Maizel,

In recent years we have witnessed an explosive appearance of communications dealing with the use of sodium dodecyl sulfate (SDS) in the field of protein chemistry, especially with respect to particle- and membrane-bound proteins. The SDS molecule combines a nonpolar hydrophobic region with a strongly polar anionic endgroup, thus mimicking, and competing with, certain membrane lipids. The peculiar structure of SDS renders this small amphipathic molecule (MW 288.4) highly suitable for complex formation both with nonpolar side chains and charged groups of amino acid residues in polypeptides of all possible sizes and shapes, without rupturing covalent bonds. The principal applications of SDS to modern protein chemistry were announced some 30 years ago. Sreenivasaya and Pirie (1938) noted that even at low SDS concentrations TMV (tobacco mosaic virus) could be separated into protein and nucleic acid fractions. This effect was paralleled by the disappearance of sedimentable virus particles. Further* Invited review.

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1971), bacteria (Holland and Tuckett, 1972), chloroplasts (Hoober, 1970), amoeba (Korn and Wright, 1973), molluscs (Gainer, 1971) to mammalian species (Kiehn and Holland, 1970; Neville and Glossman, 1971 ; Waehneldt and Neuhoff, 1972), to mention only a few. This paper aims at briefly reviewing the main aspects of SDS application to protein chemistry for the reader who is little or not at all familiar with the field. By necessity, the number of communications listed will be rather limited; therefore, many points can be treated only superficially. However, for further reading references will be made to more extensive and specialized reviews and articles whenever possible.

2. Separation of protein: determination of molecular weights

2.1. Electrophoretic systems Applying a system of 5% acrylamide, crosslinked with bisacrylamide (38: 1), Shapiro et al. (1967) were able to show that in the presence of 0.1% SDS and a neutral 0.1 M phosphate buffer 2-mercaptoethanol-reduced proteins move anodically with the logarithm of their molecular weights inversely related to the distance of migration. Although the soluble proteins used in this experiment showed widely varying intrinsic charges with isoelectric points ranging from 4 to 11 all proteins fitted the curve reasonably well. The authors suggested that the proteins are complexed with anionic SDS molecules which abolishes intrinsic charges of the native proteins, thereby forcing the pr,atein-SDS complexes to migrate towards the anode. Other workers (Weber and Osborn, 1969; Dunker and Rueckert, 1969) came to the same conclusion, using a large number of proteins of welldefined molecular v/eight in the same continu-

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ous neutral phosphate system - continuous because of identical ionic concentration and pH in gels and electrode buffers. They found, however, that the concentrations of acrylamide and bisacrylamide had to be closely observed and that proteins with molecular weights below 10-15,000 tended to show anomalous behavior in that they moved slower than expected. Swank and Munkres (1971) were able to extend the range of molecular weights down to 1,200 daltons by increasing the amount of acrylamide, crosslinker and gel length, and particularly by inclusion of 8 M urea to reduce the gel porosity. Their electrophoretic system was also a continuous one similar to that of Shapiro et al. (1967). So was that of Schnaitman (1969) who showed the existence of a large number of different protein species in microsomal and mitochondrial membranes of rat liver, for some of which he determined the approximate molecular weights by applying soluble proteins of defined molecular weights as migration markers. Laemmli (1970), in a study of SDS-solubilized structural proteins of bacteriophage T 4, used a discontinuous system based on the original procedure for soluble proteins (Davis, 1964). Laemmli's system displays a discontinuity between the ionic concentrations of electrode buffer (pH 8.3), sample and stacking gel (pH 6.8), and separation gel (pH 8.8). The electrode buffer contained glycine, thereby sandwiching the SDS-protein complexes at neutral pH as a very thin disc between the trailing glycine ion and the chloride ion moving just ahead of the protein sample. Unstacking occurs in the separation gel upon pH change, and the protein-SDS complexes are sieved in the pores of the gel matrix according to their size. An almost identical discontinuous system had been developed by Grossfeld (1968) which was used for the separation of SDS-extracted rat brain myelin proteins

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(Waehneldt and Mandel, 1970) and for membrane proteins of total mouse brain during ontogeny (Grossfeld and Shooter, 1971). Neville (1971) used a s u l f a t e - b o r a t e discontinuity for molecular weight determination of SDS-complexed soluble proteins. His system is one of several thousand multiphasic buffer systems as computed by Jovin et al. (1971). The fact that discontinuous systems have proved superior in resolving power has also been demonstrated by Glossmann and Lutz (1970) by comparing the resolution of SDS-solubilized erythrocyte membrane proteins in the continuous system of Shapiro et al. (1967) with that of the discontinuous system of Laemmli (1970).

2.2. Separation o f proteins by SDS PAGE on micro scale and macro scale S D S - P A G E is usually performed in glass tubes of 4 - 6 mm inner diameter requiring about 1 0 - 6 g of an individual protein as the lowest amount detectable with conventional staining procedures. Wilson (1971) and Gainer (1971) have developed micro electrophoretic systems in capillaries in order to study protein synthesis in single neurons of molluscs. Wilson used a continuous system, whereas Gainer applied the superior resolution of a s u l f a t e borate discontinuity, similar to that of Neville (1971). Protein quantities as low as 5 X 10 10 g could be detected. Very recently, Rtichel et al. (1974) have developed a micro S D S - P A G E with a continuous concentration gradient in the gel, ranging from lower than 1.5% to maximally 50% acrylamide. These gradient micro gels simultaneously permit the entrance of very large particles and are suitable for separating molecules in the range of several hundred daltons. Caution has to be exercised inasmuch as p r o t e i n - S D S complexes can reach a pore size under prolonged electrophoresis in which they come to a quasi

halt ("dead run") and may be partially or totally stripped of their complexing SDS molecules. This is particularly true when the upper buffer reservoir is depleted of SDS. Also, continuous buffer systems are preferable. With discontinuous buffer systems partial deloading of SDS protein complexes has been observed, leading to a confusing multitude of bands. Going to the other extreme, SDS PAGE can be scaled up to preparative dimensions. Such preparations have been performed for rat brain myelin proteins (Waehneldt, 1971), for proteins of sarcoplasmic reticulum membranes (Martonosi and Halpin, 1971), for pigmented protein from Rhodopseudomonas (Fraker and Kaplan, 1972), and for the major proteins of the human erythrocyte membrane (Tanner and Boxer, 1972).

2. 3. Reduction o f disulfide bridges For reliable determination of molecular weights of polypeptide subunits in SDS polyacrylamide gel electrophoresis ( S D S - P A G E ) it is essential that the proteins contain no disulfide bonds. Reduction to free SH-groups is performed either with 2-mercaptoethanol or with dithiothreitol. After initial reduction the reducing environment is best maintained during the total length of electrophoresis. Otherwise free SH-groups can reoxidize to give rise to dimers (Shapiro et al., 1967) and oligomers (Rustum et al., 1971; Swislocki and Tierny, 1973), as suggested by Matsuda et al. (1973), or possibly to numerous other and previously non-existing combinations of disulfides by intermolecular reshuffling of SH-groups. For further protection, free SH-groups can be blocked with an excess of iodoacetic acid or iodoacetamide, which has been performed in many instances (Shapiro et al., 1967; Pitt-Rivers and Impiombato, 1968; Arndt and Berg, 1970; Williams and Gratzer,

T. V. Waehneldt, Sodium dodeeyl sulfate in protein chemistry

1971), to mention a few. Relative fluorescence intensities of several proteins were directly proportional to the tryptophan content of the reduced SDS-protein complexes, while the same unreduced proteins showed different fluorescence intensities (Shelton and Rogers, 1971). This effect is probably due to SDS-induced changes in the tryptophan microenvironment. The advantages of reduced disulfide bridges are obvious; however, it might be desirable to electrophorese unreduced SDS-extracted membrane proteins, such as in ontogenetic studies (Grossfeld and Shooter, 1971; Waehneldt and Neuhoff, 1974), thereby revealing naturally occurring complex proteins which possibly consist of different disulfide-bonded subunits. These proteins, in turn, could be isolated and thereafter reduced to show the subunit pattern intrinsic of the original oligomeric membrane protein.

2.4. Heating and delipidation Proteolytic enzymes are quite stable in the presence of the generally denaturing SDS. Degradation to smaller polypeptides of SDS-complexed and mercaptoethanol-reduced proteins by these enzymes can be avoided by shortly heating the sample at 100°C (Pringle, 1970; Rustum et al., 1971) or by the addition of an appropriate proteolytic inhibitor. Moreover, heating of any particulate sample in 1 2% SDS (or with an excess of SDS 5-fold over the protein) has proven very effective in dissociating virus particles (Maizel, 1969) or bacterial outer membranes (Bragg and Hou, 1971). Delipidation is often applied to facilitate SDS-solubilization of membranes, using either chloroform--methanol mixtures in the case of platelet membranes (Nachman and Ferris, 1970), amoeba plasma membranes (Korn and Wright, 1973), or partial delipidation of myelin either with acetone (Agrawal

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et al., 1972) or e t h e r - e t h a n o l (Greenfield et al., 1971). It was shown, however, that delipidation of membranes prior to exposure to SDS is no absolute requirement for separation of proteins in molecular weight determinations since lipids move ahead of any protein in S D S - P A G E (Gahmberg, 1971; Waehneldt et al., 1971) (see, however, Rottem et al., 1968, using different conditions). Nevertheless, band resolution of proteins derived from delipidated microsomes was shown to be slightly better than those of undelipidated microsomes (Y Lopez and Siekevitz, 1973).

2.5. Visualization of proteins The task of visualizing protein bands in S D S - P A G E can be accomplished in several ways. A prerequisite is that proteins be immobilized in the gel matrix. Usually acidic conditions are employed, e.g., acetic acid in combination with amido black (Davis, 1964) or fast green (Greenfield et al., 1973). Both these dyes yield reproducible results up to high protein concentrations. However, the bands obtained are rarely as sharp and discrete as those found with coomassie brilliant blue (Fazekas de St. Groth et al., 1963) in mixtures of acetic a c i d - m e t h a n o l - w a t e r (Weber and Osborn, 1969) or acetic a c i d - i s o p r o p a n o l - w a t e r (Fairbanks et al., 1971). The latter methods allow the detection of 1 0 - 6 g or even less of a single protein, particularly in conjunction with discontinuous gel systems. The original staining procedure for glycoproteins with PAS (Zacharius et al., 1969) occasionally stains SDS-protein bands containing no carbohydrate. Therefore, it is advisable to eliminate SDS with acid isopropanol or acid methanol from the gels and from the protein bands prior to reaction with PAS (Fairbanks et al., 1971; Glossmann and Neville, 1971). Differential staining of phosphoproteins such as bovine caseins was effected with the cationic

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carbocyanine dye "stains all" (Green et al., 1973) which produces a blue color in contrast to no reaction or red coloration of non-phosphorylated proteins. Immediate visualization of protein has the advantage of monitoring the progress of electrophoresis in SDS PAGE. Griffith (1972) describes the covalent linkage of remazol brilliant blue R with biological material which is consequently separated by SDS PAGE. The stain is however much less sensitive than coomassie blue which can be used additionally after termination of electrophoresis. Dansylation of soluble proteins for fluorescent monitoring during S D S - P A G E was achieved by Talbot and Yphantis (1971) and by Inouye (1971), permitting the detection of 1 0 - S g and 2 X 10 7 g protein, respectively. It was only recently, however, that membrane-bound proteins could be dansylated by a gentle method, involving the ultrasonic dispersion of Dans C1 into lecithin-cholesterol micelles which were then suspended in an aqueous medium of the membranes to be dansylated (Schmidt-Ullrich et al., 1973). The gradual release of Dans CI from the lipid micelles and the direct contact with the membranes prevented the formation of inactive Dans OH with an excess of water. After dansylation S D S - P A G E proceeds as usual. This elegant method promises to be of great value for the detection of very small quantities of membrane-bound proteins and will also yield information as to the accessibility of reacting protein amino groups. 2. 6. A n o m a l i e s Besides the anomalous behavior of proteins below 1 0 - 1 5 , 0 0 0 daltons upon SDS-PAGE there are many instances in which erroneous results occur in molecular weight determinations. A substantial increase in molecular weight has been observed for maleylated isoleucyl t-RNA synthetase by Arndt and Berg

(1970) and for a series of maleylated soluble and viral proteins (Tung and Knight, 1971: 1972), with most of the increases ranging between 10 and 20% over the original protein plus attached maleic acid residues. Lysozyme, however, showed a decrease in apparent molecular weight upon maleylation ( - 7 % ) which is consistent with observations of Swank and Munkres (1971) that maleylated cytochrome c cyanogen bromide (CB) peptides increase in anionic mobility up to 62%, irrespective of a 15 22% increase in molecular weight due to reaction with maleic anhydride. Generally, the apparent molecular weights of reduced basic proteins are too high, as demonstrated for ribonuclease and lysozyme (Shapiro et ai., 1967; Dunker and Rueckert, 1969) and for histones and cytochrome c (Panyim and Chalktey, 1971). Molecular weight deviations of ribonuclease are more pronounced on gels of low concentration of acrylamide which is in agreement with observations of Ghabrial and Lister (1973) for TRV proteins. Another exception to the molecular weight-migration relationship are the two collagen ~ chains and their CB peptides. All of them exhibit linear relationships, however of consistent lower electrophoretic mobilities than standard marker proteins (Furthmayr and Timpl, 1971), which is understandable in view of the rigidity displayed by these proteins of extremely high imino acid content. Of particular interest are increases in apparent molecular weight of glycoproteins upon SDS PAGE (Bretscher, 1971; Segrest et al., 1971; Glossmann and Neville, 1971; Kobylka et al., 1972; Evans and Gurd, 1973). Here, too, an increase in acrylamide concentration helps reduce the deviation from correct molecular weight. The analogy to the behavior of maleylated "normal" proteins is obvious. In this context mention must be made of the selective reduction in apparent molecular weight of the predominant human erythrocyte membrane sialoglyco-

T. V. Waehneldt, Sodium dodeeyl sulfate in protein chemistry

protein, which upon SDS-PAGE migrates with a molecular weight of 87,000 at pH 8.3, and of 53,000 at pH 2.4 (Fairbanks and Avruch, 1972). This observation, together with the measurement of free electrophoretic mobilities and retardation coefficients (Neville, 1971; Banker and Cotman, 1972) will help establish whether molecular weight estimations of an unknown protein by SDS-PAGE are reliable or not. For comprehensive treatment of SDS PAGE and closely related topics the reader is refe,rred to excellent review papers of Maizel (1969, 1971), Guidotti (1972), Weber et al. (1972), and the exhaustive monograph on disc electrophoresis by Maurer (1971). 2. 7. Other carriers

Separation and :gross molecular weight determination of SDS-extracted membrane proteins have been performed by gel filtration through Sephadex and other carriers in the presence of SDS (Rosenberg and Guidotti, 1969; Fish et al., 1970; Lenard, 1970; H6rtnagl et al., 1971 ; Triplett et al., 1972;Capaldi, 1973), to mention only a few. These filtrations work reasonably well when simple protein mixtures have to be separated, containing one, two, or a few components with widely differing molecular weights; however, with complex mixtures all the filtration fractions show substantial overlapping of proteins of adjacent molecular size upon SDS-PAGE. This is particularly true for protein material treated with organic solvents prior to drying, which can lead to unspecific resolution by gel filtration and consequently to identical profiles on SDS-PAGE (Katzman, 1972; Dreyer et al., 1972). Neve::theless, under appropriate conditions these filtration methods are recommended for initial enrichment of a particular component to be further purified by preparative SDS-PAGE, SDS chromatography on

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hydroxylapatite (Moss and Rosenblum, 1972), or SDS controlled pore glass chromatography, as tested very promisingly for soluble molecular weight marker proteins (Collins and Hailer, 1973). It is possible to completely separate ~ and 3 chains of hemoglobin on hydroxylapatite, which cannot be resolved by SDS-PAGE. In general, elution of proteinSDS complexes from hydroxylapatite does not necessarily follow the order of molecular weight. Therefore, any combination of SDS-PAGE, SDS chromatography by exclusion filtration, hydroxylapatite, or controlled pore glass could be the choice of the future for separating complex mixtures of proteins. It would be of interest to see if membranebound proteins behave like soluble proteins on SDS controlled pore glass chromatography.

3. Binding of SDS to protein Soon after the empirical finding of Shapiro et al. (1967) that molecular weights of a sizable number of proteins could be estimated by SDS-PAGE simply by conveying strong anionic character to the complexes, interest arose as to how much SDS was bound to individual proteins. Pitt-Rivers and Impiombato (1968), using equilibrium dialysis, found that most native proteins bound 0.9-1.0 g SDS/g protein, the protein moieties of some glycoproteins included. Proteins without disulfide bonds and those with reduced disulfide bridges, however, bound around 1.4 g SDS/g protein in a rather unspecific way, solely due to the ability of SDS to unfold the proteins which is restricted by the presence of disulfide bridges. These results were confirmed by Reynolds and Tanford (1970a) who extended these observations to the finding that a monomeric concentration of SDS above 0.5 mM is sufficient for binding 1.4 g SDS/g protein in a primarily hydrophobic way. Binding of SDS

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in a micellar form was excluded. At SDS concentrations of 0.5 mM or less only 0.4 g SDS/g protein were bound. The same authors (Reynolds and Tanford, 1970b) came to the conclusion that proteins assumed the shape of a rodlike particle upon binding of SDS, the length of which varies uniquely with the molecular weight of the protein moiety, occupying 0.61 3, ( 0 . 4 g SDS bound) and 0.74A (1.4 g SDS bound) per amino acid residue. These values are about half that of an extended s-helix (1.5 A). The minor axis of their rodlike model was calculated to be 14 A (0.4 g) and 18 A (1.4 g), respectively. Although the model of a rodlike particle was also found to be valid for gel chromatography (Fish et al., 1970) it was modified to an ellipsoidal model (American football) by observations on controlled pore glass chromatography (Collins and Haller, 1973). In the ellipsoidat model increases in molecular weight would show up only by increases in length o f 1/4 that of the rigid rod, the extra length presumably being added to the diameter. Binding of more than 1.4 g SDS/g protein has been observed by Tung and Knight (1972) and by Nelson (1971) on the basis of either solubility of SDS or ultracentrifugation of SDS complexes formed. These techniques were chosen rather than equilibrium dialysis since the latter was shown to be less effective due to the extremely slow breakage of SDS micelles (see also Cassel et al., 1969). Binding of less than 1.4 g SDS/g protein has been stated, such as 0.4 g SDS for polylysylglutamic acid (Pitt-Rivers and Impiombato, 1968). Moreover, papain, pepsin and glucose oxidase bind less than 0.2 g SDS (Nelson, 1971). Nevertheless, these examples are not sufficient to cast severe doubt on the validity of molecular weight estimation by S D S PAGE. They should be regarded as a sign of caution. Moreover, there exists a delicate balance between the saturating binding o f SDS,

resulting in high negative charge, elevated molecular weight, long rigid rods (or ellipsoids) and thus increased frictional resistance on one hand, and low amounts of SDS bound, low negative charge, lower molecular weight, and increased coiling of the protein backbone, on the other. Fortuitously, by cancelling themselves out, these competing influences can lead to correct molecular weight estimation in electrophoresis (Swank and Munkres, 1971; Nelson, 1971) which has also been found for proteins in the reduced and nonreduced state (Dunker and Rueckert, 1969). Even after chemical modification of, e.g., lysozyme, producing substantial changes in charge, minute differences in mobility on SDS-PAGE were distinguishable only with the split gel technique (Dunker and Rueckert, 1969). This result is consistent with SDS swamping out relatively large charge differences. However, if SDS is bound anomalously, severe deviations will be found in the case of glycoproteins (Segrest et al., 1971). Moreover, despite "normal" binding of SDS, very large intrinsic positive charges in histones will tend to play an important role in altering the apparent molecular weight (Panyim and Chalkley, 1971). Also unusual conformations of proteins, if maintained in SDS, will lead to decreased binding of detergent and consequently to reduced migration rates on S D S - P A G E (Tung and Knight, 1972; Banker and Cotman, 1972). Therefore, proteins will only co-migrate when combining identical hydrodynamic properties with identical charge-to-mass ratios. For further exhaustive information, particularly with respect to initial binding at low SDS concentrations, the reader is referred to the monograph of Steinhardt and Reynolds (1969) and to recent papers by Katz et al. (1972), Makino et al. (1973), and Hunt and Jirgensons (1973).

T.V. Waehneldt, Sodium dodecyl sulfate in protein chemistry

4. Membrane disintegration and reaggregation When membrane preparations are sequentially treated with increasing concentrations of SDS combined with intermittent high speed centrifugation steps, selective removal of proteins, although overlapping, was observed on SDS-PAGE, such as with mycoplasma membranes (Morowitz and Terry, 1969), synaptosomal plasma membranes (Waehneldt et al., 1971), retinal rod outer segments (Virmaux et al., 1971; 1972), and rat brain myelin (WaeEneldt and Mandel, 1972). The enriched extraction extends even to different classes of protein in myelin, to lightexposed and dark-adapted outer segments and to a distinction between glycoprotein and protein in synaptosomal plasma membrane. Lipids and glycoLipids are commonly extracted with higher SDS concentrations (Mcllwain et al., 1971; Ne'eman et al., 1971; Waehneldt et al., 1971). Complete solubilization in the sense of breakage of all bonding forces intrinsic of the membrane superstructure except covalent ones is very difficult in the case of myelin (Waehneldt and Mandel, 1972) and of brain membranes in general (Waehneldt and Neuhoff, 1974). Even with SDS concentrations of 1% very small amounts of undissolved material are found in the pellets of high speed centrifugations, containing traces of protein. Possibly protein-protein bonds of aromatic nature are involved which are highly resistant to the otherwise strong hydrophobic bond-breaking capability of aliphatic SDS (Choules et al., 1973). Moreover, removal of clear supernatants closely above the pellet can lead to heavy and indiscriminate staining of the cathodal end of gels in SDS-PAGE. Therefore, several parameters have been postulated for true solubilization of membrane constituents, requiring that only covalently defined molecular entities are individually complexed with monomeric SDS

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(Kahane and Razin, t971; Grossfeld and Shooter, 1971). Although initial experiments seemed to support the idea of pre-existing lipoprotein subunits in SDS-solubilized mycoplasma membranes (Razin et al., 1965) it was later suggested that these small lipoprotein subunits were formed upon removal of 10 mM SDS by dialysis in the absence of divalent cations (Terry et al., 1967; Engelman and Morowitz, 1968a, 1968b). These lipoprotein subunits sedimented as single peaks in analytical ultracentrifugation and showed cobanding of lipid and protein in sucrose density gradient centrifugation. Addition of divalent cations to the dialysis fluid led to further aggregation of membrane-like structures. However, Kahane and Razin (1971) could demonstrate that mycoplasma membranes solubilized in 10 mM SDS displayed electrophoretic patterns in the reaggregated membranes different from those reaggregated membranes obtained by solubilization of the original membranes in 20 mM SDS. The latter were identical with the protein profiles of the original membranes. These authors state that 10 mM SDS is insufficient for complete solubilization of the membranes, thus leaving minute amounts of membrane fragments with altered protein composition available to serve as nuclei for reaggregation upon removal of SDS and addition of divalent cations. These examples may suffice to demonstratethe difficulties encountered while trying to interpret the results obtained from analysis of highly complex structures such as membranes with their interplay of bonding forces, phases, and asymmetry of numerous constituents. A superb and exhaustive review on reconstitution of biological membranes has been written by Razin (1972). A comprehensive review on the proteins of the erythrocyte membrane has appeared recently (Juliano, 1973) with chapters dealing with the reconstitution of the erythrocyte membrane and the application of

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physicochemical methods to membrane structure. The communication of Bont et al. (1969) should also be consulted for the effects of SDS on more complex rat liver plasma membrane.

5. Effect of SDS on enzymes There has been general agreement that most, if not all, enzymes are inactivated and irreversibly denatured by SDS, particularly if membrane-bound. Despite this drawback, SDS has been widely and very successfully used for dissociating oligomeric enzymes to their constituent subunits, which may consist of two (Rustum et al., 1971; Berman, 1973; Evans and Gurd, 1973), four (Deme et al., 1972; Huston et al., 1972; Berman, 1973), six (Strausbauch and Fischer, 1970; Matsuda et al., 1973), possibly 8 (Swislocki and Tierny, 1973), and of 12 polypeptides (Rosenbusch and Weber, 1971), of identical or different nature. Commonly membrane-bound enzymes are gradually inactivated by increasing SDS concentrations, as in the case of mycoplasma membranes (Ne'eman et al., 1971), where any p-nitrophenylphosphatase activity is almost immediately lost upon solubilization, in contrast to NADH oxidase which is still active at 0.4% SDS, though reduced by 75% (see also Bishop et al., 1967). Membrane-bound ATPases in erythrocyte ghosts and in Ehrlich ascites carcinoma plasma membranes are activated at low SDS concentrations to become inactive with a 4-fold increase in SDS content (Marchesi and Palade, t967; Wallach, 1969). The results so far obtained are in favor of extended studies of the activities of membrane-bound proteins at SDS concentrations substantially below those commonly employed (Makino et al., 1973). Other enzymes, such as glucose oxidase, papain, and pepsin are very resistent to SDS and retain most o f

their activity for a prolonged period of time (Nelson, 1971). In this context, the activation of proteolytic enzymes in high SDS concentrations must be mentioned again (Pringle, 1970) which are destroyed only by short heating at 100°C. Moreover, enzymes have been demonstrated to survive electrophoresis in SDS gels (Dulaney and Touster, 1970). Restoration of enzymic activity has been observed while reconstituting membranes from SDS-solubilized components, with almost complete recovery of NADH oxidase (Razin et al., 1965) and 50% recovery of light-dependent phosphorylation of ADP to ATP in chromatophore membranes (Takacs and Holt, 1971). Although elastase is inactivated by low SDS concentrations previous binding of SDS to its substrate elastin renders the complex substantially more susceptible to elastolysis by increasing its anionic charge, thus enhancing the binding of elastase to the altered complex (Kagan et al., 1972). Most promising are the experiments for complete removal of SDS which consist of passing SDS-complexed reduced enzymes through anion exchangers either in the presence (Weber and Kuter, 1971) or the absence of buffered urea (ribonuclease) (Lenard, 1971). Ribonuclease regained 100% activity after air oxidation, whereas the activity recoveries of the other, oligomeric enzymes ranged between 10 and 92%, with most of them over 50%, dependent on enzyme and conditions applied. Weber and Kuter (1971) have also reactivated these enzymes after S D S - P A G E , which is of course very encouraging with respect to restoration of SDS-solubilized membrane-bound enzymes. Here, however, cooperative effects of lipids will have to be taken into consideration (Coleman, 1973; Cordes and Gitler, 1973). Weak enzymatic activity of /3-galactosidase has been shown to be restored on gels after removal of SDS from gradient gels by prolonged electrophoresis (Ruchel et al., 1974).

T. K Waehneldt, Sodium dodecyl sulfate in protein chemistry

6. Concluding remarks Despite certain aberrations SDS has proved to be a unique tool for convenient determination of molecular weights of proteins at amounts substantially below those usually employed in ultracentrifugal analysis. Although the latter technique has recently been scaled down to at least t 0 - 5 g protein, using capillaries (Neuhoff, 1973; Neuhoff and R/Sdel, 1973), a combination of the micro disc technique in SDS gradient gels (Rtichel et al., 1974) with that of the sensitivity of the dansyl method (Schmidt-Ullrich et al., 1973) may allow detection and molecular weight determination of single proteins in amounts lower than 10 10 g, even in complex mixtures such as in membranes. Neurobiology would be invaluably promoted by such a development. Due to its lipid--like amphipathic character SDS has substantially increased out knowledge of membrane proteins and thus indirectly of membrane structure. Further progress can be expected applying SDS for disintegration and reconstitution of membranes, possibly with a structurally altered SDS molecule, bearing an aromatic ring in the w-position of the otherwise sli@tly shortened aliphatic chain. Such a compound, though certainly less soluble in aqueous media unless polar groups are attached to the aromatic function, could penetrate deeply into central regions of membrane protein,; with their high concentration of hydrophobic aromatic side chains (Capaldi and Vanderkooi, 1972; Choules et al., 1973). Finally, detailed analysis of the interaction between SDS and homopolypeptides (and limited heteropolypeptides) should yield fruitful insight into the nature and extent of the bonding forces involved. Moreover, defined homopolypeptide-lipid complexes could be formed, possibly of a membraneous struc-

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ture which in turn would serve as models for the attack of SDS on organismically synthesized membranes. This area is wide open for the researcher interested in physical chemistry.

Acknowledgement It is with pleasure that I thank my colleagues Drs. V. Neuhoff, N.N. Osborne and R. Ruchel for helpful suggestions and critical comments.

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