Between Amphiphilic and Hydrophilic Proteins in Detergent

Proc. Natl. Acad. Sci. USA Vol. 74, No. 2, pp. 529-532, February 1977 Biochemistry

Charge shift electrophoresis: Simple method for distinguishing between amphiphilic and hydrophilic proteins in detergent solution (membrane proteins/protein analysis/Triton X-100/cytochrome bs/Semliki Forest virus) ARi HELENIUS AND KAI SIMONS European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, Federal Republic of Germany Communicated by John Kendrew, November 18, 1976

ABSTRACT Seventeen hydrophilic proteins and five am- MATERIALS AND METHODS phiphilic membrane proteins were subjected to agarose gel electrophoresis in the presence of a nonionic detergent (Triton The amphiphilic membrane proteins used were microsomal X-100), a mixture of a nonionic and an anionic detergent (Triton cytochrome b5 (a generous gift from Dr. C. Lussan, Talence, X-100 and sodium deoxycholate), and a mixture of a nonionic and a cationic detergent (Triton X-100 and cetyltrimethylam- France), intestinal brush border aminopeptidase (a generous monium bromide). The electrophoretic mobility of the hydro- gift from Dr. S. Maroux, Marseille, France), membrane peni- philic proteins was unaffected in the three detergent mixtures. cillinase from Bacillus licheniformis (8), and the glycoproteins However, the mobility of the hi hilic proteins shifted an- El and E2 from Semliki Forest virus membrane (9). The fol- odally in the Triton X-100-deo a e system and cathodally lowing soluble proteins were used: the trypsin-cleaved form of in the Triton X-100-cetyltrimethylammonium bromide system the brush border aminopeptidase (a gift from Drs. S. Maroux when compared to the mobility in Triton X-100 alone. The detergent-induced shift in mobility provides a simple, rapid, and and D. Louvard, Marseille, France, ref. 10); the polar heme- sensitive method for distinguishing between hydrophilic and containing domain of cytochrome b5 (obtained by trypsin amphiphilic proteins. cleavage of the membrane form, ref. 11); the exopenicillinase (isolated from Bacillus licheniformis, ref. 12); transferrin and In the characterization of membrane proteins it is important gamma globulin (Kabi, Sweden); lysozyme, ferritin, cyto- to be able to decide whether the proteins studied possess hy- chrome c, and myoglobin (Serva, Federal Republic of Ger- drophobic domains that anchor them to the hydrocarbon in- many); chymotrypsinogen and pancreatic ribonuclease terior of the bilayer or whether they are externally bound to the (Worthington, USA); bovine serum albumin (Calbiochem, membrane. The electrophoretic screening method introduced USA); catalase and glutamate dehydrogenase (Miles, Great in this paper is based on the fundamental difference in the in- Britain); and thyroglobulin, insulin, and ovalbumin (Sigma, teraction of hydrophilic and amphiphilic proteins with "mild" USA). Triton X-100 (Rohm & Haas, USA), sodium deoxycholate detergents such as Triton X-100 (p-t-octylphenylpolyoxy- (Schwarz/Mann, USA), and cetyltrimethylammonium bromide ethyleneg-1o). It has been shown in numerous studies that (Serva, Federal Republic of Germany) were used without pu- whereas ordinary soluble proteins and peripheral membrane rification. proteins bind little or no Triton X-100, amphiphilic membrane The agarose electrophoresis experiments were performed proteins bind large amounts (usually 80-100 mol of Triton per at room temperature (250) in 1% agarose (BioRad, USA) on glass mol of protein) when solubilized from membranes (refs. 1-5; plates (11 X 20.5 cm) as described by Weeke (13), using a for reviews see refs. 6 and 7). The bound detergent forms mi- water-cooled chamber (Behringwerke, Federal Republic of celle-like clusters around the hydrophobic domains of these Germany), paper wicks, and 1 X 11 mm sample slits. The gel proteins, and usually the proteins retain their native confor- buffer was 0.05 M glycine-NaOH, 0.1 M NaCl at pH 9.0 con- mation. Several methods have been described to determine taining 5 g of Triton X-100 or 5 g of Triton X-100 and 2.5 g of Triton X-100 binding (1-3, 5). Each of these methods can as sodium deoxycholate or 5 g of Triton X-100 and 0.5 g of such be used to differentiate between amphiphilic and hy- cetyltrimethylammonium bromide per liter. The buffers in drophilic proteins, but contrary to the method described here, the electrode chambers were the same except that in the ex- they require purified protein (usually in milligram amounts) periments with Triton X-100 no detergent was added. The and radioactive detergent. plates were first electrophoresed for 15 min at 4.5 V/cm. Instead of using Triton X-100 alone we have used mixtures Samples (12 Al) were applied and electrophoresed for 2 hr at of Triton X-100 and charged detergents. When solubilized with 4.5 V/cm unless otherwise indicated. After electrophoresis the such detergent mixtures, the amphiphilic proteins form de- gels were immediately dried under an air fan (40450) and then tergent-protein complexes containing both neutral and charged autoradiographed using Kodak Royal X-Omat film, scanned detergent molecules (see ref. 6). The net charges of the com- with a Perkin-Elmer 156 double wavelength spectrophotom- plexes are thus dependent on the charge of the detergents used, eter, or stained for protein (45 min in 0.7 g of Coomassie blue, resulting in a clear-cut difference in electrophoretic mobility 450 ml of methanol, and 90 ml of acetic acid per liter; rinsed of the amphiphilic proteins when electrophoresed in cationic for 15 min in 75 ml of acetic acid and 50 ml of methanol per and anionic detergent mixtures. The electrophoretic mobility liter). Immunoelectrophoresis was performed in agarose (1 of the hydrophilic proteins, which do not interact with the g/100 ml) gels according to Scheidegger (14), except that the detergents, remains unaffected by a change in the charge of the buffer (0.06 M sodium barbital diethylbarbitate-HCI, pH 8.7) detergents used. contained Triton X-100, deoxycholate, and cetyltrimeth- 529 Downloaded by guest on October 2, 2021 530 Biochemistry: Helenius and Simons Proc. Natl. Acad. Sci. USA 74 (1977)

INSULIN BSA OVALBUMIN FERRITIN THYROGLOBULIN

c2cm

ORIGIN - l-l-4l - i I i

CATALASE GDH TRANSFERRIN MYOGLOBIN GAMMAGLOBULIN LL Mr ORIGIN -. I1 +I i- I I I { a

CHYMO- CD NO DETERGENT RIBONUCLEASE TRYPSINOGEN LYSOZYME CYTOCHROME C a TX-DOC ORIGIN -10 I~ I I i I i I I II I a TX cz3 _ TX -CTAB a_ _~ S- CO FIG. 1. Combined results from agarose gel electrophoresis of soluble proteins in the absence of detergent, in Triton X-100 (TX) and sodium deoxycholate (DOC), Triton X-100 alone, and Triton X-100-cetyltrimethylammonium bromide (CTAB). The samples applied contained 35 jig of protein in 12 Ml. Protein stain: Coomassie blue. BSA, bovine serum albumin; GDH, glutamate dehydrogenase. ylammonium bromide in the concentrations given above. The domain of cytochrome b5, the trypsin form of aminopeptidise, protein samples used for agarose electrophoresis and immu- and the bacterial exopenicillinase. Cytochrome b5 (15) and noelectrophoresis usually contained 0.7-1.5 mg of protein per membrane penicillinase (K. Simons and M. Sarvas, unpublished ml dissolved in the electrophoretic buffer containing a 4-fold results) consist of single polypeptide chains when solubilized detergent concentration over that used in the gels. The samples with Triton X-100, whereas the membrane aminopeptidase were allowed to equilibrate overnight at 00. contains three noncovalently bound polypeptide chains (16). Fig. 1 shows the electrophoretic patterns obtained for a RESULTS AND DISCUSSION number of ordinary soluble proteins in the absence of detergent We have tested 17 hydrophilic proteins and 5 amphiphilic and in the three detergent media. Only minor differences in The included acidic the electrophoretic mobility were observed. The same was true membrane proteins. hydrophilic proteins for exopenicillinase (Fig. 2), the trypsin form of aminopeptidase and basic proteins and glycoproteins, and some contained several subunits. Of the amphiphilic membrane proteins tested, (Fig. 2), and the polar fragment of cytochrome b5 (Fig. 3). In contrast, all the amphiphilic membrane proteins tested, three could be obtained in a proteolytically cleaved form that contained only the polar moiety: the polar heme-containing membrane penicillinase (Fig. 2), membrane aminopeptidase

IIANE EXOPENICIWNASE PENKMLWHASE

ORIGIN 1

TX-DOC TX TX-CTAB TX-DOC TX TX-CTAB

~~~~~~d

E TX C ORIGIN 2

I TX-CTAB AMINOPEPTIDASE MEMERANE (TRYPSIN FORM) AMWOPEPTIDASE 1cm FIG. 2. Combined results from agarose gel electrophoresis FIG. 3. Agarose electrophoresis of a mixture of cytochrome b5 (the of exopenicillinase, membrane penicillinase, the trypsin form of d-form) and trypsin-cleaved cytochrome b5 (the t-form) in Triton aminopeptidase, and membrane awminopeptidase in Triton X-100 and X-100 and sodium deoxycholate (TX-DOC), in Triton X-100 alone sodium deoxycholate (TX-DOC), in Triton X-100 alone (TX), and (TX), and in Triton X-100 and cetyltrimethylammonium bromide in Triton X-100 and cetyltrimethylammonium bromide (TX-GTAB). (TX-CTAB). Electrophoresis was performed for 75 min at 4.5 V/cm. Protein stain: Coomassie blue. The plates were dried and scanned at 415 and 450 nm. Downloaded by guest on October 2, 2021 Biochemistry:BProc.Helenius and Simons Natl. Acad. Sci. USA 74 (1977) 531

MEMBRANE PENICILLINASE

X.I 0 EXO- ORIGIN -> I I "!9 PENICILLINASE

MEMBRANE PENICILLINASE TX-DOC TX TX-CTAB EXO- FIG. 4. Combined results from agarose electrophoresis of [35Slmethionine-labeled spike glycoprotein El from Semliki Forest PENICILLINASE _ virus in the presence ofTriton X-100 and deoxycholate (TX-DOG), Triton X-100 alone (TX), and Triton X-100 and cetyltrimethylam- MEMBRANE monium bromide (TX-CTAB). The samples contained 8000 cpm in PENICILLINASE 12 Mtl (specific activity 4500 cpm/jug of protein). Electrophoresis was performed for 4 hr at 4.5 V/cm. After drying, the gels were autoradiographed for 6 days. EXO - PENICILLINASE _ FIG. 5. Immunoelectrophoresis of exopenicillinase and membrane penicillinase in the presence of Triton X-100 and sodium (Fig. 2), cytochrome b5 (Fig. 3), and the spike glycoproteins El deoxycholate (TX-DOC), Triton X-100 alone (TX), and Triton X-100 and cetyltrimethylammonium bromide (TX-CTAB). Electrophoresis (Fig. 4) and E2 (not shown) displayed a more anodal migration was performed at 3 V/cm for 90 min. The rabbit antiserum used had when electrophoresis was performed in the presence of the been raised against exopenicillinase. Triton X-100-deoxycholate mixture than in the presence of Triton X-100 alone. The migration in the Triton X-100-cetyl- is low. It should also be mentioned in trimethylammonium bromide mixture was, on the other hand, strength this context that binds amounts more cathodal than in the presence of Triton X-100 alone. In pancreatic colipase large of deoxycholate and other but it not all cases the shifts in mobility of the amphiphilic proteins were negatively charged detergents (19, 20), does bind Triton X-100 or easily detectable. The hydrophilic as well as the amphiphilic (21) positively charged deoxycholate proteins gave single bands in each electrophoretic system, with derivatives (B. Borgstrom, unpublished observation). The use of three detergent systems and the exception of aminopeptidase which was heterogeneous in (anionic, nonionic, cationic) the Triton bromide elec- helps to detect such charge-specific binding. Integral mem- X-100-cetyltrimethylammonium with trophoresis system (Fig. 2). We have interpreted the emergence brane proteins an extensive hydrophobic domain should display both an anodal shift in Triton and of the additional cathodally moving band in this case as an in- X-100-deoxycholate dication of partial dissociation of the subunit structure. 5 a cathodal shift in Triton X-100-cetyltrimethylammonium Fig. bromide as compared to their mobility in Triton X-100 shows the immunoelectrophoretic patterns obtained using both alone. forms of penicillinase in the three detergent mixtures. The shifts The charge shift electrophoresis be for in the membrane penicillinase mobility could be seen easily, may easily adapted whereas the remained unshifted. Similar results preparative purposes and for other gel media. When combined exopenicillinase with sensitive to it can were obtained with the trypsin and the membrane forms of methods detect the protein bands also aminopeptidase, and with the membrane proteins of Semliki be used in the study of trace amounts of protein (see Figs. 4 and Forest virus using the respective antisera (not shown). 5). Specific detection using antibodies, biological activities, etc. in Rather than using deoxycholate and cetyltrimethylammo- make it feasible to analyze proteins complex mixtures (unpublished results). In such mixtures a clear-cut shift in one of nium bromide alone (which may have given a larger shift in indicates either the mobility for the amphiphilic proteins), we have used these the protein components that protein itself is or is of an in combination with an excess of Triton amphiphilic that the protein part amphiphilic charged detergents are X-100. This was in order to keep the structure of protein-de- protein complex. When solubilized whole membranes an- it is to include an excess of in tergent complexes as constant as possible in all three detergent alyzed important detergent both systems (see ref. 17). Furthermore, cetyltrimethylammonium the sample and the gel to ensure maximal separation of lipid bromide is known to be a denaturant when used alone. How- and protein. If necessary, the lipids and the proteins can be in ever, when mixed with sufficient Triton X-100, its chemical separated beforehand using sucrose gradient centrifugation potential drops below that required for massive binding and the presence of detergent (3). Different dissociation states of denaturation (see ref. 6). The fact that the migration of the oligomeric proteins may sometimes occur in the three different hydrophilic proteins remained unchanged and that the anti- detergent media (see Fig. 2). Our preliminary data indicate that prior crosslinking of the proteins can be used to eliminate this body-antigen reactions were not affected indicated, indeed, that the cetyltrimethylammonium bromide present did not problem. drastically denature the proteins tested. We thank Bodil Holle and Hilkka. Virta for their skillful assis- In preliminary experiments performed at lower ionic tance. M the basic strength (0.025 Tris1HCl, pH 9.0) proteins cyto- 1. Helenius, A. & Simons, K. (1972) J. Biol. Chem. 247, 3656- chrome c, lysozyme, and chymotrypsinogen exhibited clear-cut 3661. anodal shifts in the Triton X-100-deoxycholate system but no 2. Makino, S., Reynolds, J. A. & Tanford, C. (1973) J. Biol. Chem. difference in the Triton X-100-cetyltrimethylammonium 248,4926-4932. bromide system when compared to the mobility in Triton X-100 3. Simons, K., Helenius, A. & Garoff, H. (1973) J. Mol. Bwol. 80, alone. This observation suggests that basic proteins can bind 119-133. deoxycholate electrostatically (see ref. 18), especially if the ionic 4. Clarke, S. (1975) J. Biol. Chem. 250,5459-5469. Downloaded by guest on October 2, 2021 532 Biochemistry: Helenius and Simons Proc. Nati. Acad. Sci. USA 74 (1977)

5. Fries, E. (1976) Blochim. Blophys. Acta, 455,928-936. 103-110. 6. Helenius, A. & Simons, K. (1975) Biochim. Biophys. Acta 415, 15. Visser, L., Robinson, N. C. & Tanford, C. (1975) Biochemistry 29-79. 14, 1194-1199. 7. Tanford, C. & Reynolds, J. A. (1976) Biochlm. Blophys. Acta, 457, 16. Maroux, S. & Louvard, D. (1976) Biochim. Biophys. Acta 419, 133-170. 189-195. 8. Sawai, T. & Lampen, J. 0. (1974) J. Biol. Chem. 249, 6288- 17. Tanford, C. (1973) The Hydrophobic Effect (John Wiley and 6294. Sons, New York), pp. 814-5. 9. Helenius, A., Fries, E., Garoff, H. & Simons, K. (1976) Biochim. 18. Rowley, R. R. & Wainio, W. W. (1958) J. Am. Chem. Soc. 80, Biophys. Acta, 436,319-334. 4384-4386. 10. Maroux, S., Louvard, D. & Baratti, J. (1973) Biochim. Biophys. 19. Borgstr6m, B. & Donner, J. (1975) J. Lipid Res. 16,287-292. Acta, 321,282-295. 20. Charles, M., Sari, H., Entressangles, B. & Desnuelle, P. (1975) 11. Ito, A. & Sato, R. (1968) J. Biol. Chem. 243,4922-4930. Biochem. Biophys. Res. Commun. 65,740-745. 12. Pollock, M. R. (1965) Blochem. J. 94,666-675. 21. Borgstr6m, B., Donner, J. & Erlanson, C. 01974) in Advances in 13. Weeke, B. (1973) "A manual of quantitative immunoelectro- Bile Acid Research, eds. Matern, S., Hackensmith, Back, P. J. & phoresis," Scand. J Immunol. 2, Suppl. no. 1, 25-3. Gerok, W. (F. K. Schattauer Verlag, Stuttgart-Newr York), pp. 14. Scheidegger, J. J. (1955) Int. Arch. Allergy Appl. Immunol. 7, 213-217. Downloaded by guest on October 2, 2021