ANALYTICAL

BIOCHEMISTRY

95, 201-208 (1979)

Electrofocusing of Integral Membrane Zwitterionic and Nonionic LEONARD

M. HJELMELAND,

DANIEL

W. NEBERT,

Developmental Pharmacology Branch, and *Endocrinology Institute of Child He&h and Human Development, National

Proteins in Mixtures Detergent9 AND ANDREAS

of

CHRAMBACH*

and Reproduction Research Brunch, National Institutes of Health, Bethesda. Maryland 20014

Received November 1, 1978 In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubiiized proteins was preserved. A mixture of the zwitterionic detergent, SB,,, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing integral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (PI range 7- 11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of pattern constancy, these conditions were achieved within 15 h, 0-4”C, at 200 V in 6-cm gels of 5% T/15% CBiS with 0.1 N H&SO, and 0.1 N KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.

The fractionation of multiple forms of cytochrome P-450 from liver endoplasmic reticulum is fraught with several problems. Most of these problems are common to the purification of all membrane proteins, and are related generally to achieving soluble species which retain biological activity or native structure in protein subunits (l-3). In addition, there appear to be multiple forms (at least eight) of cytochrome P-450 in this tissue, and this multiplicity of species creates specific problems in the purification of these proteins (4). The need to separate proteins differing mainly in net charge would suggest some form of charge fractionation as the most suitable separation method. To date, although ion-exchange chromatography has been used for preparative purposes (5), electrofocusing has received little attention (6, 7). The several advantage of electrofocusing ’ This paper is dedicated to the memory of Dr. Alvin Nason.

compared with ion-exchange chromatography have been discussed (8), and yet the problems encountered in dealing with membrane proteins have obviated the use of this method. Specifically, only the anionic detergents SDS and sodium cholate have proven capable of completely solubilizing membranes which contain cytochrome P450. (“Complete solubilization” in this context refers not only to preparation of soluble complexes by the criterion of centrifugation at 105,OOOg for 1 h, but also to disaggregation of soluble protein aggregates.) These detergents obviously modify the charge properties of proteins to which they are bound and are therefore not well suited for use in charge fractionation methods. In our own experience, SDS also appears to interfere with the formation of basic pH gradients, presumably through interaction with basic carrier ampholytes. Zwitterionic detergents offer a solution to the problem of preserving the surface net 201

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C 1979 by Academac Press. Lnc. of reproductmn ,n an,’ form rs\er\ed

202

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NEBERT

AND

CHRAMBACH

charge of solubilized proteins. Since these l-propane sulfonic acid (SB,,) was synthecompounds possess both cationic and an- sized by the procedure of Gonenne (10). available from ionic groups, their binding to proteins does SB,, is also commercially Calbiochem under the name Zwittergent not alter the net charge of the macromoleTMs14 (Catalog No. 693017). cule. In addition, zwitterions at their isoIsoelectric focusing on polyacrylamide electric points are expected to have no net electrophoretic mobility, and should thus be gel (ZFPA). IFPA was carried out in 2% ideal for electrofocusing systems (9). It was Ampholine [pZ range 7-11, 5% T, 5% CBis, 20% (v/v) glycerol, 0.05% Triton X-100, also originally thought that certain zwitterionic detergents were generally nondenatur0.05% SB,,]. Ampholine was prepared by ing in their interaction with membrane pro- mixing the commercially available pZ ranges teins (10). These properties prompted us to 7-9 (40%) and 9-11 (20%) to give a final concentration of 1% each. Conditions of investigate the use of zwitterionic detergents and IFPA were those prein the solubilization and electrofocusing of polymerization membrane proteins from mouse liver endo- viously described (12,13); 0.015% K2S208, 5 x 10p4% riboflavin, and 0.05% TEMED plasmic reticulum. were used as initiators. Anolyte (top reservoir) was 0.1 N HzS04, catholyte 0.1 N MATERIALS AND METHODS KOH. Samples were applied in 20% glyProteins. Liver microsomes from C57BL/ cerol, 2% Ampholine (pZ range 6-8), 1 mM 6N mice obtained from the Veterinary Re- DTT, and 1 mM EDTA. Samples were oversources Branch, NIH, were prepared as de- layered with the same solution as used for scribed (6), and solubilized at 1 mg/ml pro- preparation of the sample except that the tein [based on the Lowry procedure (1 l)] glycerol concentration was 10%. A regin 10 mM Tris acetate, pH 7.4, containing ulated power (LKB power supply No. 2103) 20% (v/v) glycerol, 1 mM dithiothreitol of 0.25 W/tube (0.27 cm2 gel surface area) (DTT),2 1 mM ethylenediamine tetraacetate was maintained until the voltage reached (EDTA), 1% (v/v) Triton X-100, 1% (w/v) 200; then, regulated voltage was maintained pl-range 6-8 at 200 for 14 h. Thereafter, 500 V was mainSB,I, and 2% Ampholine, (LKB), at 4°C just prior to use. The solu- tained for 1 additional h. Gels were fixed bilization procedure consisted of making an and stained by either Procedure D of Vesterapproximately 2 mg/ml microsomal suspen- berg (14) (designated as I) or by the followsion in buffered glycerol, DTT, EDTA, and ing procedure (designated as II) the developAmpholine, and final adjustment of protein ment of which is described in a separate concentration to 1 mg/ml after addition of communication (L. M. Hjelmeland, and A. detergents. Chrambach, in preparation). Gels were Detergents. Triton X-100 from Sigma was placed into a lo-fold volume excess of 0.1% used. N-Tetradecyl-Nfl-dimethyl3-aminoCoomassie brilliant blue G-250 in 10% TCA, 1 M urea for 8-16 h at room temperature. * Abbreviations used: Bis, N,N’-methylenebisacrylThe staining solution is made by dissolving amide; %C, crosslinking agent (g) x lOO/%T; CMC, the dye in 1 M urea, followed by addition critical micelle concentration; DATD, NW-diallylof solid TCA to 10% and filtration through tartardiamide; DTT, dithiothreitol; EDTA, ethylenediWhatman No. 1 filter paper by gravity. The amine tetraacetate; IFPA, isoelectric focusing on polyacrylamide gel; SB,,, N.N-dimethyl-N-tetradecyl-3staining solution is stored at room temperaamino-1-propanesulfonic acid; SDS, sodium dodecyl ture. Gels are destained in 10% TCA (sevsulfate; %T, total gel concentration [acrylamide eral changes) over a period of at least 2 days. + crosslinking agent (g/l00 ml)]; TCA, trichloroacetic Determination of pH gradients was made acid; TEMED, N,N,N’,N’-tetramethylethylenedieither electronically (15) or manually, after amine.

ZWITTERIONIC

DETERGENT

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ELECTROFOCUSING

sectioning of gels by razor blade into 5-mm segments and suspension of gel slices in 1 ml 0.01 M KCl. Spectrophotometric assay for cytochrome P-450. Cytochrome P-450 was assayed

spectrophotometrically

as described (16).

RESULTS Solubilization and Denaturation Cytochrome P-450 by SB,,

of

a-.

---A

0

LOG (% SE,.)

Mouse liver microsomes when reduced with sodium dithionite and saturated with carbon monoxide (Fig. 1, top panel) exhibit an optical difference spectrum with a Soret maximum at 450 nm characteristic of native cytochrome P-450, whereas the denatured enzyme is characterized by an absorption maximum at 420 nm with a corresponding decrease in the 450 absorption (17). De-

0.T B

/

0.

450 "Ill

O.OOl/

0.2

0.4 PROTEIN

0.6

0.6

1.0

tmg/mlb

FIG. 2. Loss of heme from cytochrome P-450 as a function of SB,, concentration: Nativeness is expressed and quantitated as “bound heme” on the basis of the spectrophotometric assay (Fig. 1). (A) Sigmoidat dependence of bound heme on the log of SB,, concentration at differing protein concentrations: Open circles, 1 mglml; closed circles, 0.5 mgiml; open squares, 0.25 mg/ml; closed squares, 0.125 mglml. (B) Plot of SB,, concentrations required for 50% heme loss as a function of protein concentration.

0.

s f g 0. g t

0.

400

420

I I I , I I 1 440 450 480 500

WAVELENGTH

inm)

FIG. 1. Difference spectra of dithionite reduced vs [reduced plus carbon monoxide] states of cytochrome P-450, providing a spectrophotometric assay for native (peak at 450 nm) and denatured (peak at 420 nm) enzyme. Top panel: Microsomal preparation containing 1 mg/ml protein. Bottom panel: The same preparation containing 0.1% SB,,. Loss of heme was quantitated using the known extinction coefficients for the species absorbing at the 450- and 420-nm Soret bands to estimate bound heme (21).

naturation also involves the loss of bound heme from the protein When microsomes were partially solubilized by relatively low levels of SB14, partial denaturation by these criteria was evidenced (Fig. 1, bottom panel). The quantitative degree of denaturation can be expressed in terms of percentage bound heme, on the basis of the known extinction coefficients of the 420- and 450nm species. The extent of heme loss from cytochrome P-450 is sigmoidally related to SB,, concentration at any one protein concentration (Fig. 2A). The quantitative rela-

204

HJELMELAND,

NEBERT

AND CHRAMBACH

gregation by the criterion of electrofocusing gels improve, expense of denaturation. amounts of SBII mixed with led to significant denaturation

entrance into but at the Even small Triton X-100 (Fig. 3).

Electrofocusing in the presence of SB,,

O.‘A 0,

400420440480400600 WAVELENGTH

Polymerization of both 5 %T, 5 %CBis and 5 %T, 15 %CDATD gels containing up to 0.1% of Triton TX-100 and SB14, and 20% glycerol proceeded readily under standard conditions. Wall adherence of the detergent containing gels appeared to be satisfactory. Under the normal conditions of electrofocusing, SB,, does not concentrate at a single isoelectric point, but instead remains evenly distributed across the pH gradient in a manner similar to “poor carrier ampholytes” (l&19). This result was easily visualized by soaking focused gels in 10% trichloroacetic acid, which precipitated SB,, in the gel giving an opaque zone. The pH gradients which were formed in the pres-

Inm)

FIG 3. Loss of heme from cytochrome P-450 as a function of the concentrations of SBll and Triton X-100 in mixed micelles: Heme loss was evaluated spectrophotometrically as described in the Legend to Fig. 1. (A) Microsomes at 1 mg/ml protein concentration containing 1% Triton X-100 and 0.001% SB,,. (B) Microsomes from the same preparation ontaining 1% Triton X-100 and 0.01% SB,,.

0 12 hrs A 15 hrs 018 hrs

PH

tion between the extent of heme loss, due to SB,4, and protein concentration in the microsomal preparation is shown in Fig. 2B. At any protein concentration, solubilization was attended by a proportional degree of denaturation . Solubilization and Denaturation of Cytochrome P-450 in Mixtures of SB,, and Triton X-100

Triton X-100 alone was not capable of sufficiently solubilizing microsomes to allow for electrofocusing. When SB,, was admixed to Triton X-100, solubilization and disag-

77

Slice

5

6

7

8

Number

FIG. 4. Stability of pH gradients in IFPA (pI range 7- 11) in the presence of 0.05% SB,,: IFPA (5 %T, 15 Y66g,s. 20% glycerol, 0-4”C, 200 V) was carried out with 0.05% SB,, and 0.05% Triton X-100 in the gel, and 0.1 N H,SO, and KOH as anolyte (top reservoir) and catholyte, respectively.

ZWITTERIONIC

CONTROL I

DETERGENT

INDUCED

I

205

ELECTROFOCUSING

CONTROL

It

4

SAMPLE STAIN

FIG. 5. Gel patterns of mouse liver microsomal preparations in IFPA (0.05% SB,,, 0.05% Triton X-100): Microsomai preparations, either control mice or mice induced with 3-methylcholanthrene (22), were solubilized and disaggregated in SB,, and Triton X-100 (see Materials and Methods), and applied (250 &gel of 0.27 cm* surface area) to IFPA as described under Materials and Methods. Gels were stained by either procedure I or II, using Coomassie brilliant blue G-250. as indicated in the figure. Lines denote the major bands. Arrows indicate induced proteins.

206

HJELMELAND,

NEBERT

ence of SB,, appeared to be remarkably stable, as evidenced by the series of pH gradients measured at different times depicted in Fig. 4. Basic proteins of mouse liver microsomes, however, did not enter the electrofocusing gel at “nonrestrictive” pore sizes, even when solubilized by a lo-fold (w/v) excess of SBi4. Electrofocusing in the Presence of Mixtures of SB,, and Triton X-100

When electrofocusing was carried out under conditions identical to those described above, with the addition of Triton X-100 in the same concentration as SB,, in both the gel and the solubilization medium, basic proteins readily entered the gel (Fig. 5). In an attempt to optimize the relative concentrations of SB,, and Triton X-100, a series of experiments was carried out varying the composition of the gel and the solubilizing medium. In each case, the ratio of the two detergents was kept the same in the gel and sample phases (Table 1). An all-or-none effect with respect to the mixing of these detergents was observed. All mixtures above a certain critical concentration gave similar gel patterns, whereas either detergent alone, or very low gel concentrations of the mixed detergents, failed to give focused protein zones.

AND

CHRAMBACH TABLE

1

VARIATION OF THE PERCENTAGE OF TRITON AND SB,, IN ORDER TO OPTIMIZE RELATIVE CONCENTRATIONS Triton

X-100

THEIR

X-100

(%)

SB,, (%I

0.0

0.01

0.05

0.10

0.0 0.01 0.05 0.10

-

-

-

-

+a

+ +

a Designates

entry

of proteins

+ +

into the gel.

of Svensson (l&19), i.e., its pl-pK, difference is large (approximately 7). Like other poor carrier amphofytes, this compound does not focus into a sharp peak at its isoelectric point, but remains evenly distributed in the gel during the lifetime of a normal focusing pH gradient. In addition, the stability of pH gradients formed in the presence of SB,, seems enhanced when compared with gradients formed in the absence of this compound. The mechanism of pH gradient stabilization by SB,, is possibly related to the bridging of conductivity gaps which normally arise in the course of electrofocusing experiments. All of these properties would indicate that SB14, or some other zwitterionic detergent, should be uniquely applicable to electrofocusing. The original report of focusing in the DISCUSSION presence of zwitterionic detergents (9) also claimed that these agents were generally The recent introduction of zwitterionic nondenaturing in their interaction with memdetergents into electrofocusing (9) appeared brane proteins. SB,, in concentrations sufpromising for the fractionation of intrinsic ficient to fully solubilize endoplasmic membrane proteins, since these detergents however, also completely appeared to have solubilizing power far reticulum, in excess of nonionic detergents, while at denatures cytochrome P-450. We feel that the denaturing properties of SB,, are most the same time lacking electrophoretic mobility in their isoelectric states. As a con- likely representative of the general class of sequence, zwitterionic detergents are also n-alkyl zwitterions, and are related to the nonconductors and thus do not give rise to flexible nature of the hydrocarbon tail in these molecules (1,20). Allen and Humphsignificant current and the Joule heating that accompanies electrofocusing in the ties’ finding to the contrary (9) is presumpresence of ionic detergents. SB,, is a ably due to the use of detergent concentration inadequate to effect full soiubilization “poor carrier ampholyte” by the criterion

ZWITTERIONIC

DETERGENT

of membranes. Their study employed a detergent:protein ratio of 1:6, while our own results as well as the work of Gonenne and Ernst (10) indicate that ratios in excess of 10: 1 are required. Figure 2 demonstrates that critical phenomena related to detergent concentration are also dependent on protein concentration. This finding obviously suggests that detergent protein ratios should be used as an experimental variable instead of detergent concentration, and this in turn suggests that enough detergent in the micellar form must be present to dissolve membrane constituents on a stoichiometric basis (1). The finding that mixed micelles composed of Triton X-100 and SB,* were superior to either detergent alone was quite surprising. Both the critical micelle concentration (Triton X-100 = 0.24 mM, SB,, = 0.6 M) and the aggregation number (Triton X-100 = 140, SBll = 80) are similar for both detergents (1). The CMC and aggregation number of the mixed micelle is reasonably expected to assume intermediate values. The shape of mixed micelles, however, is often quite different from the globular shape of normal micelles, and this may have some effect on the solubilizing power of these species. The zwitterionic polar group of SB,, may also have a specific effect on the hydrophilic portion of the mixed micelle, rendering this species a better solubilizing medium for proteins. In any case, the origin of this phenomenon remains unknown at the present time. One of the commonly encountered difficulties in IFPA in the presence of detergents concerns staining and destaining of protein zones. Fundamentally, the antagonism between the use of solubilizers (detergents) and fixatives (acids) is involved. Hydrophobic membrane proteins are by definition more soluble than water soluble proteins in the hydrophobic destaining media employed by most current methods. This leads to selective loss of hydrophobic proteins during destaining in the absence

207

ELECTROFOCUSING

of effective fixatives; this problem can be observed by comparing the stained gels in Fig. 5. Successful staining, however, also depends on detergent removal from the gel under conditions of minimal protein solubilization, because detergents bind most dyes quite avidly and produce a high background. This removal is aided by consideration of the critical micelle concentrations of the detergents used. A workable procedure for staining electrofocusing gels containing SB,,-Triton X-100 mixtures has been developed along those lines (see Materials and Methods). In conclusion, our expectations concerning the use of zwitterionic detergents in electrofocusing were partially met. Although we found SB,, to be strongly denaturing in contrast to claims in the literature, this detergent in combination with Triton X- 100 allowed us, for the first time, to focus the basic proteins from mouse liver endoplasmic reticulum. It appears therefore that although the zwitterionic sulfobetaine group is a useful hydrophilic moiety, other hydrophobic groups should be considered. Work is presently in progress to synthesize sulfobetaines with rigid hydrophobic domains in the expectation that these detergents may preserve the native structures of solubilized proteins. REFERENCES I. Hjelmeland. L. M., Nebert, D. W., and Chrambath, A. (1978) in Electrophoresis ‘78 (Catsimpoolas, N., ed.) Elsevier. Amsterdam/New York/North-Holland, Amsterdam, in press.‘1 2. Newby, A. C.. Rodbell, M.. and Chrambach. A. (1978)

Arch.

Biochmn.

Biophys.

3. Newby, A. C., and Chrambach, chcm.

190, 109- 117.

A. (1978) Bio-

.I.. 177, 623-630.

4. Guengerich. F. P. (1977)5. Eiol. Chrm. 252,39703979.

5. Coon, M. J., Van der Hoeven, T. A., Dahl, S. B., and Hauger. D. A. (1978) in Methods in Enzymology (Fleischer, S., and Packer, L.. eds.), Vol. 52. Part C, pp. 109-l 17, Academic Press, New York. s Available upon request.

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6. Ingleman-Sundberg, M., and Gustaffson, J. A. (1977) FEBS Lett. 74, 103-106. 7. Warner, M., VellaLaMarca, M., and Neims, A. H. (1978) Drug Merab. Disp. 6, 353-362. 8. Chrambach, A. and Nguyen, N. Y. (1978) in Electrophoresis ‘78 (Catsimpoolas, N., ed.), Elsevier, North-Holland, Amsterdam/New York, pp. 3- 18. 9. Allen, J. C., and Humphries, C. (1975) in Isoelectric Focusing (Arbuthnott, J. P., and Beeley, J. A., eds.), pp. 347-354, Butterworths, London. 10. Gonenne, A., and Ernst, R. (1978)Anal. Biochem. 87,28-38.

11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275.

12. Chrambach, A., Jovin, T. M., Svendsen, P. J., and Rodbard, D. (1976) in Methods of Protein Separation (Catsimpoolas, N., ed.), Vol. 2, pp. 27-161, Plenum, New York.

AND CHRAMBACH 13. Doerr, P., and Chrambach, A. (1971) Anal. Biothem. 42, %- 107. 14. Vesterberg, O., Hansen, L., and Sjosten, A. (1977) Biochim. Biophys. Acta 491, 160-166. 15. Chidakel, B. E., Nguyen, N. Y., and Chrambath, A. (1977) Anal. Biochem. 7, 216-225. 16. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239,2370-2378.

17. Imai, Y., and Sato, R. (1967) Eur. J. Biochem. 1, 419-426. 18. Svensson, H. (1962) Acta Chem. Stand. 16, 456-466.

19. Righetti, P. G., and Chrambach, A. (1978) Anal. Biochem. 90,633-643. 20. Tanford, C. (1973) The Hydrophobic Effect, Wiley, New York. 21. Nishibayash, H., and Sato, R. (1967) J. Biochem. 61,491-496. 22.

Gielen, J. E., Goujon, F. M., and Nebert, D. W. (1972)J. Biol. Chem. 247, 1125-1137.