Molecular weight estimation of Triton X-100 solubilized proteins by polyacrylamide gel electrophoresis

ANALYTICAL

BIOCHEMISTRY

72, 1t3- 122 t 1976)

Molecular Weight Estimation of Triton X-100 Soiubiiized Proteins by Polyacryiamide Gel Electrophoresis VINCENT J. HEARING, WALTER G. KLINGLER, THOMAS M. EKEL, AND PAUL M. MONTAGUE Dermatology

Eranch, National Cuncer lnstitate, National Bethesda, Maryland 2OOi4

Institutes of Heaith,

Received May 29, 197.5: accepted February

10, 1976

A polyacrylamide gel elec&ophoresis system is described which employs the nonionic detergent T&on X- 100 as a protein solvent. Aside from the obvious advantages of this detergent over ionic detergents and urea in the preservation of protein structure and function, it is demonstrated that the ancillary benefits of gel electrophoresis. i.e., molecular size and charge determination, are possible.

Polyacrylamide gel electrophoresis (PAGE) is often the method of choice for the separation and characterization of various biological macromolecules. Since electrophoretic migration depends on both the size and the charge of the molecule, these physical parameters can often be estimated by a study of their electrophoretic behavior under differing conditions. Several methods have been developed for the estimation of the molecular size of proteins, employing anionic detergents (l-5), cationic detergents (6,7), and urea (8,9) as solvents; unfortunately, these methods depend on the denaturation of the molecules under study, resulting in the loss of both structure and biological function. Several studies (10,ll) have demonstrated the operability of molecular size estimation in the absence of protein denaturants, but these techniques employed only ‘soluble’ proteins as markers, and did not attempt to use any of the ‘insoluble’ proteins, which represent a large class of proteins of biological interest. Triton X-100, a nonionic detergent, is widely used to solubilize ‘insoluble’ macromolecules, such as ribosomal and membrane proteins (12- 14); it has an advantage over other detergents in that it preserves the three dimensional structure of proteins, and thus their enzymatic and/or immunological activity (15- 18). Several investigators have shown the feasibiIity of carrying out gel electrophoresis on proteins solubilized with nonionic detergents (12,19-23) and this report describes a PAGE system employing Triton X-100 for protein solubilization which allows molecular size estimations.

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ET AL.

METHODS All gel electrophoretic procedures were carried out using the rigorously controlled conditions outlined for quantitative PAGE by Rodbard and Chrambach (11). The Tris:glycine buffer system (11, No. A, separation pH 9.45) was used; the %T (acrylamide and ~,~‘-methylene-bisac~lamide concentration) was varied between 4 and 15%, while the %C (% NJV’-methylene-bisacrylamide/%T) was held constant at 3% in the separation gel. The concentration gel was maintained at 5%T, 2O%C. Routinely, no detergent was added to the gel; in exploratory studies, the polymerization mixtures contained 0.1% Triton X- 100. Both upper and lower gels were photopolyme~zed with riboflavin (50 pg.00 ml) and potassium persulfate (1 S mg/lO ml). Protein samples were solubilized at I mg/ml in 1% (15 mM, critical micelle concentration = .24 mM) Triton X-100 for several hours, then an equal volume of 20% (w/v) sucrose: 2x upper buffer in 1% Triton X-100 was added. Approximately 5 fig of each protein were applied to the gels, which were subjected to electrophoresis at 2 mA/tube at 2O*C until the bromphenolblue tracking dye neared the bottom of the gels; the gels were then removed and cut off at the marker dye front. Following fixation in 12.5% (w/v) TCA for 10 min, the gels were stained with Coomassie blue G for approximately 15 min (24), and stored in 7.5% (v/v) acetic acid; some gels containing Triton X-100 were stained in 0.1% (w/v) Amidoblack (25)* or 0.1% (w/v) Fast green FCF (26), in 7.5% acetic acid. Relative mobilities of the bands, calculated against that of the bromphenolblue, were used for statistical analysis of retardation coefficient (KJ and electrophoretic free mobility (Y,,); molecular size and charge data were estimated by computer programs designed by Rodbard and Chrambach (1 I ,27). For SDS gels, the Tris/glycine buffer system was used as above, and both upper buffer and samples contained 0.02 M SDS as previously described (4). For urea gels, all solutions contained 8 M urea with 0.1 M 2mercaptoethanol in the sample. A minimum of 10 independent data points were used to establish the KR and Y0 values for each standard protein. RESULTS In order to determine the usefulness of this gel system in the estimation of various physical parameters of polypeptides, standard proteins to be studied were selected which varied widely as to molecular size, conformation, and the presence or absence of metals and prosthetic groups; these are listed in Table 1. Triton X-100 does not affect the linear relationship of (K,# vs radius of solubilized proteins (Fig. I). It has been shown previously that the presence of Triton X-100 did not alter the sedimentation velocities of normally water-soluble proteins (13); the KB and Y0 of these proteins were

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TABLE GENERAL

Number 1 2 3 4 5 6 7 8 9 10 11 12 13 I4 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

115

X-100 PAGE SYSTEM

CHARACTERIZATION

1 OF STANDARD

PROTEINS

Protein

Tissue sourcea

Shapeh

Insulin dimer n-factogtobuhn A Myoglobin Chymotrypsin Trypsin inhibitor Chymotrypsinogen Carbonic anhydrase Phosvitin Carboxypeptidase B n-tactogiobuhn Pepsin Ovalbumin Fetuin Keratin la Keratin lb Albumin Transferrin D-amino acid oxidase Phosphohpase C D-amino acid oxidase Tyrosinase Lactate dehydrogenase Albumin dimer Transferrin dimer Aldolase Catalase Transferrin trimer Acetylchohnesterase Fibrinogen Phosphorylase A Ferritin Apoferritin Thyroglobulin

bovine pancrea@ milkd sperm whalec bovine pancrea& soybeand bovine pancreasd bovine erythrocyted egg vitehind hog pancreasd milkd hog stomach mucosa? e& fetal calf serumd calf hoof’ calf hooff bovine serumc human, iron freed Neurospora, mitochondriag Cl. welchiid Neurospora, solubleg mushroomd beef heartd bovine serumc human, iron freed rabbit muscled bovine liverd human, iron freed electric eeld bovined rabbit muscled horse* horseb bovined

G G G G G G G F G G G G F F F G F G G G G G G F G G F G F G G G G

Group

Metal

S Fe

Zn Zn

S Zn cu Zn S Zn S S S Fe S

a Superscript indicates source; b Mannheim; c Schwartz-Mann; d Sigma; p Worthington; f gift from P. Steinert; g gift from M. Rosenfeld. A Shape: (F) fibrillar, (G) globular; Group: Prosthetic group, (S) sugars; Metal: associated metals.

identical, regardless of the presence or absence of Triton X-100 in the sample. Furthermore, the ability of a protein to bind small amounts of Triton X- 100 did not cause a significant change in either the KR or Y,, (e.g., protein No. 16,23,28, and 33). The absence of Triton X-100 in the gels did not present a solubility problem with any of the proteins studied, but since it is nonionic in nature, Triton X-100 will not migrate into the gel unless bound by the protein. For this reason, other proteins may encounter

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FIG. la. Plot of (K,# vs the effective molecular radius for the standard proteins. Dashed line = 99% confidence limits.

I

b” FIG. lb. Same data as in Fig. la, replotted as KE vs the molecular weight of the standard proteins. Dashed line = 9% confidence limits; Dotted line = best linear fit.

solubility problems once out of the sample area; this could be avoided by the incorporation of the detergent into the gels. The presence of 0.1% Triton X-100 in the gels did not affect the mobility of any of the proteins we examined, although when fixed in TCA, the gels become somewhat opaque, and destaining takes 24 hr. Staining of these gels with Fast green or Amidoblack will avoid this problem. The retardation coefficient (KR) and electrophoretic free mobiiity (Y,,) were calculated for each of these proteins from Ferguson plots (log Rm vs%T) calculated by weighted least-squares linear regression (11). The square root of the slope (KR) of this plot has been shown to be propor-

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117

X-100 PAGE SYSTEM TABLE

2

PHYSICAL CHARACTERISTICS OF STANDARD PROTEINS BY TRITON X-100 GEL ELECTROPHORESIS Molecular weight* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

,031 ,039 ,037 ,045 .046 ,046 ,051 .049 .056 .055 ,058 .058 .073 ,078 ,074 .078 .086 .097 ,093 .I05 ,107 .115 .120 .I08 .I33 ,131 ,154 ,140 ,181 ,209 .209 ,207 .298

1.55 1.83 .39 .71 1.90 .94 .77 1.73 1.16 1.87 1.65 1.86 2.25 1.41 1.24 2.59 1.59 1.77 2.62 2.56 3.39 1.56 .62 2.01 2.38 2.12 1.26 4.83 4.87 4.87 7.97

1.15 ( .98) 1.75 ( 1.71) 1.78 ( 1.55) 2.16 ( 2.30) 2.27 ( 2.35) 2.32 ( 2.37) 3.00 ( 3.00) 3.39 ( 2.72) 3.43 ( 3.59) 3.50 ( 3.53) 3.50 ( 3.87) 4.50 ( 3.91) 5.06 ( 6.06) 5.80 ( 6.91) 5.80 ( 6.26) 6.70 ( 6.95) 7.40 ( 8.35) 10.00 (10.30) 10.00 ( 9.68) 12.50 (11.80) 12.50 (12.43) 13.30 (14.20) 13.40 (15.43) 14.80 (12.71) 15.80 (18.48) 22.00 (18.00) 22.20 (24.20) 23.00 (20.40) 34.00 (32.10) 40.00 (41.10) 46.00 (41.30) 46.00 (40.40) 67.00 (75.50)

Radiusc 1.50 (1.42) 1.73 (1.71) 1.74 (1.66) 1.85 (1.89) 1.88 (1.90) 1.90 (1.91) 2.06 (2.07) 2.15 (2.00) 2.16 (2.19) 2.i7 (2.18) 2.17 (2.25) 2.36 (2.26) 2.46 (2.61) 2.57 (2.73) 2.57 (2.64) 2.70 (2.73) 2.79 (2.90) 3.08 (3.13) 3.08 (3.05) 3.32 (3.28) 3.32 (3.32) 3.39 (3.47) 3.40 (3.56) 3.51 (3.34) 3.59 (3.79) 4.01 (3.75) 4.02 (4.14) 4.07 (3.91) 4.64 (4.55) 4.90 (5.21) 5.13 (5.22) 5.13 (5.19) 5.81 (6.34)

Valenced 4.7 6.6 1.4 2.8 7.8 3.9 3.6 8.5 5.8 9.4 8.3 10.5 13.4 8.9 7.9 17.7 11.4 -. 14.7 24.4 24.6 32.6 15.9 6.5 24.9 29.5 26.9 19.5 81.5 88.4 88.4 176.3

a Refers to protein number in Table 1. * Molecular weights in daltons x 10m4(estimates in parentheses). c Effective molecular radius in nanomoles (estimates in parentheses). ‘I -Protons/molecule.

tional to radius of globular proteins in a variety of PAGE systems (11). In Fig. la, the linear plot of (K# vs the radius is shown for all 33 marker proteins, along with the 9% confidence limits for the line. Excellent correspondence is observed throughout the range of this study, from 1.5 to 6.0 nm, for all types of proteins examined. As has been demon-

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TABLE STANDARD

Protein type All Globular Fibrous MetalIoproteins Glycoproteins

All Globular Fibrous Metalloproteins Glycoproteins

R

CURVES

FOR DIFFERENT

PIota

Y intercept

RAD RAD RAD RAD RAD

- .75.5 (.078) -.738 (.081) -.849 (.273) -.803 (.159) -.636 (. 164)

MW MW MW MW MW

3 CLASSES

Slope 12.47 12.44 12.68 12.63 12.08

(.25) (.25) (.86) (.50) (.48)

Y intercept (x 10-Z)

Slope (X 10-S)

-1.12 -1.10 -1.12 - 1.15 -1.21

2.49 2.51 2.35 2.47 2.55

(.ll) (.12) (.21) (.32) (.17)

(.09) (.lO) (. 19) (.29) (. 12)

OF PROTEINS

r

R

.994 .995 .986 .995 .994

.13 .I2 .16 .ll .15

r

(x 10-S)

.981 .983 .981 ,962 .991

.31 .33 .23 .42 .28

R

=RAD = (K#z vs radius; MW = KR vs molecular weight; r = correlation = square root of residual variance; NL = curve significantly nonlinear.

NL NL NL NL coefficient;

strated in other gel systems (5,28,29), the relationship of the KR vs molecular weight was found to be significantly nonlinear in this gel system between 10,000 and700,OOO (Fig. lb). It has been shown (27) that (K,J* vs protein radius is statistically a superior way for the calculation of molecular weight and radius data for globular proteins; linearity of the (K,J* vs radius plot (Fig. la), is consistent with the notion that theTriton X-100 solubilized proteins are globular. It is of special interest to note that the only KR vs molecular weight plot which has no significant nonlinearity is that for the fibrous proteins (Table 3), as is predicted by the Ogston theory (27,30). In Table 2 the physical characteristics of the standard proteins determined by the Triton gel system are listed. The rigorous statistical treatment given these results (discussed in 11 and 27) leads to fairly large 9% confidence limits (see Fig. 1) when compared to other methods of molecular size estimation by PAGE. However, as pointed out (11,27), this reflects the strict statistical analysis given the data rather than a failure in the accuracy of the technique. The estimate of the effective radius of each protein was accurate, with the error averaging just over 23% and ranging from 0 to 7%. The error about the molecular weight estimates averaged just over &9% and ranged up to 20%. The various classes of proteins studied had random distribution around the plot of (K& vs the molecular radius, regardless of their shape, the presence of polysaccharides, or the presence of metals. When this plot was estimated using the programs of Rodbard and Chrambach (11,27) by unweighted least-squares linear regression for the various classes of proteins, the general parameters of the curve were insignificantly changed.

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X-100 PAGE SYSTEM

These curves are defined in Table 3. Thus, if an unknown protein can be shown to fall into one of these subclasses, the best fitting curve can be utilized, but in the absence of such information, the general curve drawn for all proteins is reliable. Use of the more accurate curve in each case leads to less than a 2% increase in the accuracy of molecular size measurements. It was of interest to study the mobility characteristics in this PAGE system of proteins known to bind extremely large quantities of Triton X-100. It has been shown that several membrane proteins are in this class (16,23,31), and one of the best characterized is bovine rhodopsin. This protein (a gift from Dr. P. O’Brien) has a molecular weight of 35,000 and binds approximately 70 mol of Triton X-lOO/mol protein (yielding a total molecular weight of 7gOOO; see 18,3 1,32). The KR of rhodopsin was .079, indicating an estimated molecular weight of 71,000. Thus, as would be expected, proteins which bind Triton X-100 migrate as a function of their total molecular size. To determine the relative accuracy of the various methods of PAGE molecular size measurement, several polypeptides were characterized by SDS, urea, and Triton X-100 electrophoresis, using the same buffer system. Both SDS and urea gave slightly more accurate estimates of molecular weights than did Triton X- 100 (Table 4). The average error was &5% for SDS, 24% for urea and ? 10% for Triton X-100. As an illustration of the use of this PAGE system to solubilize an “insoluble” protein and determine its molecular weight, we have localized several tyrosinase isomers in a preparation of murine melanosomal proteins by the DOPA reaction (33, see 34 for further details and discussion). These proteins are soluble only in SDS, urea, or Triton X-100; although these samples contained more than 20 protein species, two major DOPA-positive bands are demonstrable. These have Kg of .081 and .084. The estimated molecular weights of these bands, 74,000 and 80,000, re-

TABLE COMPAIUSONOFPOLYACRYLAMIDE Protein Myoglobin Chymotrypsin Pepsin Ovalbumin Albumin

Molecular weight 17800 21600 35000 45000 67000

4

GEL SYSTEMSFORMOLECULAR WEIGHT ESTIMATION SDS’ 18900 22300 31200 45600 69000

( 6) ( 31 (11) ( 1) ( 3)

Urea 18000 21400 30400 45300 69500

( 1) ( 1) (13) ( 11 ( 4)

Triton x-100 15500 23000 38700 39100 69500

(13) ( 6) (11) (131 ( 4)

a Molecular weight estimates using gel system noted, percent error of the estimate yielded by each gel system is listed in parentheses.

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spectively, agree well with the previously (80,000) for mm-me tyrosinase (T-l ; 35).

published

molecular

weight

DISCUSSION This study was initiated primarily to develop a PAGE system which employed conditions: (1) sufficiently “harsh” to solubilize the generally insoluble proteins, (2) “mild” enough to preserve the structural and functional integrity of the molecules under study, and (3) which allowed the ancillary benefits of PAGE such as molecular weight and charge estimation. The nonionic detergent Triton X-100 was a particularly advantageous choice of solvent, due to its excellent protein solubilizing properties, the preservation of protein configuration, and its nonionic nature which allows the protein’s intrinsic charge to be a factor in electrophoretic migration. One drawback of ionic detergent solubilization and electrophoresis is the masking of individual protein charge, resulting in the lack of resolution of proteins with similar molecular size. The advantage of Triton X- 100 electrophoresis over that with anionic detergents in the resolution of a maximal number of protein species has been shown (12). The advantages of Triton X-100 as a solvent for proteins and subsequent PAGE include good solubilization with no denaturation of proteins (13,15,36,37), fairly accurate molecular size determinations, and rapid fixation and staining (within 30 min) with little or no destaining. In addition, the nonionic nature of the detergent allows the intrinsic charge of the protein to be expressed, and thus charge isomers can be resolved. The conditions of Triton X-100 solubilization can be altered for the selective solubilization of proteins, by varying ionic strength, time, etc. (13). Lastly, the problems encountered in ionic detergent gel electrophoresis as discussed below do not affect the calculations involved here. The disadvantages of the Triton X-100 technique include the insolubility of some proteins in Triton X-100 and the accompanying removal of lipids which may have to be added back to restore function to some proteins. It has been shown (32,38) that for total delipidation of lipoproteins to take place, it is important to keep the ratio of Triton X-100 to protein high. The advantages of PAGE molecular weight estimation using ionic detergents include excellent solubilization and greater accuracy of molecular weight determinations (generally ~5% or better). The disadvantages include the disappearance of the protein due to subunit dissociation, the denaturation of the subunits, the susceptibility to various types of inaccurate molecular weight estimates due to the presence of sugars, differences in detergent preparations, the inability of SDS in some cases to provide an equalized charge density on the molecular surface due to folding of the protein or forms of steric hindrance (5,39-41), and the inferior fixation of proteins, which is concomitent with their more

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121

X-100 PAGE SYSTEM

thorough solubilization. One of the reasons ionic detergent PAGE systems can not measure high molecular weight proteins with great confidence is the fact that because of the strongly dissociating conditions, few high molecular weight markers are available for the calibration of the standard curve. Triton X-100 does not dissociate proteins into their subunits; on the contrary, it is a favorable environment for normal protein aggregation, and thus provides for many high molecular weight markers as used in this study. PAGE in the presence of urea has advantages over ionic detergents in that the intrinsic charges of proteins are retained and allow for greater resolution of protein species; however, urea is not as good a protein solvent as Triton X-100, and again usually results in the loss of protein conformation and function. Because of the preservation of protein structure and function realized with this technique, use of purified protein preparations are not necessary for the measurements described herein, providing some method of identifying the protein under study is available. Included among these techniques are enzymatic function, immunologic reactivity, reactivity in histochemical tests, or the demonstration of the presence of prosthetic groups. The pH of the gel system used here is rather high (9.45); it was chosen to allow most proteins to migrate towards the anode. However, the potential use of Triton X-100 in other compatible PAGE buffer systems should allow analogous estimations of the molecular size of proteins. Thus a PAGE system employing Triton X-100 as solvent could be tailored for any type of protein simply by varying pore size (%C), pH, stacking, and unstacking limits for maximal resolution. It would also be feasible to adapt this PAGE system to the study of integral membrane proteins which bind large amounts of Triton X-100. As was shown with rhodopsin in this study, one can easily determine the total molecular size of the protein-detergent complex. If one also wishes to determine the amount of Triton X-100 bound to a particular protein, the method described by Clarke (31) is compatible with this gel system. ACKNOWLEDGMENTS The authors wish to thank Drs. Andreas Chrambach and Peter Steinert for their valuable assistance with the manuscript, Dr. David Rodbard for the use of his computer programs, and Drs. Peter Steinert, Paul O’Brien, and Melvin Rosenfeld for their gifts of protein samples.

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