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Effect of fatty acids on the movement and staining of membrane proteins in polyacrylamide gel electrophoresis
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECT OF FATTY ACIDS ON THE MOVEMENT AND STAINING OF MEMBRANE PROTEINS IN POLYACRYIAMIDE GEL ELECTROPHORESIS
June Mo Fessenden-Raden Section of Biochemistry and Molecular Biology Division of Biological Seiences~ Cornell University Ithaca 3 New York 14850
R e c e i v e d J a n u a r y 3, 1972 Summary The presence of fatty acids in preparation of membrane proteins can cause a change in the apparent mobility and staining characteristics of the proteins as well as induce the appearance of multiple bands in polyacrylamide gel electrophoresis.
Ever since it was first described by Ornstein (I) and Davis (2) polyacrylsmide gel electrophoresis has become a much-used tool in biochemistry. The use of Coomassie brilliant blue in trichloroacetic acid (3) to fix and visualize proteins rather than other stains such as amido black has been extensively employed because of its sensitivity (3, 4).
It is known, however~
that proteins vary in their ability to interact with Coomassie brilliant blue (5)-
This study was prompted by the observation that at equivalent protein
concentrations a coupling factor 6 preparation devoid of fatty acids did not stain as intensely with C0omassie brilliant blue as a preparation with fatty acids and exhibited less bands.
Methods Polyacrylamide gel electrophoresis was run as described by Davis (2) with or without a stacking gel.
The gels were fixed and stained ~ith Coomassie
1347 Copyright © 1972, by Academic Press, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
brilliant blue R in i0~ trichloroacetic acid as described by Chrambach~ et al (3).
Polyacrylamide gel e!eetrophoresis in the presence of sodium dodecyl-
sulfate (SDS) was carried out as described (6). Densitometer traces were ob~ tained at 560 m~ on a recording Gilford spectrophotometer with a linear transport for gel scanning.
Stained gels were photographed by transmitted light
with a Polaroid MP3XL camera using TYPE 57 film and an 25A filter.
Results and Discussion With Coomassie brilliant blue as the protein stain~ polyacrylamide gel electrophoresis of a purified preparation of the heat-stable coupling factor 6 (F6) from bovine heart mitochondria (7) revealed several bands (Fig. IA).
0.5 A
F6
0.4 0.3
dye
02
E
fronl
0
0 ~0
B. Extracted F6 0.2
I'<:[ Ld OJ (..) Z
0
0 0.6
.~ 0.5
C. E x t r a c t e d F6 + Oleic A c i d
0.4
0,5
0.2
0.1
Fig. 1. Densitometer traces of various F~ preparations. A) F~, B) Fg ext - ~ t e d with iso-octane to remove fatty a~ids, C) extracted F6~plus lOVnmoles of oleic acid (30 ~g of protein) was applied per gel. Electrophoresis (anode at the right), staining~ and recording were carried out as described under "Methods."
1348
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Electrophoresis of F 6 on gels prepared at different acrylamide concentration as described by Hedrick and Smith (8) indicated that the multiple bands of F 6 represented proteins of similar size but varying in charge.
SDS gel electro-
phoresis, how~ver~ also revealed multiple bands. It wag subsequently discovered that preparations of F 6 contained widely varying amounts of fatty acids.
After extraction with iso-octane, F 6 gave a
negative rhodamine 6G test for fatty acids (9), yet was still fully functional as a coupling factor in oxidative phosphorylation.
When this extracted prep~-
ration of F 6 was analyzed by gel electrophoresis it wag discovered that a) there was only a single band; and b) the band did not stain well with Coomassie brilliant blue.
(Compare Fig. IA and 1B).
to F 6 prior to electrophoresis~
When fatty acids were added back
the pattern seen in Fig. IC was obtained.
Fig. 2. Electrophoretograms of various fatty acids. A) stearic, B) oleic, ~-~idic, D ) l i n o l e i % E) blank. Each fatty acid wag applied in excess (50 nmoles) to assure enough density in the bands for photographing. Electrophoresis (anode at the bottom), staining and photography were carried out as described uader "Methods."
1349
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Both the number of bands and their stain intensity increased.
Fig. iC shows
F 6 with oleic acid but several other fatty acids tested revealed
a) an in-
crease in the staining capacity of the major band; b) the appearance of either new distinct bands or of a diffuse area of stain; and c) a consistent decrease in the mobility of the major band. When purified fatty acids were electrophoresed in the absence of any protein they could be visualized with Coomassie brilliant blue (Figs. 2 and 3).
Unsaturated cis-fatty acids consistently ran at or closely behind the
dye front (Figs. 2B~ D. 3B) while the trans-fatty acids did not penetrate nearly as far into the gel (Figs. 2C~ BC). much more variable.
The saturated fatty acids were
These differing mobilities would have to be explained in
ter~r~ of differences in shape since stearic aeid~ oleic acid an~ elaidic acid have virtually the same molecular weight and Charge.
0.2
i
A. Stearic Acid
/7
,o%
//
Elaidie acid might be
]
dye/
0.1
o B. Oleic Acid E 0.3
c
o
£0 0.2 t-hi O,I (,.) Z
<[
L-
oa o u_)O I.~
1
;. Elaidic Acid
i
0.4 ().3 0.2 OJ 0
Fig. 3. Densitometer traces Of various fatty acids. A) steari% B) olei% C--~ elaidic (!0 nmoles each) were applied. Electrophoresis, staining~ and recording were carried out as described under "Methods."
1350
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
expected to have the lowest mobility since it is least compact~ whereas the cis-fatty acid is the most compact.
Likewis% the absence of a double bond
would allow ~aturated fatty acids to be the most variable in shape. terns shown were obtained without a stacking gel.
All pat-
However~ the presence of a
stacking gel did not change the patterns obtained either with the fatty acids alone or proteins plus fatty acids.
The stacking gel did reduce somewhat the
smearing often encountered with some fatty acids (See Fig. 3A). The most dramatic effect of a fatty acid on a protein was seen with Fz (i0).
This purified protein from bovine heart mitochondria showed a single
major band on gel electrophoresis (Fig. 4A).
The addition of oleic acid to Fi
caused a decrease in this major band and the appearance of several faster
1.6 A. L4
o2 )P
dye front $
Ol J £ c O~ 0c.D 0.4 t~
I---
A B. F I + 5 nmoles
OleicAcid ~
~
1
0.5 W <) Z 0.2 G~ OK
0o9 G~
OI 0 0.4 05
C. FI+10nmoles OleicAcid
//l!
0.2
0
Fig. 4. Densitometer traces showing the effect of o!eic acid on the electrophoresis of FI. A) F1 (i ~g)~ B) F1 (i ~g) plus 5 nmoles of oleic acid~ C) Fl (i ~g) plus i0 nmoles of oleic acid. Electrophoresis~ staining~ and recording were carried out as described under "Methods."
1351
Vol. 46, No. 3, 1972
moving bands.
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
(Fig. 4B).
At i0 nmoles of o!eic acid the original band had
disappeared and a gel pattern entirely different from that of the original Fl appeared (Fig. 4C). Preliminary results with SDS gel electrophoresis has shown that h e r %
too~
F 6 freed of fatty acids exhibited fewer bands and did not stain as intensely with Coomassie brilliant blue as the non-extracted F 6 (Fig. 5).
The patterns
did not vary significantly dependent on whether the SDS (final concentration = i~) was added to the protein at 23 ° for 18 hrs., or I00 ° for ! min, or merely added immediately prior to electrophoresis without heating.
It would appear
0.9
A. F6
/\
0,8
dye
0.7 06 06 E c
i
04
0 u3 F<
0.5 0.2
Z < 0.1 m 0 <
0 0.5
B. Extrocted F6
04 0.~ 02.L OI
0
~
. Densitometer traces of various F~preparations on SDS gels. A) F6, extracted with iso-octane to remove fatty acids (30 #g of protein) was applled per gel. Electrophoresis~ staining~ and recording were carried out as described under "Methods."
1352
Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
that the presence of fatty acids may in some manner hinder the interaction between protein and SDS and thus cause multiple bands°
Further studies with
several other proteins are presently being undertaken. Although these dramatic effects have been shown only with membrane proteins, it is likely that any protein that will interact with fatty acids will be influenced in its staining with Coomassie brilliant blue, its charge, and its mobility characteristics.
Acknowledgement s This investigation was supported by grant AM-II715 from the National Institute of Arthritis and Metabolic Diseases and undertaken during the tenure of Career Development Award AM-12614 from the National Institute of Arthritis and Metabolic Diseases. The excellent technical assistance of Mrs. Janice Bruns is gratefully acknowledged.
REFERENCES
i. 2. 3. 4. 5. 6. 7. 8. 9. i0.
0rnstein, L., Ann. N. Y. Acad. Sci. 121, 321 (1964). Davis, B. J., Ann. N. Y. Aead. Sei. 121, 404 (1964). Chrambach, A., Reisfeld, R. A., Wyckoff, M., and Zaccari, J., Anal. Biochem. 20, 150 (1967). Fazekas de St. Groth, S., Webster, R. G., and Datyner, A., Bioehim. Biophys Acta, 71, 377 (1963). Sehnaitman, C. A., Proc. Nat'l Acad. Sci. (U.S.) 63, 412 (1969). Weber, K. and Osborn, M., J. Biol. Chem. 244, 440~--(1969). Fessenden-Raden, J. M., J. Biol. Chem. (in press). Hedriek, J. L., and Smith, A. J., Arch. Biochem. Biophys. 126 , 155 (1968). Anderson, M., and McCarty, R., Anal. Biochem. (in press). Horstman, L. L., and Racker, E., J. Biol. Chem. 245, 1336 (1970).
1353
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECT OF FATTY ACIDS ON THE MOVEMENT AND STAINING OF MEMBRANE PROTEINS IN POLYACRYIAMIDE GEL ELECTROPHORESIS
June Mo Fessenden-Raden Section of Biochemistry and Molecular Biology Division of Biological Seiences~ Cornell University Ithaca 3 New York 14850
R e c e i v e d J a n u a r y 3, 1972 Summary The presence of fatty acids in preparation of membrane proteins can cause a change in the apparent mobility and staining characteristics of the proteins as well as induce the appearance of multiple bands in polyacrylamide gel electrophoresis.
Ever since it was first described by Ornstein (I) and Davis (2) polyacrylsmide gel electrophoresis has become a much-used tool in biochemistry. The use of Coomassie brilliant blue in trichloroacetic acid (3) to fix and visualize proteins rather than other stains such as amido black has been extensively employed because of its sensitivity (3, 4).
It is known, however~
that proteins vary in their ability to interact with Coomassie brilliant blue (5)-
This study was prompted by the observation that at equivalent protein
concentrations a coupling factor 6 preparation devoid of fatty acids did not stain as intensely with C0omassie brilliant blue as a preparation with fatty acids and exhibited less bands.
Methods Polyacrylamide gel electrophoresis was run as described by Davis (2) with or without a stacking gel.
The gels were fixed and stained ~ith Coomassie
1347 Copyright © 1972, by Academic Press, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
brilliant blue R in i0~ trichloroacetic acid as described by Chrambach~ et al (3).
Polyacrylamide gel e!eetrophoresis in the presence of sodium dodecyl-
sulfate (SDS) was carried out as described (6). Densitometer traces were ob~ tained at 560 m~ on a recording Gilford spectrophotometer with a linear transport for gel scanning.
Stained gels were photographed by transmitted light
with a Polaroid MP3XL camera using TYPE 57 film and an 25A filter.
Results and Discussion With Coomassie brilliant blue as the protein stain~ polyacrylamide gel electrophoresis of a purified preparation of the heat-stable coupling factor 6 (F6) from bovine heart mitochondria (7) revealed several bands (Fig. IA).
0.5 A
F6
0.4 0.3
dye
02
E
fronl
0
0 ~0
B. Extracted F6 0.2
I'<:[ Ld OJ (..) Z
0
0 0.6
.~ 0.5
C. E x t r a c t e d F6 + Oleic A c i d
0.4
0,5
0.2
0.1
Fig. 1. Densitometer traces of various F~ preparations. A) F~, B) Fg ext - ~ t e d with iso-octane to remove fatty a~ids, C) extracted F6~plus lOVnmoles of oleic acid (30 ~g of protein) was applied per gel. Electrophoresis (anode at the right), staining~ and recording were carried out as described under "Methods."
1348
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Electrophoresis of F 6 on gels prepared at different acrylamide concentration as described by Hedrick and Smith (8) indicated that the multiple bands of F 6 represented proteins of similar size but varying in charge.
SDS gel electro-
phoresis, how~ver~ also revealed multiple bands. It wag subsequently discovered that preparations of F 6 contained widely varying amounts of fatty acids.
After extraction with iso-octane, F 6 gave a
negative rhodamine 6G test for fatty acids (9), yet was still fully functional as a coupling factor in oxidative phosphorylation.
When this extracted prep~-
ration of F 6 was analyzed by gel electrophoresis it wag discovered that a) there was only a single band; and b) the band did not stain well with Coomassie brilliant blue.
(Compare Fig. IA and 1B).
to F 6 prior to electrophoresis~
When fatty acids were added back
the pattern seen in Fig. IC was obtained.
Fig. 2. Electrophoretograms of various fatty acids. A) stearic, B) oleic, ~-~idic, D ) l i n o l e i % E) blank. Each fatty acid wag applied in excess (50 nmoles) to assure enough density in the bands for photographing. Electrophoresis (anode at the bottom), staining and photography were carried out as described uader "Methods."
1349
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Both the number of bands and their stain intensity increased.
Fig. iC shows
F 6 with oleic acid but several other fatty acids tested revealed
a) an in-
crease in the staining capacity of the major band; b) the appearance of either new distinct bands or of a diffuse area of stain; and c) a consistent decrease in the mobility of the major band. When purified fatty acids were electrophoresed in the absence of any protein they could be visualized with Coomassie brilliant blue (Figs. 2 and 3).
Unsaturated cis-fatty acids consistently ran at or closely behind the
dye front (Figs. 2B~ D. 3B) while the trans-fatty acids did not penetrate nearly as far into the gel (Figs. 2C~ BC). much more variable.
The saturated fatty acids were
These differing mobilities would have to be explained in
ter~r~ of differences in shape since stearic aeid~ oleic acid an~ elaidic acid have virtually the same molecular weight and Charge.
0.2
i
A. Stearic Acid
/7
,o%
//
Elaidie acid might be
]
dye/
0.1
o B. Oleic Acid E 0.3
c
o
£0 0.2 t-hi O,I (,.) Z
<[
L-
oa o u_)O I.~
1
;. Elaidic Acid
i
0.4 ().3 0.2 OJ 0
Fig. 3. Densitometer traces Of various fatty acids. A) steari% B) olei% C--~ elaidic (!0 nmoles each) were applied. Electrophoresis, staining~ and recording were carried out as described under "Methods."
1350
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
expected to have the lowest mobility since it is least compact~ whereas the cis-fatty acid is the most compact.
Likewis% the absence of a double bond
would allow ~aturated fatty acids to be the most variable in shape. terns shown were obtained without a stacking gel.
All pat-
However~ the presence of a
stacking gel did not change the patterns obtained either with the fatty acids alone or proteins plus fatty acids.
The stacking gel did reduce somewhat the
smearing often encountered with some fatty acids (See Fig. 3A). The most dramatic effect of a fatty acid on a protein was seen with Fz (i0).
This purified protein from bovine heart mitochondria showed a single
major band on gel electrophoresis (Fig. 4A).
The addition of oleic acid to Fi
caused a decrease in this major band and the appearance of several faster
1.6 A. L4
o2 )P
dye front $
Ol J £ c O~ 0c.D 0.4 t~
I---
A B. F I + 5 nmoles
OleicAcid ~
~
1
0.5 W <) Z 0.2 G~ OK
0o9 G~
OI 0 0.4 05
C. FI+10nmoles OleicAcid
//l!
0.2
0
Fig. 4. Densitometer traces showing the effect of o!eic acid on the electrophoresis of FI. A) F1 (i ~g)~ B) F1 (i ~g) plus 5 nmoles of oleic acid~ C) Fl (i ~g) plus i0 nmoles of oleic acid. Electrophoresis~ staining~ and recording were carried out as described under "Methods."
1351
Vol. 46, No. 3, 1972
moving bands.
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
(Fig. 4B).
At i0 nmoles of o!eic acid the original band had
disappeared and a gel pattern entirely different from that of the original Fl appeared (Fig. 4C). Preliminary results with SDS gel electrophoresis has shown that h e r %
too~
F 6 freed of fatty acids exhibited fewer bands and did not stain as intensely with Coomassie brilliant blue as the non-extracted F 6 (Fig. 5).
The patterns
did not vary significantly dependent on whether the SDS (final concentration = i~) was added to the protein at 23 ° for 18 hrs., or I00 ° for ! min, or merely added immediately prior to electrophoresis without heating.
It would appear
0.9
A. F6
/\
0,8
dye
0.7 06 06 E c
i
04
0 u3 F<
0.5 0.2
Z < 0.1 m 0 <
0 0.5
B. Extrocted F6
04 0.~ 02.L OI
0
~
. Densitometer traces of various F~preparations on SDS gels. A) F6, extracted with iso-octane to remove fatty acids (30 #g of protein) was applled per gel. Electrophoresis~ staining~ and recording were carried out as described under "Methods."
1352
Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
that the presence of fatty acids may in some manner hinder the interaction between protein and SDS and thus cause multiple bands°
Further studies with
several other proteins are presently being undertaken. Although these dramatic effects have been shown only with membrane proteins, it is likely that any protein that will interact with fatty acids will be influenced in its staining with Coomassie brilliant blue, its charge, and its mobility characteristics.
Acknowledgement s This investigation was supported by grant AM-II715 from the National Institute of Arthritis and Metabolic Diseases and undertaken during the tenure of Career Development Award AM-12614 from the National Institute of Arthritis and Metabolic Diseases. The excellent technical assistance of Mrs. Janice Bruns is gratefully acknowledged.
REFERENCES
i. 2. 3. 4. 5. 6. 7. 8. 9. i0.
0rnstein, L., Ann. N. Y. Acad. Sci. 121, 321 (1964). Davis, B. J., Ann. N. Y. Aead. Sei. 121, 404 (1964). Chrambach, A., Reisfeld, R. A., Wyckoff, M., and Zaccari, J., Anal. Biochem. 20, 150 (1967). Fazekas de St. Groth, S., Webster, R. G., and Datyner, A., Bioehim. Biophys Acta, 71, 377 (1963). Sehnaitman, C. A., Proc. Nat'l Acad. Sci. (U.S.) 63, 412 (1969). Weber, K. and Osborn, M., J. Biol. Chem. 244, 440~--(1969). Fessenden-Raden, J. M., J. Biol. Chem. (in press). Hedriek, J. L., and Smith, A. J., Arch. Biochem. Biophys. 126 , 155 (1968). Anderson, M., and McCarty, R., Anal. Biochem. (in press). Horstman, L. L., and Racker, E., J. Biol. Chem. 245, 1336 (1970).
1353