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Isoelectric focusing of myosin in dilute polyacrylamide gels
ANALYTICAL
40, 345-350 (1971)
BIOCHEMISTRY
Isoelectric
JAMES AND
Biology
Focusing of Myosin Polyacrylamide Gels
in Dilute
R. FLORINI, ROSARIA P. BRIVIO, BARBARA-ANNE M. BATTELLE
Department
and Biochemistry Syracuse, New
Comtittee, York 13EIO
Syracuse University.
Received August 28, 1970
We recently described a procedure for disc electrophoresis of myosin and myosin derivatives in dilute polyacrylamide gels (1). Although we and others have found the method to be useful, there is often rather substantial precipitation as the protein sample becomes concentrated in a very thin band at the beginning of the electrophoresis; one cannot be certain that there is no selective loss of individual proteins in the sample. Apparently even 9 M urea does not completely prevent aggregation of myosin at the top of the polyacrylamide columns. Isoelectric focusing avoids this difficulty because protein samples are introduced uniformly throughout the gel column and individual proteins reach high concentrations only when they have arrived at regions of the gel corresponding to their isoelectric points. Consequently we have adapted the procedure of Catsimpoolas (2) to the fractionation of myosin in dilute polyacrylamide gels. Although the procedure is reasonably straightforward, we encountered some technical problems in adapting it for use in dilute polyacrylamide; our solutions to these problems may be helpful to other workers. In addition, we wish to call attention to our conclusion that isoelectric focusing is preferable to disc electrophoresis as a method for analysis of small quantities of myosin. METHODS
Acrylamide and bisacrylamide were purchased from Eastman Organic Chemicals and recrystallized from chloroform and acetone, respectively, as described by Loening (3). (We have noted that complete removal of chloroform is essent’ial for successful polymerization of dilute acrylamide solutions.) The sodium salt of anilinonaphthalene sulfonate was purchased from Eastman; it was recrystallized as described by Hartman 345
346
FIBRIN1
ET
AL.
and Udenfriend (4). Bromophenol blue was purchased from Allied Chemicals, and ampholites from LKB. Myosin was prepared from chick skeletal muscle as described previously (1) except that the final purification was on DEAE-Sephadex as described by Richards et al. (5). Heavy meromyosin was prepared by the method of Barany and Oppenheimer (6). Small subunits were separated from myosin by treatment with IX1 and sodium citrate as specified by Gershman and Dreizen (7). Although some variation was required to allow for differences in samples, our procedure is best described in terms of the following standard conditions: For each 10 ml of gel, 1.0 ml of 26% (w/v) acrylamide, 1.0 ml of 1.6% (w/v) bisacrylamide (freshly dissolved), 5 ,ul of TEMED, 0.5 ml of 40% Ampholine ampholytes of the desired pH range, and 4.86 gm of urea were mixed with H,O to give a total volume of 8.9 ml. To this was added 1.0 ml of protein sample (0.5 to 1.25 mg in 9 M urea) followed by 0.1 ml of 10% (w/v) ammonium persulfate. The mixture was mixed, degassed quickly under reduced pressure (water aspirator), and transferred to 90 X 5 mm glass tubes. Polymerization was allowed to proceed in the dark at room temperature for 30 min. This procedure gives 2.6% polyacrylamide gels containing 100 to 250 J*g of protein sample per column, all in 9 M urea. Isoelectric focusing was done in a Canalco model 66 apparatus using a Heathkit model IP-17 power supply. The upper (anode) bath was filled with 2% (v/v) concentrated phosphoric acid and the lower (cathode) bath with 4% (v/v) ethylenediamine. Voltage was adjusted quickly to obtain an initial current of 5 mA per tube; this fell rapidly to 0.5 mA per tube during the first hour. Under our conditions, these current levels were obtained with a voltage between 200 and 250. Focusing was continued for a total of 8 hr except as specified in Fig. 1. Current was recorded by simply connecting a Heath EUW-20 recorder in parallel across the terminals of the milliameter in the power supply. With
this arrangement,
the millivolt
full-scale
setting
of the recorder
corresponded exactly to the current (in milliamperes) causing full-scale deflection of the recorder; it was thus very convenient to read and record rather low current levels. All separations in 9 M urea were carried out at room temperature, and the analyses in 12 M urea was at 50” ; elevation of temperature was required by the limited solubility of urea. Gel columns were removed from the glass tubes as described by Davis (8) and stained with bromophenol blue by the method of Awdeh (9). With this procedure, care must be taken to avoid loss of bands in destaining. Storage of the destained gels in 7% acetic acid slows (but does not completely prevent) fading of the bands. We routinely photo-’
ISOELECTRIC
FOCUSING
OF
347
MYOSIpi
PH8
PH5
Gels containing FIG. 1. Time course of isolectric focusin g in polyacrylamide. approximately 150 pg myosin were prepared and analyzed as described under “Methods.” All samples were started at the same time; columns were removed and stained after focusing for the time indicated.
graph all gels within 12-24 hr of destaining. The ANS procedure of Hartman and Udenfriend (4) can be used for more rapid visualization of protein bands. RESULTS
Figure 1 shows the time course of isoelectric focusing of myosin in Although the current fell to approx9 M urea in 2.6% polyacrylamide. imately 0.5 mA per tube within 1 hr and subsequently remained steady at this level, it is clear that focusing was not complete before 8 hr. No sharper focusing of the heavy band in the middle of the gel was observed when separations were continued as long as 18 hr. It is clear from these results that stabilization of current at a low level is not a valid indication that focusing is complete; apparently the large myosin molecules do not migrate rapidly enough to contribute detectably to current flow. As might be expected, the pH range of the ampholytes included in the gel had some effect on the patterns of bands observed (Fig. 2)
FIG. 2. Isoelectric focusing of myosin and heavy meromyosin over tlvo ampholyte pH ranges. Gels containing 200 gg myosin or 175 ~g heavy meromyosin (HMMJ were analyzed in gels containing ampholytes of the indicated nominal pH ranges.
348
FIDRINI
ET
AL.
Both intact myosin and heavy meromyosin exhibited wider distribution of bands when ampholytes with a narrow nominal pH range were used to establish a more shallow pH gradient. In addition, the broad central band was more widely distributed, suggesting substantial heterogeneity in this portion of the preparation. Measurement of the pH gradient actually obtained in the gels showed that the nominal pH ranges of the ampholytes must be regarded as only a general indication of pH in the gels, as shown in Fig. 3. It should be noted that these data were obtained with a gel from which the protein sample had been omitted; presumably the amphoteric properties of the proteins would further distort the pH gradient. Clearly the isoelectric point of a protein cannot be estimated simply from its position in the acrylamide gel column. PH
a-
10
. 20
GEL SLICE Fm. 3. Actual pH gradient in an a&amide gel column after isoelectric focusing. A gel was prepared by the procedure described in the text, using ampholytes with a nominal pH range of 5-g. No protein sample was added to the mixture. The gel was focused for 8 hr and cooled to -10” in a freezer; the frozen gel was cut into 1.5 mm slices using a block assembled from stainless-steel razor blades with Teflon spacers. Slices were transferred to small test tubes; 2.0 ml degassed deionized HSO was added and the tubes flushed with NE gas and stoppered. After 18 hr, pH’s were measured using a Sargent S-30070-10 combination electrode with a Corning model 7 pH meter.
Figure 4 shows that most of the bands at the lower pH end of the gel are contributed by the small subunits of myosin. It is striking that none of the LiCl-sodium citrate soluble materials overlap the major bands near the center of the gel which presumably contain the large subunits of myosin. Figure 4 also demonstrates that the apparent heterogeneity of the myosin subunits cannot be attributed simply to partial dissociation and reassociation in 9 M urea; repetition of the analyses in
ISOELECTRIC
FOCUSING
OF
MYOSIN
349
FIG. 4. Isoelectric focusing of small subunits of myosin: (A) small subunits of myosin and (B) intact myosin were analyzed in 9 M urea; (C) intact myosin leas analyzed in 1244 urea at 50”
12 M urea at 50” gave results very similar to those obtained at lower urea concentration and temperature. Cystamine-exchanged (10) myosin and myosin reduced with 0.5 mM dithiothreitol also showed similar heterogeneous patterns (at least two bands corresponding to the large subunits). DISCUSSION
In our experience, analysis of myosin preparations by isoelectric focusing in dilute polyacrylamide provides substantial advantages over analysis by disc electrophoresis in urea (1) ; electrophoresis in sodium dodecyl sulfate has also been shown (11) to give excellent results. The most important advantage of isoelectric focusing is the elimination of precipitation as the sample enters the gel. In addition, relatively dilute samples can be analyzed conveniently by this technique. In our opinion, isoelectric focusing and disc electrophoresis in sodium dodecyl sulfate should be regarded as usefully complementary methods for the analysis of myosin preparations; they both allow analyses of small and large subunits of myosin in the same gel column; as shown in Fig. 4, the isoelectric points of the small subunits differ substantially from those of the large subunits. Isoelectric focusing, like disc electrophoresis, can be done only at low ion concentrations, so substantial amounts of urea must be added to keep myosin in solution. Thus these analyses involve separated subunit’s rather than intact myosin. Although one might attribute the apparent heterogeneity of the purified myosin preparations to partial dissociation of various subunit combinations or to aggregation via disulfide bridges, the heterogeneity was not eliminated by the treatment of myosin with
350
FLORINI
ET
AL.
urea concentrations reported to give complete dissociation (12) or by the treatment of myosin with thiol protecting reagents. There are a number of possible technical and theoretical explanations for this heterogeneity, but an exploration of these possiblities is beyond the scope of this simple technical paper. SUMMARY
Isoelectric focusing of myosin in dilute polyacrylamide gels containing 9 M urea offers several advantages over disc electrophoresis of myosin under similar conditions. There is no loss of protein sample by precipitation at the top of the gel, relatively dilute samples can be used, and both small and large subunits can be analyzed on a single gel column. Like disc electrophoresis, isoelectric focusing indicates the presence of substantial heterogeneity in both large and small subunits of myosin. ACKNOWLEDGMENTS This work was supported by grants from the Muscular of America and the American Heart Association.
Dystrophy
Associations
REFERENCES 1. FLORINI,
J. R., AND BFZIVIO,R. P., And. Biochem. 30, 358 (1969). 2. CATSIMPOOLAS, N., Bbchim. Btiphys. Acta 175, 214 (1969). 3. LOENINQ, U. W., B&hem. J. 102,, 251 (1967). 4. HARTMAN, B. K., AND UDENFRIEND, S., Anal. Biochem. 30, 391 (1969). 5. RICHARDS, E. G., CHUNG, C. S., MENZEL, D. B., AND OLC~TT, H. S., Biochemistry
6, 529 (1967). 6. BARYAN, K., AND OPPENHEIMER, H., Nature 213, 626 (1967). 7. GERSHMAN, L. C., AND DREIZEN, P., Bbchemistrg 9, 1977 (1970). 8. DAVIS, B. J., Ann. N. Y. Acad. Sci. ,121, 404 (1964). 9. AWDEH, Z. L., Sci. Took 16, 42 (1969). 10. WEEDS, A. G., AND HARTLEY, B. S., Biochem. J. 107, 531 (1968). 11. PATERSON, B., AND ~OHMAN, R. C., Biochemistry 9, 4094 (1970). 12. SMALL, P. A., HARBINQTON, W. F., AND KIELLEY, W. W., B&him. Biophys. Acta 49, 462 (1961).
40, 345-350 (1971)
BIOCHEMISTRY
Isoelectric
JAMES AND
Biology
Focusing of Myosin Polyacrylamide Gels
in Dilute
R. FLORINI, ROSARIA P. BRIVIO, BARBARA-ANNE M. BATTELLE
Department
and Biochemistry Syracuse, New
Comtittee, York 13EIO
Syracuse University.
Received August 28, 1970
We recently described a procedure for disc electrophoresis of myosin and myosin derivatives in dilute polyacrylamide gels (1). Although we and others have found the method to be useful, there is often rather substantial precipitation as the protein sample becomes concentrated in a very thin band at the beginning of the electrophoresis; one cannot be certain that there is no selective loss of individual proteins in the sample. Apparently even 9 M urea does not completely prevent aggregation of myosin at the top of the polyacrylamide columns. Isoelectric focusing avoids this difficulty because protein samples are introduced uniformly throughout the gel column and individual proteins reach high concentrations only when they have arrived at regions of the gel corresponding to their isoelectric points. Consequently we have adapted the procedure of Catsimpoolas (2) to the fractionation of myosin in dilute polyacrylamide gels. Although the procedure is reasonably straightforward, we encountered some technical problems in adapting it for use in dilute polyacrylamide; our solutions to these problems may be helpful to other workers. In addition, we wish to call attention to our conclusion that isoelectric focusing is preferable to disc electrophoresis as a method for analysis of small quantities of myosin. METHODS
Acrylamide and bisacrylamide were purchased from Eastman Organic Chemicals and recrystallized from chloroform and acetone, respectively, as described by Loening (3). (We have noted that complete removal of chloroform is essent’ial for successful polymerization of dilute acrylamide solutions.) The sodium salt of anilinonaphthalene sulfonate was purchased from Eastman; it was recrystallized as described by Hartman 345
346
FIBRIN1
ET
AL.
and Udenfriend (4). Bromophenol blue was purchased from Allied Chemicals, and ampholites from LKB. Myosin was prepared from chick skeletal muscle as described previously (1) except that the final purification was on DEAE-Sephadex as described by Richards et al. (5). Heavy meromyosin was prepared by the method of Barany and Oppenheimer (6). Small subunits were separated from myosin by treatment with IX1 and sodium citrate as specified by Gershman and Dreizen (7). Although some variation was required to allow for differences in samples, our procedure is best described in terms of the following standard conditions: For each 10 ml of gel, 1.0 ml of 26% (w/v) acrylamide, 1.0 ml of 1.6% (w/v) bisacrylamide (freshly dissolved), 5 ,ul of TEMED, 0.5 ml of 40% Ampholine ampholytes of the desired pH range, and 4.86 gm of urea were mixed with H,O to give a total volume of 8.9 ml. To this was added 1.0 ml of protein sample (0.5 to 1.25 mg in 9 M urea) followed by 0.1 ml of 10% (w/v) ammonium persulfate. The mixture was mixed, degassed quickly under reduced pressure (water aspirator), and transferred to 90 X 5 mm glass tubes. Polymerization was allowed to proceed in the dark at room temperature for 30 min. This procedure gives 2.6% polyacrylamide gels containing 100 to 250 J*g of protein sample per column, all in 9 M urea. Isoelectric focusing was done in a Canalco model 66 apparatus using a Heathkit model IP-17 power supply. The upper (anode) bath was filled with 2% (v/v) concentrated phosphoric acid and the lower (cathode) bath with 4% (v/v) ethylenediamine. Voltage was adjusted quickly to obtain an initial current of 5 mA per tube; this fell rapidly to 0.5 mA per tube during the first hour. Under our conditions, these current levels were obtained with a voltage between 200 and 250. Focusing was continued for a total of 8 hr except as specified in Fig. 1. Current was recorded by simply connecting a Heath EUW-20 recorder in parallel across the terminals of the milliameter in the power supply. With
this arrangement,
the millivolt
full-scale
setting
of the recorder
corresponded exactly to the current (in milliamperes) causing full-scale deflection of the recorder; it was thus very convenient to read and record rather low current levels. All separations in 9 M urea were carried out at room temperature, and the analyses in 12 M urea was at 50” ; elevation of temperature was required by the limited solubility of urea. Gel columns were removed from the glass tubes as described by Davis (8) and stained with bromophenol blue by the method of Awdeh (9). With this procedure, care must be taken to avoid loss of bands in destaining. Storage of the destained gels in 7% acetic acid slows (but does not completely prevent) fading of the bands. We routinely photo-’
ISOELECTRIC
FOCUSING
OF
347
MYOSIpi
PH8
PH5
Gels containing FIG. 1. Time course of isolectric focusin g in polyacrylamide. approximately 150 pg myosin were prepared and analyzed as described under “Methods.” All samples were started at the same time; columns were removed and stained after focusing for the time indicated.
graph all gels within 12-24 hr of destaining. The ANS procedure of Hartman and Udenfriend (4) can be used for more rapid visualization of protein bands. RESULTS
Figure 1 shows the time course of isoelectric focusing of myosin in Although the current fell to approx9 M urea in 2.6% polyacrylamide. imately 0.5 mA per tube within 1 hr and subsequently remained steady at this level, it is clear that focusing was not complete before 8 hr. No sharper focusing of the heavy band in the middle of the gel was observed when separations were continued as long as 18 hr. It is clear from these results that stabilization of current at a low level is not a valid indication that focusing is complete; apparently the large myosin molecules do not migrate rapidly enough to contribute detectably to current flow. As might be expected, the pH range of the ampholytes included in the gel had some effect on the patterns of bands observed (Fig. 2)
FIG. 2. Isoelectric focusing of myosin and heavy meromyosin over tlvo ampholyte pH ranges. Gels containing 200 gg myosin or 175 ~g heavy meromyosin (HMMJ were analyzed in gels containing ampholytes of the indicated nominal pH ranges.
348
FIDRINI
ET
AL.
Both intact myosin and heavy meromyosin exhibited wider distribution of bands when ampholytes with a narrow nominal pH range were used to establish a more shallow pH gradient. In addition, the broad central band was more widely distributed, suggesting substantial heterogeneity in this portion of the preparation. Measurement of the pH gradient actually obtained in the gels showed that the nominal pH ranges of the ampholytes must be regarded as only a general indication of pH in the gels, as shown in Fig. 3. It should be noted that these data were obtained with a gel from which the protein sample had been omitted; presumably the amphoteric properties of the proteins would further distort the pH gradient. Clearly the isoelectric point of a protein cannot be estimated simply from its position in the acrylamide gel column. PH
a-
10
. 20
GEL SLICE Fm. 3. Actual pH gradient in an a&amide gel column after isoelectric focusing. A gel was prepared by the procedure described in the text, using ampholytes with a nominal pH range of 5-g. No protein sample was added to the mixture. The gel was focused for 8 hr and cooled to -10” in a freezer; the frozen gel was cut into 1.5 mm slices using a block assembled from stainless-steel razor blades with Teflon spacers. Slices were transferred to small test tubes; 2.0 ml degassed deionized HSO was added and the tubes flushed with NE gas and stoppered. After 18 hr, pH’s were measured using a Sargent S-30070-10 combination electrode with a Corning model 7 pH meter.
Figure 4 shows that most of the bands at the lower pH end of the gel are contributed by the small subunits of myosin. It is striking that none of the LiCl-sodium citrate soluble materials overlap the major bands near the center of the gel which presumably contain the large subunits of myosin. Figure 4 also demonstrates that the apparent heterogeneity of the myosin subunits cannot be attributed simply to partial dissociation and reassociation in 9 M urea; repetition of the analyses in
ISOELECTRIC
FOCUSING
OF
MYOSIN
349
FIG. 4. Isoelectric focusing of small subunits of myosin: (A) small subunits of myosin and (B) intact myosin were analyzed in 9 M urea; (C) intact myosin leas analyzed in 1244 urea at 50”
12 M urea at 50” gave results very similar to those obtained at lower urea concentration and temperature. Cystamine-exchanged (10) myosin and myosin reduced with 0.5 mM dithiothreitol also showed similar heterogeneous patterns (at least two bands corresponding to the large subunits). DISCUSSION
In our experience, analysis of myosin preparations by isoelectric focusing in dilute polyacrylamide provides substantial advantages over analysis by disc electrophoresis in urea (1) ; electrophoresis in sodium dodecyl sulfate has also been shown (11) to give excellent results. The most important advantage of isoelectric focusing is the elimination of precipitation as the sample enters the gel. In addition, relatively dilute samples can be analyzed conveniently by this technique. In our opinion, isoelectric focusing and disc electrophoresis in sodium dodecyl sulfate should be regarded as usefully complementary methods for the analysis of myosin preparations; they both allow analyses of small and large subunits of myosin in the same gel column; as shown in Fig. 4, the isoelectric points of the small subunits differ substantially from those of the large subunits. Isoelectric focusing, like disc electrophoresis, can be done only at low ion concentrations, so substantial amounts of urea must be added to keep myosin in solution. Thus these analyses involve separated subunit’s rather than intact myosin. Although one might attribute the apparent heterogeneity of the purified myosin preparations to partial dissociation of various subunit combinations or to aggregation via disulfide bridges, the heterogeneity was not eliminated by the treatment of myosin with
350
FLORINI
ET
AL.
urea concentrations reported to give complete dissociation (12) or by the treatment of myosin with thiol protecting reagents. There are a number of possible technical and theoretical explanations for this heterogeneity, but an exploration of these possiblities is beyond the scope of this simple technical paper. SUMMARY
Isoelectric focusing of myosin in dilute polyacrylamide gels containing 9 M urea offers several advantages over disc electrophoresis of myosin under similar conditions. There is no loss of protein sample by precipitation at the top of the gel, relatively dilute samples can be used, and both small and large subunits can be analyzed on a single gel column. Like disc electrophoresis, isoelectric focusing indicates the presence of substantial heterogeneity in both large and small subunits of myosin. ACKNOWLEDGMENTS This work was supported by grants from the Muscular of America and the American Heart Association.
Dystrophy
Associations
REFERENCES 1. FLORINI,
J. R., AND BFZIVIO,R. P., And. Biochem. 30, 358 (1969). 2. CATSIMPOOLAS, N., Bbchim. Btiphys. Acta 175, 214 (1969). 3. LOENINQ, U. W., B&hem. J. 102,, 251 (1967). 4. HARTMAN, B. K., AND UDENFRIEND, S., Anal. Biochem. 30, 391 (1969). 5. RICHARDS, E. G., CHUNG, C. S., MENZEL, D. B., AND OLC~TT, H. S., Biochemistry
6, 529 (1967). 6. BARYAN, K., AND OPPENHEIMER, H., Nature 213, 626 (1967). 7. GERSHMAN, L. C., AND DREIZEN, P., Bbchemistrg 9, 1977 (1970). 8. DAVIS, B. J., Ann. N. Y. Acad. Sci. ,121, 404 (1964). 9. AWDEH, Z. L., Sci. Took 16, 42 (1969). 10. WEEDS, A. G., AND HARTLEY, B. S., Biochem. J. 107, 531 (1968). 11. PATERSON, B., AND ~OHMAN, R. C., Biochemistry 9, 4094 (1970). 12. SMALL, P. A., HARBINQTON, W. F., AND KIELLEY, W. W., B&him. Biophys. Acta 49, 462 (1961).