- Email: [email protected]
The application of high-voltage thin-layer electrophoresis to the separation of peptides, nucleotides, and proteins
ANALYTICAL
BIOCHEMISTRY
18, 72-80 (1967)
The Application of High-Voltage Thin-Layer Electrophoresis to the Separation of Peptides, Nucleotides, and Proteins1 A. M. TOMETSKO” The Diviaims of Biochemists Brookhaven National
AND
N. DELIHAS
and Microbiology, Medical Research Center, Laboratory, Upton, New York 11973
Received July 20, 1966
The migration of molecules during electrophoresis can be markedly influenced by interactions with the supporting media. An example of such an interaction is the adsorption of proteins to cellulose. The adsorption of insulin to Whatman 3MM paper during electrophoresis has been reported (1) and we have observed similar binding of the S-sulfa-B-chain of insulin to Whatman 3MM paper at acidic PH. In the latter case this adsorption was overcome by using an acidic buffer containing 8 M urea (2, 3). Interactions such as these might also be minimized if more inert supporting media were available. The thin-layer technique permits great flexibility in choosing proper supporting media. With thin-layer electrophoresis (TLE) ,s low molecular weight compounds (e.g., amino acids and nucleoside phosphates) have been separated on various supporting media including kieselgel G (4), kieselguhr G (4)) aluminum oxide G (4)) silica gel (5), and cellulose (6, 7). The results of Miiller-Eberhard (8) and Bocci (9) with preparative zone electrophoresis suggest that Pevikon C-870 (PVC), a copolymer of polyvinyl chloride and polyvinyl acetate, might be a favorable supporting medium for TLE of macromolecules. We would like to report the use of cellulose powder and PVC (8, 9) as the supporting medium for the separation of large peptides, nucleotides, and proteins by high-voltage TLE as well as the application of TLE to the quantitative estimation of nucleotides from an RNA digest. 1 This research was supported by the United States Atomic Energy Commission. ‘Present address: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York. *Abbreviations: TLE, thin-layer electrophoresis; TLC, thin-layer chromatography; PVC, Pevikon C470; B-SSO, S-sulfa-B-chain of insulin; A-SSOa, S-sulfo-Achain of insulin; Up, Gp, Ap, and Cp signify uridine, guanosine, adenosine, and cytidine 2’,3’-monophosphates, respectively; RNA, ribonucleic acid. 72
SEPARATION
BY
THIS-LAYER
ELECTROl’HORESIb
73
MATERIALS
All reagents were of analytical grade. Electrophoresis was carried out on a Wieland-Pfleiderer Pherograph (Brinkman Instrument Company). The supporting materials were Whatman cellulose powder CFll (W. D. R. Balston, Ltd.), MN 300 cellulose powder (Macherey and Nagel) , and Pevikon C-870 (Mercer Chemical Corporation). Thin-layer plates were prepared with a spreader obtained from Brinkman Instrument Company. Glucagon and nucleotide markers were purchased from Calbiothem and insulin from Mann Research Laboratories, Inc. Twice-crystallized trypsin and chromatographically homogeneous chymotrypsinogen A were obtained from Worthington Biochemical Corp. The S-sulfo-A- and B-chains of insulin were prepared by oxidative sulfitolysis of natural insulin and were isolated by a new method (3,lO). METHODS
Plate Preparation: Quantities giv’en below were suficient to prepare two plates (20 x S9 cm). In the preparation of thin-layer plates, a
mixture of PVC (90 gm) and water (75 ml) was vigorously agitated for 2 min in a Waring Blendor. The mixture was immediately transferred to the spreader set at 0.5 mm and was quickly applied to two plates (20 X 39 cm). This operation should be carried out swiftly before sedimentation can occur in the spreader. The plates were then air-dried. Following the application of the sample, the plates were wetted by spraying with the appropriate buffer using a Universal Spray Kit. This spraying was continued until the white supporting media became gray. Electrophoresis was carried out for the desired time. When the plates were dried and sprayed with the Pauly reagent (11)) the peptides and proteins appeared as orange or pink spots. In preparing cellulose powder plates, cellulose powder (Whatman CFll or Macherey and Nagel MN-300) (20 gm) was suspended in water (135 ml) and the mixture was vigorously agitated in a Waring Blendor. This mixture was transferred to a spreader and was applied to two plates (20 X 39 cm), giving a cellulose thickness of 0.5 mm. Thx samples were applied to the dry plates, with the exception of nucleotides and KGB digests of RNA, which were applied to wet plates. Nucleotide samples were generally applied in 0.01 to 0.05 ml volumes. \Vhen samples were banded, as much as 0.1 ml/6 cm could be applied to 0.5 mm cellulose without loss of resolution. Following the electrophoresis run the peptide material was developed by spraying with Pauly reagent (11) . whereas nucleotide spots were visible under ultraviolet light,
74
TOMETSKO
AND
DELIHAS
Nucleotides were eluted from cellulose by scraping the material from the plate, adding distilled water (1 ml), mixing for 2 min, and centrifuging out the cellulose powder. Optical densities were read against blanks from adjacent areas on the plate. Electrophoresis Procedure: Following the application of the sample and wetting, the plates were placed in the Pherograph, the wicks and upper glass plate were fixed into position, and the electrodes were then connected. The cooling unit was maintained at -5°C throughout the run to counteract the heating effects. Even at this temperature considerable heating occurred at high voltage (4200V) as seen from the condensation droplets which accumulate on the upper plate. When excessive wetting was avoided, this condensation did not affect the sample migration. During the nucleotide separation using an acetic acid-ammonium hydroxide buffer, some evaporation occurred. Overheating and subsequent plate charring were overcome by decreasing the voltage to 3000 V or lower. The concentration and volume of the sample spotted also influence the resolution. Thus, for peptide material good resolution was obtained if 2-10 ~1 of a 5 mg/ml solution was spotted. With nucleotides it was necessary to overcome background absorbance; consequently, a volume less than 5 ~1 was spotted for the quantitative measurement of nucleotides of the order of 5 fig. RESULTS Electrophoresti of Proteins and Peptides: The interaction of macromolecules with the supporting media during electrophoresis can drastically influence their migration. As shown in Figure 1, when insulin and glucagon were applied to cellulose and electrophoresis was carried out, migration was retarded and the peptide material found in an elongated Pauly-positive spot extending to the origin. However, when similar conditions were applied to these samples on thin-layer plates of PVC both insulin and glucagon showed considerable mobility with very little trailing. Since previous results indicated that the migration of the S-sulfo-Bchain of insulin is influenced by adsorption phenomena, it was of interest to test the mobility of the insulin chains on PVC. As shown in Figure 2, both S-sulfo-A- and B-chains of insulin displayed considerable mobility during electrophoresis on PVC in both acidic and alkaline buffers. Thus, PVC seems to be much more inert as a supporting medium for electrophoresis of proteins and large peptides than cellulose. These promising results then led us to attempt the resolution of a mixture of known components. A mixture of insulin, glucagon, and S-sulfo-B-chain was spotted (Fig. 3). The mixture was resolved into three components, each com-
SEPARATION
INSULIN
PEVIKON
BY
GLUCAGON
870
THIN-LAYER
75
ELECTROPHORESIS
GLUCAGON
INSULIN
CELLULOSE
1. Migration of insulin and glucagon during TLE on PVC and cellulose (MN-?&O). Electrophoretic conditions: 4200V (140 V/cm) ; current, 25 mA; time, 26 mm; buffer, 1M acetic acid. Amount applied: 10 ~1 of a 10 mg/ml solution. Distance migrated on PVC: insulin,15.5cm; glucagon,12.6cm. FIG.
ponent migrating the same dist#ance fro111the origin as the individual components. In order to test the inertness of PVC to larger proteins, chymotrypsin-
ogen A and trypsin were next subjected to electrophoresis on a PVC thin layer. These proteins were chosen since they gave elongated spots extending from the origin with very little resolution when electrophoresis was carried out on thin-layer cellulose (CFll) plates or Whatman 3MM paper. It was found (Fig. 4) that chymotrypsinogen A moved as a single component while trypsin was resolved into two major and one minol components at acidic pH. The electrophoretic heterogeneity of crystalline trypsin has recently been reported (12). Electrophoresis of Nucleotides: The migration of the four major riho-
76
TOMETSKO
A-SSO;
AND
DELIHAS
a-ssoi
A- SSO,
a- so;
+ pH 10.0
pH 2.5
FIQ. 2. Migration of S-sulfo- A and B-chains of insulin during TLE on PVC at acidic and alkaline pH. Electrophoretic conditions: 3300 V (110 V/cm) ; current, 20-25 mA; time, I6 min; buffers, 1 M acetic acid (pH 2.5) and 0.01 M NBHCO, adjusted to pH 10.0 with NKOH. Distance migrated: (at pH 2.5) A-SSO,, -6.0 cm, B-SSOs, 5.8 cm (at pH 10.0) A-SS08, -11 cm, B-SSOs, -4.2 cm.
nucleotides on thin-layer cellulose MN-300 is shown in Figure 5. Although the movement is similar on thin-layer cellulose and Whatman 3MM paper (13)) TLE has several distinct advantages. For example, the separation of nucleotides from a KOH digest of RNA is rapid, requiring approximately one-third as much time as with paper. With TLE, a complete separation can be achieved within 15 min at 3200 V. The low degree of spreading of spots during migration permits the detection of small quantities (24 pg) of nucleotides. Thus, the application of 3 ~1 of Up gave a spot with a 0.7 cm diameter which spreads to a diameter of 1.2 cm after migrating 12 cm. Another advantage of TLE is the low background absorbancy obtained after elution of the nucleotides from the plates with water. For example, the OD/cm” of cellulose (0.5 mm thick) eluted from the plate was 0.010 at 300 rnp, 0.025 at 280 w, 0.030 at 260 m,F,, and 0.035 at 240 rnp.
SEPARATIOS
INSULIN
BY
THIN-LAYER
e-sso;
ELECTROPHORESIS
MIXTURE
77
GLUCAGON
FIG. 3. Resolution of mixture of insulin, glucagon, and B-SSO, by TLE on a PVC thin layer. Electrophoretic conditions: 3400V (110 V/cm) ; current, 18 mA; time, 20 min; buffer, 1 M acetic acid. Amount applied: 4 bl of a solution containing 15 mg insulin, 10 mg glucagon, and 10 mg B-SSO, per ml. Zkstance migrated: insulin, 18 cm ; glucagon, 14.5 cm; and B-SSO,, 9.5 cm.
The electrophoretic mobility of each nucleotide and its recovery from a cellulose MN-300 thin-layer plate is listed in Table 1. When electrophoresis was carried out for 45 min at 2800 V, the nucleotides displayed considerable migration with the fastest moving component. I?,,, migrating 13 cm from the origin. The high recovery of nucleotides following electrophoresis is another advantage of TLE. Thus, when 0.660 OD unit of A, was applied to the origin and subjected to electrophoresis, the recovery following elution was 0.670 OD unit. Similar recoveries were obtained for the ot,her nucleotides. DISCUSSIOK
With high-voltage TLE it is possible to separate and detect minute amounts of chemically different macromolecules in a relatively short
78
TOMETSKO
CHWXRYPSINOSEN
AND
DELIHAS
TRYPSIN
+
FIQ. 4. Migration of chymotrypsinogen A and crystalline trypsin during TLE on a PVC thin layer. Ekctrophoretic conditions: 3300V (110 V/cm); current, 22 mA; time, 21 min; buffer, 1 M acetic acid. Amount applied: 4 pl of a solution containing 20 mg protein per ml. Distance migrated: chymotrypsinogen A, 19 cm; trypsin components, 18.7, 15.2, and 11 cm.
Often the separation and resolution are influenced by interactions between the molecules and the supporting medium. We have overcome adsorption effects by employing Pevikon C-870 as the supporting material rather than cellulose. The interactions of macromolecules with cellulose materials was responsible for decreased mobility of the model compounds used in this study. When the more inert supporting material, PVC, was used instead of cellulose, increased mobility, separation, and resolution of the model compounds was obtained. TLE on a cellulose support is a rapid, accurate method for separating and quantitatively estimating nucleotides. This technique supplements existing methods of estimating small quantities of nucleotides (1447). There is also additional separating capacity if thin-layer chromatography time.
SEPARATIOK
BY
THIN-LAYER
MAFjKERS _ I I I I
79
ELECTROPHORESIS
RNA
DIGEST
,
I
GP
“P
+ pn
3.1
Fra. 5. Separation of nucleotides from a KOH digest of RNA by TLE on cellulose MN-366. Electrophoretic conditions: 2800 V (93 V/cm) ; current. 30-50 mA ; time, 45 min; buffer, 5% acetic acid adjusted to pH 3.1 with NH,OH.
is run in a second dimension. The separation of oligonucleotides from a ribonuclease digest of RNA by two dimensional paper electrophoresia and chromatography (18) can be accomplished using two-dimensional TLE and TLC in approximately one-fifth the time (19). Electrophoretic
Mobility
Distance, cm
RI (rel. UP)
Nucleotide
UP
13.0
GP .kP CP
10.1
5.3 3.5
1.00 0.78 0.41 0.27
TARLE 1 and Recovery of Nucleotides from Cellulose MN-300 % recovery
OD applied0
1.29 1.40 0.618 0.147
zk 0.03 (13) + 0.05 (6) + ,004 (5) + 0.006 (10)
1.34 1.37 0.614 0.144
+ 0.04 (14) fO.O1 (9) f .034 (9) * 0.015 (15)
103.8 98.0 99.4 98.0
n 0.5 mm cellulose; buffer; 5% acetic acid adjusted to pH 3.1 with ammonium hydroxide. Electrophoresis was carried out at 2800 V, 30-60 maA, 45 min. b 0.01 ml of the above samples applied to 0.5 mm cellulose; elution with Hz0 before electrophoresis. Similar recoveries were obtained following electrophoresis. Values in parenthesis represent number of determinations.
80
TOMETSKO
AND
DELIHAS
SUMMARY
The migration of large peptides, nucleotides and proteins during thinlayer electrophoresis has been studied using cellulose and Pevikon C-870 (PVC), a copolymer of polyvinyl chloride and polyvinyl acetate, as the supporting medium. PVC provides an inert support which overcomes the adsorption effects that occur between macromolecules and cellulose. Peptides and proteins of various molecular weights all exhibited good mobility and high resolution when electrophoresis was carried out on PVC thin-layer plates. Nucleotides separate rapidly and with good resolution when cellulose is used as the supporting medium. TLE affords a good method for quantitatively estimating microgram quantities of nucleotides in RNA. ACKNOWLEDGMENTS The authors would like to thank Jean Bertman for technical assistance and Dr. P. G. Katsoyannis and Dr. L. D. Hamilton for their encouragement during this work. REFERENCES 1. BERSON, S. A., AND YALOW, R. S., in “The Hormones” (G. Pincus, K. V. Thimann, and E. B. Astwood, eds.), p. 587. Academic Press, New York, 1964. 2. DIXON, G. H., AND WARDLAW, A. C., Nature 188,721 (1966). 3. KATSOYANNIS, P. G., Brookhaven National Laboratory Symposium on “Structure and Function of Polypeptide Hormones: Insulin,” Am. J. Med. 40, 652 (1966). 4. HONEGGER, C. G., Helv. Chim. Acta 44, 173 (1961). 5. RITSCHARD, W. J., J. Chromatog. 16, 327 (1964). 6. YARON, A., AND SOBER, H. A., Anal. Biochem. 12, 173 (1965). 7. BIELESKI, R. L., Anal. Biochm. 12,230 (1965). 8. MILLER-EBERHARD, H. J., Stand. J. Clin. Lab. Invest. 12, 33 (1960). 9. Bocq V., J. Chromatog. 8, 218 (1962). 10. KATSOYANNIS, P. G., AND TOMETSKO, A. M., unpublished data. 11. PAULY, H., Z. Physiol. Chem. 94,427 (1915). 12. GANROT, P. O., Acta Chem. &and. 20, 175 (1966). 13. MARKEIAM, R., AND SMITH, J. D., Nature 168,406 (1951). 14. KATZ, S., AND COMB, D. G., J. Biol. Chem. 238, 3065 (1963). 15. GEBICKI, J. M., AND FREED, S., Anal. Biochem. 13, 565 (1965). 16. ZAK, B., AND WEINER, C. M., J. Chromatog. 13, 255 (1964). 17. RANDERATH, E., AND RANDERATH, K., And. Biochem. 12,83 (1965). 18. RUSHINZKY, G. W., AND KNIQHT, C. A., Virology 11, 236 (1960)., 19. DELIHAS, N., unpublished data.
BIOCHEMISTRY
18, 72-80 (1967)
The Application of High-Voltage Thin-Layer Electrophoresis to the Separation of Peptides, Nucleotides, and Proteins1 A. M. TOMETSKO” The Diviaims of Biochemists Brookhaven National
AND
N. DELIHAS
and Microbiology, Medical Research Center, Laboratory, Upton, New York 11973
Received July 20, 1966
The migration of molecules during electrophoresis can be markedly influenced by interactions with the supporting media. An example of such an interaction is the adsorption of proteins to cellulose. The adsorption of insulin to Whatman 3MM paper during electrophoresis has been reported (1) and we have observed similar binding of the S-sulfa-B-chain of insulin to Whatman 3MM paper at acidic PH. In the latter case this adsorption was overcome by using an acidic buffer containing 8 M urea (2, 3). Interactions such as these might also be minimized if more inert supporting media were available. The thin-layer technique permits great flexibility in choosing proper supporting media. With thin-layer electrophoresis (TLE) ,s low molecular weight compounds (e.g., amino acids and nucleoside phosphates) have been separated on various supporting media including kieselgel G (4), kieselguhr G (4)) aluminum oxide G (4)) silica gel (5), and cellulose (6, 7). The results of Miiller-Eberhard (8) and Bocci (9) with preparative zone electrophoresis suggest that Pevikon C-870 (PVC), a copolymer of polyvinyl chloride and polyvinyl acetate, might be a favorable supporting medium for TLE of macromolecules. We would like to report the use of cellulose powder and PVC (8, 9) as the supporting medium for the separation of large peptides, nucleotides, and proteins by high-voltage TLE as well as the application of TLE to the quantitative estimation of nucleotides from an RNA digest. 1 This research was supported by the United States Atomic Energy Commission. ‘Present address: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York. *Abbreviations: TLE, thin-layer electrophoresis; TLC, thin-layer chromatography; PVC, Pevikon C470; B-SSO, S-sulfa-B-chain of insulin; A-SSOa, S-sulfo-Achain of insulin; Up, Gp, Ap, and Cp signify uridine, guanosine, adenosine, and cytidine 2’,3’-monophosphates, respectively; RNA, ribonucleic acid. 72
SEPARATION
BY
THIS-LAYER
ELECTROl’HORESIb
73
MATERIALS
All reagents were of analytical grade. Electrophoresis was carried out on a Wieland-Pfleiderer Pherograph (Brinkman Instrument Company). The supporting materials were Whatman cellulose powder CFll (W. D. R. Balston, Ltd.), MN 300 cellulose powder (Macherey and Nagel) , and Pevikon C-870 (Mercer Chemical Corporation). Thin-layer plates were prepared with a spreader obtained from Brinkman Instrument Company. Glucagon and nucleotide markers were purchased from Calbiothem and insulin from Mann Research Laboratories, Inc. Twice-crystallized trypsin and chromatographically homogeneous chymotrypsinogen A were obtained from Worthington Biochemical Corp. The S-sulfo-A- and B-chains of insulin were prepared by oxidative sulfitolysis of natural insulin and were isolated by a new method (3,lO). METHODS
Plate Preparation: Quantities giv’en below were suficient to prepare two plates (20 x S9 cm). In the preparation of thin-layer plates, a
mixture of PVC (90 gm) and water (75 ml) was vigorously agitated for 2 min in a Waring Blendor. The mixture was immediately transferred to the spreader set at 0.5 mm and was quickly applied to two plates (20 X 39 cm). This operation should be carried out swiftly before sedimentation can occur in the spreader. The plates were then air-dried. Following the application of the sample, the plates were wetted by spraying with the appropriate buffer using a Universal Spray Kit. This spraying was continued until the white supporting media became gray. Electrophoresis was carried out for the desired time. When the plates were dried and sprayed with the Pauly reagent (11)) the peptides and proteins appeared as orange or pink spots. In preparing cellulose powder plates, cellulose powder (Whatman CFll or Macherey and Nagel MN-300) (20 gm) was suspended in water (135 ml) and the mixture was vigorously agitated in a Waring Blendor. This mixture was transferred to a spreader and was applied to two plates (20 X 39 cm), giving a cellulose thickness of 0.5 mm. Thx samples were applied to the dry plates, with the exception of nucleotides and KGB digests of RNA, which were applied to wet plates. Nucleotide samples were generally applied in 0.01 to 0.05 ml volumes. \Vhen samples were banded, as much as 0.1 ml/6 cm could be applied to 0.5 mm cellulose without loss of resolution. Following the electrophoresis run the peptide material was developed by spraying with Pauly reagent (11) . whereas nucleotide spots were visible under ultraviolet light,
74
TOMETSKO
AND
DELIHAS
Nucleotides were eluted from cellulose by scraping the material from the plate, adding distilled water (1 ml), mixing for 2 min, and centrifuging out the cellulose powder. Optical densities were read against blanks from adjacent areas on the plate. Electrophoresis Procedure: Following the application of the sample and wetting, the plates were placed in the Pherograph, the wicks and upper glass plate were fixed into position, and the electrodes were then connected. The cooling unit was maintained at -5°C throughout the run to counteract the heating effects. Even at this temperature considerable heating occurred at high voltage (4200V) as seen from the condensation droplets which accumulate on the upper plate. When excessive wetting was avoided, this condensation did not affect the sample migration. During the nucleotide separation using an acetic acid-ammonium hydroxide buffer, some evaporation occurred. Overheating and subsequent plate charring were overcome by decreasing the voltage to 3000 V or lower. The concentration and volume of the sample spotted also influence the resolution. Thus, for peptide material good resolution was obtained if 2-10 ~1 of a 5 mg/ml solution was spotted. With nucleotides it was necessary to overcome background absorbance; consequently, a volume less than 5 ~1 was spotted for the quantitative measurement of nucleotides of the order of 5 fig. RESULTS Electrophoresti of Proteins and Peptides: The interaction of macromolecules with the supporting media during electrophoresis can drastically influence their migration. As shown in Figure 1, when insulin and glucagon were applied to cellulose and electrophoresis was carried out, migration was retarded and the peptide material found in an elongated Pauly-positive spot extending to the origin. However, when similar conditions were applied to these samples on thin-layer plates of PVC both insulin and glucagon showed considerable mobility with very little trailing. Since previous results indicated that the migration of the S-sulfo-Bchain of insulin is influenced by adsorption phenomena, it was of interest to test the mobility of the insulin chains on PVC. As shown in Figure 2, both S-sulfo-A- and B-chains of insulin displayed considerable mobility during electrophoresis on PVC in both acidic and alkaline buffers. Thus, PVC seems to be much more inert as a supporting medium for electrophoresis of proteins and large peptides than cellulose. These promising results then led us to attempt the resolution of a mixture of known components. A mixture of insulin, glucagon, and S-sulfo-B-chain was spotted (Fig. 3). The mixture was resolved into three components, each com-
SEPARATION
INSULIN
PEVIKON
BY
GLUCAGON
870
THIN-LAYER
75
ELECTROPHORESIS
GLUCAGON
INSULIN
CELLULOSE
1. Migration of insulin and glucagon during TLE on PVC and cellulose (MN-?&O). Electrophoretic conditions: 4200V (140 V/cm) ; current, 25 mA; time, 26 mm; buffer, 1M acetic acid. Amount applied: 10 ~1 of a 10 mg/ml solution. Distance migrated on PVC: insulin,15.5cm; glucagon,12.6cm. FIG.
ponent migrating the same dist#ance fro111the origin as the individual components. In order to test the inertness of PVC to larger proteins, chymotrypsin-
ogen A and trypsin were next subjected to electrophoresis on a PVC thin layer. These proteins were chosen since they gave elongated spots extending from the origin with very little resolution when electrophoresis was carried out on thin-layer cellulose (CFll) plates or Whatman 3MM paper. It was found (Fig. 4) that chymotrypsinogen A moved as a single component while trypsin was resolved into two major and one minol components at acidic pH. The electrophoretic heterogeneity of crystalline trypsin has recently been reported (12). Electrophoresis of Nucleotides: The migration of the four major riho-
76
TOMETSKO
A-SSO;
AND
DELIHAS
a-ssoi
A- SSO,
a- so;
+ pH 10.0
pH 2.5
FIQ. 2. Migration of S-sulfo- A and B-chains of insulin during TLE on PVC at acidic and alkaline pH. Electrophoretic conditions: 3300 V (110 V/cm) ; current, 20-25 mA; time, I6 min; buffers, 1 M acetic acid (pH 2.5) and 0.01 M NBHCO, adjusted to pH 10.0 with NKOH. Distance migrated: (at pH 2.5) A-SSO,, -6.0 cm, B-SSOs, 5.8 cm (at pH 10.0) A-SS08, -11 cm, B-SSOs, -4.2 cm.
nucleotides on thin-layer cellulose MN-300 is shown in Figure 5. Although the movement is similar on thin-layer cellulose and Whatman 3MM paper (13)) TLE has several distinct advantages. For example, the separation of nucleotides from a KOH digest of RNA is rapid, requiring approximately one-third as much time as with paper. With TLE, a complete separation can be achieved within 15 min at 3200 V. The low degree of spreading of spots during migration permits the detection of small quantities (24 pg) of nucleotides. Thus, the application of 3 ~1 of Up gave a spot with a 0.7 cm diameter which spreads to a diameter of 1.2 cm after migrating 12 cm. Another advantage of TLE is the low background absorbancy obtained after elution of the nucleotides from the plates with water. For example, the OD/cm” of cellulose (0.5 mm thick) eluted from the plate was 0.010 at 300 rnp, 0.025 at 280 w, 0.030 at 260 m,F,, and 0.035 at 240 rnp.
SEPARATIOS
INSULIN
BY
THIN-LAYER
e-sso;
ELECTROPHORESIS
MIXTURE
77
GLUCAGON
FIG. 3. Resolution of mixture of insulin, glucagon, and B-SSO, by TLE on a PVC thin layer. Electrophoretic conditions: 3400V (110 V/cm) ; current, 18 mA; time, 20 min; buffer, 1 M acetic acid. Amount applied: 4 bl of a solution containing 15 mg insulin, 10 mg glucagon, and 10 mg B-SSO, per ml. Zkstance migrated: insulin, 18 cm ; glucagon, 14.5 cm; and B-SSO,, 9.5 cm.
The electrophoretic mobility of each nucleotide and its recovery from a cellulose MN-300 thin-layer plate is listed in Table 1. When electrophoresis was carried out for 45 min at 2800 V, the nucleotides displayed considerable migration with the fastest moving component. I?,,, migrating 13 cm from the origin. The high recovery of nucleotides following electrophoresis is another advantage of TLE. Thus, when 0.660 OD unit of A, was applied to the origin and subjected to electrophoresis, the recovery following elution was 0.670 OD unit. Similar recoveries were obtained for the ot,her nucleotides. DISCUSSIOK
With high-voltage TLE it is possible to separate and detect minute amounts of chemically different macromolecules in a relatively short
78
TOMETSKO
CHWXRYPSINOSEN
AND
DELIHAS
TRYPSIN
+
FIQ. 4. Migration of chymotrypsinogen A and crystalline trypsin during TLE on a PVC thin layer. Ekctrophoretic conditions: 3300V (110 V/cm); current, 22 mA; time, 21 min; buffer, 1 M acetic acid. Amount applied: 4 pl of a solution containing 20 mg protein per ml. Distance migrated: chymotrypsinogen A, 19 cm; trypsin components, 18.7, 15.2, and 11 cm.
Often the separation and resolution are influenced by interactions between the molecules and the supporting medium. We have overcome adsorption effects by employing Pevikon C-870 as the supporting material rather than cellulose. The interactions of macromolecules with cellulose materials was responsible for decreased mobility of the model compounds used in this study. When the more inert supporting material, PVC, was used instead of cellulose, increased mobility, separation, and resolution of the model compounds was obtained. TLE on a cellulose support is a rapid, accurate method for separating and quantitatively estimating nucleotides. This technique supplements existing methods of estimating small quantities of nucleotides (1447). There is also additional separating capacity if thin-layer chromatography time.
SEPARATIOK
BY
THIN-LAYER
MAFjKERS _ I I I I
79
ELECTROPHORESIS
RNA
DIGEST
,
I
GP
“P
+ pn
3.1
Fra. 5. Separation of nucleotides from a KOH digest of RNA by TLE on cellulose MN-366. Electrophoretic conditions: 2800 V (93 V/cm) ; current. 30-50 mA ; time, 45 min; buffer, 5% acetic acid adjusted to pH 3.1 with NH,OH.
is run in a second dimension. The separation of oligonucleotides from a ribonuclease digest of RNA by two dimensional paper electrophoresia and chromatography (18) can be accomplished using two-dimensional TLE and TLC in approximately one-fifth the time (19). Electrophoretic
Mobility
Distance, cm
RI (rel. UP)
Nucleotide
UP
13.0
GP .kP CP
10.1
5.3 3.5
1.00 0.78 0.41 0.27
TARLE 1 and Recovery of Nucleotides from Cellulose MN-300 % recovery
OD applied0
1.29 1.40 0.618 0.147
zk 0.03 (13) + 0.05 (6) + ,004 (5) + 0.006 (10)
1.34 1.37 0.614 0.144
+ 0.04 (14) fO.O1 (9) f .034 (9) * 0.015 (15)
103.8 98.0 99.4 98.0
n 0.5 mm cellulose; buffer; 5% acetic acid adjusted to pH 3.1 with ammonium hydroxide. Electrophoresis was carried out at 2800 V, 30-60 maA, 45 min. b 0.01 ml of the above samples applied to 0.5 mm cellulose; elution with Hz0 before electrophoresis. Similar recoveries were obtained following electrophoresis. Values in parenthesis represent number of determinations.
80
TOMETSKO
AND
DELIHAS
SUMMARY
The migration of large peptides, nucleotides and proteins during thinlayer electrophoresis has been studied using cellulose and Pevikon C-870 (PVC), a copolymer of polyvinyl chloride and polyvinyl acetate, as the supporting medium. PVC provides an inert support which overcomes the adsorption effects that occur between macromolecules and cellulose. Peptides and proteins of various molecular weights all exhibited good mobility and high resolution when electrophoresis was carried out on PVC thin-layer plates. Nucleotides separate rapidly and with good resolution when cellulose is used as the supporting medium. TLE affords a good method for quantitatively estimating microgram quantities of nucleotides in RNA. ACKNOWLEDGMENTS The authors would like to thank Jean Bertman for technical assistance and Dr. P. G. Katsoyannis and Dr. L. D. Hamilton for their encouragement during this work. REFERENCES 1. BERSON, S. A., AND YALOW, R. S., in “The Hormones” (G. Pincus, K. V. Thimann, and E. B. Astwood, eds.), p. 587. Academic Press, New York, 1964. 2. DIXON, G. H., AND WARDLAW, A. C., Nature 188,721 (1966). 3. KATSOYANNIS, P. G., Brookhaven National Laboratory Symposium on “Structure and Function of Polypeptide Hormones: Insulin,” Am. J. Med. 40, 652 (1966). 4. HONEGGER, C. G., Helv. Chim. Acta 44, 173 (1961). 5. RITSCHARD, W. J., J. Chromatog. 16, 327 (1964). 6. YARON, A., AND SOBER, H. A., Anal. Biochem. 12, 173 (1965). 7. BIELESKI, R. L., Anal. Biochm. 12,230 (1965). 8. MILLER-EBERHARD, H. J., Stand. J. Clin. Lab. Invest. 12, 33 (1960). 9. Bocq V., J. Chromatog. 8, 218 (1962). 10. KATSOYANNIS, P. G., AND TOMETSKO, A. M., unpublished data. 11. PAULY, H., Z. Physiol. Chem. 94,427 (1915). 12. GANROT, P. O., Acta Chem. &and. 20, 175 (1966). 13. MARKEIAM, R., AND SMITH, J. D., Nature 168,406 (1951). 14. KATZ, S., AND COMB, D. G., J. Biol. Chem. 238, 3065 (1963). 15. GEBICKI, J. M., AND FREED, S., Anal. Biochem. 13, 565 (1965). 16. ZAK, B., AND WEINER, C. M., J. Chromatog. 13, 255 (1964). 17. RANDERATH, E., AND RANDERATH, K., And. Biochem. 12,83 (1965). 18. RUSHINZKY, G. W., AND KNIQHT, C. A., Virology 11, 236 (1960)., 19. DELIHAS, N., unpublished data.