ANALYTICAL
69, 278-282
BIOCHEMISTRY
SHORT Protein
Staining
(1975)
COMMUNICATIONS and
on the Same
Gel
pH Gradient
Determination
in Isoelectric
Focusing
The apparent isoelectric point of a component focused on polyacrylamide gels is normally estimated by extrapolating a pH gradient determined on one gel to another gel which has been stained for protein in order to locate the position of the component (1). The pH gradient is determined by slicing the gel transversely and reading the pH of the eluate after soaking the segments for l-2 hr in a small amount of degassed water. It is assumed that the gradients in both gels are identical. Alternatively, an antimony microelectrode has been used to measure pH gradients directly in unsectioned gels (2). Similar techniques have been applied to polyacrylamide gel slabs and are reviewed by Vesterberg (3). Righetti and Drysdale (4) have recently reviewed these and other aspects of isoelectric focusing in gels. I report here a very precise method for the determination of a protein “isoelectric point” that can be accomplished with a single gel. The technique is demonstrated with yeast phosphoglycerate kinase and the very low density lipoprotein (VLDL) fraction from human plasma. MATERIALS
AND
METHODS
Phosphoglycerate kinase (EC 2.7.2.3) was prepared in pure form from “active dried yeast” as described (5). VLDL was isolated from the plasma of an individual with Type III hyperlipoproteinemia (6) according to the ultracentrifugation method of Hatch and Lees (7). Ampholine as a 40% (w/v) solution was purchased from LKB-Produkter AB, Bromma Sweden. Focusing was accomplished in polyacrylamide gels polymerized with 0.05% (w/v) ammonium persulfate (8) with an MRA Corp., Boston, Mass., gel isoelectric focusing system, M 137-A. Phosphoglycerate kinase was focused in 4% (w/v) acrylamide gels with 2% (w/v) pH 7-9 ampholine and the VLDL was focused in 3% (w/v) acrylamide gels with 1% (w/v) pH 3-10 ampholine. All gels were made with an acrylamide: bisacrylamide ratio of 38: 1. Under the preparation and polymerization conditions used it was found that the VLDL would not enter the gel if the acrylamide concentration (including the “bis” component) was greater than 3%. In addition, 3% acrylamide gels with 278 Copyright 0 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
279
1% (w/v) ampholine polymerized to a desirable consistency only if the acrylamide and bisacrylamide solutions were mixed just prior to polymerization. All gels were prefocused for 30 min at 4°C and 0.5 mA per tube until a voltage of 400 V was reached; thereafter a constant potential of 400 V was applied. Samples were applied in volumes of 20 ~1 or less in 25% (w/v) sucrose plus 1% (w/v) pH 3-10 ampholine layered under 15 ~1 of 20% sucrose (w/v) plus 8% (w/v) pH 3-10 ampholine. After 16 hr the gels were removed from the apparatus. Certain of these conditions were arbitrary. To stain a complete gel for phosphoglycerate kinase protein the anodal end of the gel was gently forced a few millimeters out of the end of the tube and marked with india ink. The excess ink was washed off and the gel extruded into a 12.5% (w/v) trichloroacetic acid solution. Staining was accomplished with Coomassie Blue R-250 as described by Allen et al. (1974), a procedure that did not require removal of the carrier ampholytes prior to staining. The apparent isoelectric point of phosphoglycerate kinase was determined as follows. Several gels to which approximately 2 pg or more of the enzyme had been added were focused simultaneously. One gel was extruded into a 12.5% (w/v) trichloroacetic acid solution and the position of the precipitated band noted. The other gels were then extruded from their tubes and an area encompassing 1 cm on either side of the position of the enzyme, as estimated from the gel in the trichloroacetic acid, was transversely cut into sections of 3.1 mm each with a razor blade. The pH gradient was determined in the conventional way by placing each section in 0.5 ml of degassed double distilled water for a maximum of 2 hr. The pH of each solution was determined at room temperature on the expanded scale of a Radiometer model 26 pH meter and the eluate was decanted from the gel segment. Each individual segment was stained for protein (10) and the apparent isoelectric point was taken to be the pH of the segment that contained the protein. The stained gel slice was easily recognized if 5-10 pg of protein was applied. Lipoprotein staining of entire gels was accomplished with Sudan Black B as described (11). The apparent isoelectric point of the major VLDL component was determined by the general technique outlined above using Sudan Black B to stain the individual segments. RESULTS
AND
DISCUSSION
Figure 1 shows appropriately stained isoelectric focused gels of phosphoglycerate kinase (left) and VLDL (very low density lipoprotein) (right). Figure 2 shows the pH gradients achieved with 2% pH 7-9 and 1% pH 3- 10 ampholytes under the described conditions. The data for the pH 3-10 ampholytes represent the averages of three gels. The ex-
280
SHORT
COMMUNICATIONS
Fro. 1. Polyacrylamide gel isoelectric focusing of phosphoglycerate kinase on pH range fraction from the plasma of an individual with Type I11 7-9 ampholyte (left) and VLDL hyperlipoproteinemia on pH range 3-10 ampholyte (right)
1
5
IO I5 20 SEGMENT NUMBER
2j
3b
FIG. 2. Open circles are the pH gradient established in 8.4-cm long gels when 1% (w/v) of pH range 3-10 ampholyte was added. Each point of the 3-10 gradient is the average of segments from three different gels: the pH ranges for segments 9 and 22 are indicated and were the two largest, excluding the first three segments. Closed circles are the pH gradient established in 8.4-cm long gels when 2% (w/v) of pH range 7-9 ampholyte was added.
SHORT
COMMUNICATIONS
281
cellent reproducibility of the gradients is exemplified by the standard error in pH of segments 9 and 22 from the three gels (Fig. 2), which compared to other segments is maximal. Note that under the conditions and focusing time used here the gradients achieved with pH range 7-9 and pH range 3-10 ampholytes were 6.2-8.3 and 4.0-8.2, respectively. These conditions yielded linear pH gradients for all the other ranges, save 3-10. No difficulty was found with identifying the stained gel slices providing 5-10 pg of protein was applied. On occasion, the colour may have been distributed between two segments in which case the proportion of colour in each segment was visually estimated and the pH value of each segment was weighted accordingly. The average pH range encompassed by each segment (determined from the data in Fig. 2) was found to be 0.08 for the pH 6.1 to pH 8.2 range gel and 0.18 for the pH 4.1 to pH 8.1 range gel. The apparent isoelectric point of yeast phosphoglycerate kinase determined as described was 7.02 ? 0.07 SD for six determinations. The apparent isoelectric point of the major VLDL component (most anodal) present in the plasma of a Type III hyperlipoproteinemic subject was found to be 5.44. This value is the average from three gel segments with a range from 5.38 to 5.48. The accuracy of these isoelectric points is, at least partially, assured by the fact that the values for hemoglobin when focused in this same system are identical to literature values when focusing is stopped at 12 hr or continued for 24 hr, although this justification cannot be rigorously applied to VLDL. In addition, the current through the gels, as measured by an ammeter was found to reach a minimum well within 12 hr. One source of inaccuracy could be that the pH values of the segments were read at 22°C and focusing was at 5°C although Ui (12) found that the pH of ampholyte solutions did not vary with temperature. The method used to stain the individual segments of the gels in which phosphoglycerate kinase was focused (10) was very insensitive but this could be improved by using the same method that was employed on the entire gel. This latter method (9), although more tedious, is faster. The use of a pH gradient determined on one gel to determine the isoelectric point of a protein focused on another gel will lead to errors since many of the gel staining methods result in either shrinkage or expansion of the gels, and although a correction can be introduced this will undoubtedly lead to inaccuracies and a lack of precision. The technique described in this communication allows a gel on which a pH gradient has been determined to be subsequently stained for protein. A specific enzyme stain could be used in place of the protein staining procedure to locate the component of interest in the presence of
282
SHORT COMMUNICATIONS
other proteins. Thus, it is possible to determine an apparent isoelectric point of a protein from a single isoelectric focused gel. ACKNOWLEDGMENTS This work was financially assisted by the University of Alberta Hospital Special Services and Research Committee. The technical assistance of Mrs. Valerie Bowlen and Mrs. Ann Williams is gratefully acknowledged. Dr. William Godolphin supplied the VLDL for this investigation.
REFERENCES 1. Gainer, H. (1972) Anal. Biochem. 51, 646-650. 2. Beeley, J. A., Stevenson, S. M., and Beeley. J. G. (1972) Biochim. Biophys. Acta 285, 293-300. 3. Vesterberg, 0. (1972) Biochim. Biophys. Acta 257, 11-19. 4. Righetti, P. G. and Drysdale, J. W. (1974) J. Chromatogr. 98 27 1-32 I. 5. Stinson, R. A. (1974) Biochemistry 13, 4523-4529. 6. Fredrickson, D. S. and Levy, R. 1. (1972) in Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds.), 3rd Ed., p. 581, McGraw-Hill, New York. 7. Hatch, F. T. and Lees, R. S. (1968) Advan. Lipid Res. 6, l-68. 8. Righetti, P. and Drysdale, J. W. (1971) Biochim. Biophys. Acta 236, 17-18. 9. Allen, R. C., Russell, A. H., and Talamo, R. C. (1974) Amer. J. C/in. Pathol. 62, 732-739. 10. Malik, N. and Berrie, A. (1972) Anal. Biochem. 49, 173-183. 11. Godolphin, W. J. and Stinson, R. A. (1974) C/in. Chim. Acta 56, 97-103. 12. Ui, N. (1971) Biochim. Biophys. Acta 229, 567-581. ROBERT Department of Pathology Medical Laboratory Science University of Alberta Edmonton, Alberta T6G 2G3 Received August 14, 1974: accepted April 29, 1975
A. STINSON
69, 278-282
BIOCHEMISTRY
SHORT Protein
Staining
(1975)
COMMUNICATIONS and
on the Same
Gel
pH Gradient
Determination
in Isoelectric
Focusing
The apparent isoelectric point of a component focused on polyacrylamide gels is normally estimated by extrapolating a pH gradient determined on one gel to another gel which has been stained for protein in order to locate the position of the component (1). The pH gradient is determined by slicing the gel transversely and reading the pH of the eluate after soaking the segments for l-2 hr in a small amount of degassed water. It is assumed that the gradients in both gels are identical. Alternatively, an antimony microelectrode has been used to measure pH gradients directly in unsectioned gels (2). Similar techniques have been applied to polyacrylamide gel slabs and are reviewed by Vesterberg (3). Righetti and Drysdale (4) have recently reviewed these and other aspects of isoelectric focusing in gels. I report here a very precise method for the determination of a protein “isoelectric point” that can be accomplished with a single gel. The technique is demonstrated with yeast phosphoglycerate kinase and the very low density lipoprotein (VLDL) fraction from human plasma. MATERIALS
AND
METHODS
Phosphoglycerate kinase (EC 2.7.2.3) was prepared in pure form from “active dried yeast” as described (5). VLDL was isolated from the plasma of an individual with Type III hyperlipoproteinemia (6) according to the ultracentrifugation method of Hatch and Lees (7). Ampholine as a 40% (w/v) solution was purchased from LKB-Produkter AB, Bromma Sweden. Focusing was accomplished in polyacrylamide gels polymerized with 0.05% (w/v) ammonium persulfate (8) with an MRA Corp., Boston, Mass., gel isoelectric focusing system, M 137-A. Phosphoglycerate kinase was focused in 4% (w/v) acrylamide gels with 2% (w/v) pH 7-9 ampholine and the VLDL was focused in 3% (w/v) acrylamide gels with 1% (w/v) pH 3-10 ampholine. All gels were made with an acrylamide: bisacrylamide ratio of 38: 1. Under the preparation and polymerization conditions used it was found that the VLDL would not enter the gel if the acrylamide concentration (including the “bis” component) was greater than 3%. In addition, 3% acrylamide gels with 278 Copyright 0 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
279
1% (w/v) ampholine polymerized to a desirable consistency only if the acrylamide and bisacrylamide solutions were mixed just prior to polymerization. All gels were prefocused for 30 min at 4°C and 0.5 mA per tube until a voltage of 400 V was reached; thereafter a constant potential of 400 V was applied. Samples were applied in volumes of 20 ~1 or less in 25% (w/v) sucrose plus 1% (w/v) pH 3-10 ampholine layered under 15 ~1 of 20% sucrose (w/v) plus 8% (w/v) pH 3-10 ampholine. After 16 hr the gels were removed from the apparatus. Certain of these conditions were arbitrary. To stain a complete gel for phosphoglycerate kinase protein the anodal end of the gel was gently forced a few millimeters out of the end of the tube and marked with india ink. The excess ink was washed off and the gel extruded into a 12.5% (w/v) trichloroacetic acid solution. Staining was accomplished with Coomassie Blue R-250 as described by Allen et al. (1974), a procedure that did not require removal of the carrier ampholytes prior to staining. The apparent isoelectric point of phosphoglycerate kinase was determined as follows. Several gels to which approximately 2 pg or more of the enzyme had been added were focused simultaneously. One gel was extruded into a 12.5% (w/v) trichloroacetic acid solution and the position of the precipitated band noted. The other gels were then extruded from their tubes and an area encompassing 1 cm on either side of the position of the enzyme, as estimated from the gel in the trichloroacetic acid, was transversely cut into sections of 3.1 mm each with a razor blade. The pH gradient was determined in the conventional way by placing each section in 0.5 ml of degassed double distilled water for a maximum of 2 hr. The pH of each solution was determined at room temperature on the expanded scale of a Radiometer model 26 pH meter and the eluate was decanted from the gel segment. Each individual segment was stained for protein (10) and the apparent isoelectric point was taken to be the pH of the segment that contained the protein. The stained gel slice was easily recognized if 5-10 pg of protein was applied. Lipoprotein staining of entire gels was accomplished with Sudan Black B as described (11). The apparent isoelectric point of the major VLDL component was determined by the general technique outlined above using Sudan Black B to stain the individual segments. RESULTS
AND
DISCUSSION
Figure 1 shows appropriately stained isoelectric focused gels of phosphoglycerate kinase (left) and VLDL (very low density lipoprotein) (right). Figure 2 shows the pH gradients achieved with 2% pH 7-9 and 1% pH 3- 10 ampholytes under the described conditions. The data for the pH 3-10 ampholytes represent the averages of three gels. The ex-
280
SHORT
COMMUNICATIONS
Fro. 1. Polyacrylamide gel isoelectric focusing of phosphoglycerate kinase on pH range fraction from the plasma of an individual with Type I11 7-9 ampholyte (left) and VLDL hyperlipoproteinemia on pH range 3-10 ampholyte (right)
1
5
IO I5 20 SEGMENT NUMBER
2j
3b
FIG. 2. Open circles are the pH gradient established in 8.4-cm long gels when 1% (w/v) of pH range 3-10 ampholyte was added. Each point of the 3-10 gradient is the average of segments from three different gels: the pH ranges for segments 9 and 22 are indicated and were the two largest, excluding the first three segments. Closed circles are the pH gradient established in 8.4-cm long gels when 2% (w/v) of pH range 7-9 ampholyte was added.
SHORT
COMMUNICATIONS
281
cellent reproducibility of the gradients is exemplified by the standard error in pH of segments 9 and 22 from the three gels (Fig. 2), which compared to other segments is maximal. Note that under the conditions and focusing time used here the gradients achieved with pH range 7-9 and pH range 3-10 ampholytes were 6.2-8.3 and 4.0-8.2, respectively. These conditions yielded linear pH gradients for all the other ranges, save 3-10. No difficulty was found with identifying the stained gel slices providing 5-10 pg of protein was applied. On occasion, the colour may have been distributed between two segments in which case the proportion of colour in each segment was visually estimated and the pH value of each segment was weighted accordingly. The average pH range encompassed by each segment (determined from the data in Fig. 2) was found to be 0.08 for the pH 6.1 to pH 8.2 range gel and 0.18 for the pH 4.1 to pH 8.1 range gel. The apparent isoelectric point of yeast phosphoglycerate kinase determined as described was 7.02 ? 0.07 SD for six determinations. The apparent isoelectric point of the major VLDL component (most anodal) present in the plasma of a Type III hyperlipoproteinemic subject was found to be 5.44. This value is the average from three gel segments with a range from 5.38 to 5.48. The accuracy of these isoelectric points is, at least partially, assured by the fact that the values for hemoglobin when focused in this same system are identical to literature values when focusing is stopped at 12 hr or continued for 24 hr, although this justification cannot be rigorously applied to VLDL. In addition, the current through the gels, as measured by an ammeter was found to reach a minimum well within 12 hr. One source of inaccuracy could be that the pH values of the segments were read at 22°C and focusing was at 5°C although Ui (12) found that the pH of ampholyte solutions did not vary with temperature. The method used to stain the individual segments of the gels in which phosphoglycerate kinase was focused (10) was very insensitive but this could be improved by using the same method that was employed on the entire gel. This latter method (9), although more tedious, is faster. The use of a pH gradient determined on one gel to determine the isoelectric point of a protein focused on another gel will lead to errors since many of the gel staining methods result in either shrinkage or expansion of the gels, and although a correction can be introduced this will undoubtedly lead to inaccuracies and a lack of precision. The technique described in this communication allows a gel on which a pH gradient has been determined to be subsequently stained for protein. A specific enzyme stain could be used in place of the protein staining procedure to locate the component of interest in the presence of
282
SHORT COMMUNICATIONS
other proteins. Thus, it is possible to determine an apparent isoelectric point of a protein from a single isoelectric focused gel. ACKNOWLEDGMENTS This work was financially assisted by the University of Alberta Hospital Special Services and Research Committee. The technical assistance of Mrs. Valerie Bowlen and Mrs. Ann Williams is gratefully acknowledged. Dr. William Godolphin supplied the VLDL for this investigation.
REFERENCES 1. Gainer, H. (1972) Anal. Biochem. 51, 646-650. 2. Beeley, J. A., Stevenson, S. M., and Beeley. J. G. (1972) Biochim. Biophys. Acta 285, 293-300. 3. Vesterberg, 0. (1972) Biochim. Biophys. Acta 257, 11-19. 4. Righetti, P. G. and Drysdale, J. W. (1974) J. Chromatogr. 98 27 1-32 I. 5. Stinson, R. A. (1974) Biochemistry 13, 4523-4529. 6. Fredrickson, D. S. and Levy, R. 1. (1972) in Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds.), 3rd Ed., p. 581, McGraw-Hill, New York. 7. Hatch, F. T. and Lees, R. S. (1968) Advan. Lipid Res. 6, l-68. 8. Righetti, P. and Drysdale, J. W. (1971) Biochim. Biophys. Acta 236, 17-18. 9. Allen, R. C., Russell, A. H., and Talamo, R. C. (1974) Amer. J. C/in. Pathol. 62, 732-739. 10. Malik, N. and Berrie, A. (1972) Anal. Biochem. 49, 173-183. 11. Godolphin, W. J. and Stinson, R. A. (1974) C/in. Chim. Acta 56, 97-103. 12. Ui, N. (1971) Biochim. Biophys. Acta 229, 567-581. ROBERT Department of Pathology Medical Laboratory Science University of Alberta Edmonton, Alberta T6G 2G3 Received August 14, 1974: accepted April 29, 1975
A. STINSON
