ANALYTICAL
BIOCHEMISTRY
78,
287-294 (1977)
Electrofocusing in Natural pH Gradients by Buffers: Gradient Modification
Formed
Natural pH gradients formed in buffers (1) can be shifted in slope or in parallel along the pH scale by addition or substitution of buffers to obtain, for each particular fractionation problem, a gradient in which the species of interest occupies the center position. Similar to the natural pH gradients formed in Ampholine (2). the gradients formed in buffer decay concomitantly with a cathodic drift and progressive acidification of the pH gradient from the anodic end (3). Reproducibility of the pH gradient in buffer electrofocusing was found to be adequate. The stability of pH gradients with time is not affected by addition of 10 amine (basic) buffers to the standard mixture of IO amphoteric buffers.
Buffer electrofocusing (BEF) has recently been introduced (1) as an alternative to isoelectric focusing in pH gradients formed by Ampholine (2). In both cases, the pH gradient is formed “naturally” (2), i.e., by the electric field rather than by use of a gradient maker. While, at this time, Ampholine pH gradients made of 50-500 amphoteric constituents (43) excel in linearity of pH gradient over those made from as few as 10 buffers, the buffer gradients appear to have advantages in temporal stability when made at high (0.1 M) concentration, in two-dimensional electrofocusing-PAGE experiments, and in the ease of staining in addition to economic advantages. In principle, they also appear to have advantages in flexibility of gradient design to suit the particular requirements of a fractionation. This report will provide evidence for this concept. Another theoretically important property of buffer pH gradients was that they could be made, although with imperfect linearity and possibly with slight deviations from monotonic pH variation [Fig. 9 of Ref. (6)], from nonamphoteric buffers (1). It appeared of interest to us to investigate whether improved temporal stability or linearity can be obtained for pH gradients formed by a mixture of amphoteric and nonamphoteric buffers. The initial study (1) has shown that the BEF gradients decay in a similar fashion to pH gradients established by Ampholine, with progressive acidification of the gradient during gradient decay, thus ruling out Ampholine-specific reactions as the possible causes of gradient instability. This study will show further similarity between Ampholine and buffer gradients in the cathodic drift of protein zones. MATERIALS
AND METHODS
1. Proteins. A preparation of calf ovarian cyclic AMP-dependent protein kinase was obtained as previously described (7). 287 Copyright 0 lY77 by Academic Pres,, Inc. All right5 of reproduction in any form reserved.
ISSN tXW3.2697
288
SHORT COMMUNICATIONS
BUFFER MIXTURE NONE BASIC
0 SLICE NUMBER
10
20
30
40
50
TIME lhoursl
FIG. 1. Left panel: pH gradients formed in BEF by a mixture of amphoteric buffers prior to and subsequent to the addition of basic nonamphoteric buffers. Gel concentration = 10% T, 2% Csis; buffer concentration = 0.1 M; catholyte = 0.2 N KOH; anolyte = 0.2 N H,SO,; current = 1 mA per tube; Duration of BEF = 48 hr. Right panel: time curve of the initial rate of decrease of current at constant voltage (200 V) per 6-mm i.d. gel under the conditions listed above.
2. Gel B&U mixtures. (A) The amphoteric (standard) buffer mixture was prepared as previously described (1). The standard gel buffer was modified by: (B) addition, in equal proportions, of the mixture of 10 nonamphoteric amine (basic) buffers prepared as described (1); (C) addition, to a final concentration of 0.1 M, of pyridine (pK, = .5..50), picoline (pK, = 6.21), histidine (pKZ = 6.35), and Bis-(2-hydroxyethyl)-imino-tris(hydroxymethyl) methane (Bistris; pK, = 6.88); and (D) addition of 0.1 M histidine, Bistris, lactic acid (pK = 3.84), and propionic acid (pK = 4.86).’ 3. BEF in Gel Buffers. BEF was carried out in gel buffers A, B, C, and D in polyacrylamide gel of 10% T, 2% CBis. In BEF of the kinase preparation, a gel concentration of 5% T and 15% CDATD was used, and preelectrofocusing (20-hr duration) preceded application of the proteins. Polymerization was carried out at 0-4°C in the Pyrex PAGE apparatus as described (8). Conditions of BEF were similar to those used previously in IFPA (3,9). Duration of BEF ranged from 16 to 186 hr. At various intervals, gel tubes were withdrawn from the apparatus and gels were sliced and allowed to diffuse into 0.5 ml of 0.02 M KC1 overnight in vucuo over NaOH to adsorb CO, prior to pH analysis (3). RESULTS
1. Upward parallel shift of pH gradients in BEF along the pH scale by addition of 10 basic buffers to the standard amphoteric gel buffer mixture. A pH gradient was formed in BEF. A mixture of the standard assortment of amphoteric buffers and 10 basic buffers was used as the gel buf1 Abbreviations: %T, acrylamide + crosslinking agent (g)/lOO ml; Bis. N.N’-methylenebis-acrylamide; %C, crosslinking agent/acrylamide + crosslinking agent (g); DATD, N,N’diallyltartardiamide; pK,. pK of amino group at 0°C.
289
SHORT COMMUNICATIONS
FIG. 2. pH Gradients formed in BEF of a mixture of amphoteric buffers prior to and subsequent to the addition of either basic and neutral or neutral and acidic components. Gel concentration = 10% T, 2% Csis. The conditions and procedure of electrofocusing are listed in Fig. I.
fer. Electrofocusing was carried out for 48 hr. The addition of the basic buffers (a) extended the pH range covered by the “linear” pH gradient from (4.5-5.5) to (6.5-8.5); (b) caused the pH gradient to shift at the cathodic end from 6.5 to 9.5, while, at the anodic end, the pH remained constant at 2.4-3.2; (c) shifted the linear segment of the pH gradient upward by 2-3 pH units; (d) did not improve the linearity of the pH gradient; and (e) caused the current to decrease, as a function of time, at a slower rate than observed with the standard buffers alone (Fig. 1). The addition did not prevent, however, the attainment of a very low final current at the steady-state, suitable for BEF. 2. Similar parallel shift of pH gradients in BEF by addition of t+lvo basic and two neutral buffers to the standard arnphoteric gel buffer
10
20
30
40
50
60
SLICENUMBER
FIG. 3. pH Gradients formed with a mixture of amphoteric and acidic buffers as a function of time of electrofocusing. Conditions of experiment as in Fig. 1. Duration of BEF = 12. 17, 20, 24. 30. and 58 hr, respectively.
SHORT COMMUNICATIONS
290
-.
36
68
92
112
20
20
20
20
16
48
72
92
With Pmtoin
FIG. 4. Cathodic drift of kinase protein bands: gel concentration = 5% T, 15% CDATD; buffer concentration = 0.05 M; duration of BEF = 36, 68, 92, and 112 hr. Conditions of BEF as described in Fig. 1.
291
SHORT COMMUNICATIONS
PH
I
I
I
I
I
10
20
30
40
50
60
SLICE NUMBER
FIG. 5. pH Gradients formed in BEF repetitively with a mixture of amphoteric buffers under a single set of conditions. Duration of BEF = 48 hr; buffer concentration = 0.1 M; current = 1 mA per tube; gel concentration = 5% T, 15% CDATD.Other conditions as in Fig. I.
mixture. The addition of two basic and two neutral buffers to the standard amphoteric buffer mixture (a) extended the pH gradient in the linear range from (4.5-5.5) to (5.8-7.5); (b) shifted the pH gradient at the basic end from 6.5 to 7.8 and maintained it at the acidic end at pH 2.4-2.2; (c) shifted the linear part of the pH gradient by 1.5 to 2 pH units upward; and (d) rendered the pH gradient more nonlinear (Fig. 2). 3. Increase in the slope of pH gradients in BEF by addition of two neutral and/or two acidic buffers to the standard amphoteric gel buffer mixture. The addition of two neutral (histidine and Bistris) and two acidic (propionic and lactic acids) compounds to the standard gel buffer mixture (a) extended the linear range of the pH gradient from (4.5-5.5) to (3.0-7.5); (b) reduced the linearity of the pH gradient; and (c) shifted the pH gradient at the basic end from 6.4 to 7.8 and maintained it at the acidic end at pH 2.4-2.2 (Fig. 2). The addition of two acidic buffers (propionic and lactic acids) alone to the standard buffer mixture had no effect on the pH gradient. 4. pH Gradient instability, progressive acidi$cation, and cathodic drift of protein in BEF. BEF was conducted in gel buffer D for durations up to 112 hr (Figs. 3 and 4) and resulted in (a) instability of the pH gradient with time as previously observed (1); (b) progressive acidification with time starting at the anodic end (Fig. 3); and (c) a progressive cathodic migration of proteins constituting the kinase preparation (Fig. 4). 5. Reproducibility of pH gradients in BEF. BEF was repetitively carried out, at different times, in the standard buffer mixture using a single duration of electrofocusing and otherwise identical conditions. The pH gradients appeared reproducible within 1 pH unit, both with regard to the displacement along the pH axis and the slope of the pH gradient. The extent of the pH range was constant (Fig. 5).
292
SHORT COMMUNICATIONS
DISCUSSION
pH Gradients formed in BEF (1) promised, in addition to economy, increased flexibility in the choice of both the pH range and the slope of pH gradient. BEF also promised advantages of gradient stability, reproducibility, and applicability of fast no-background protein-staining procedures. Also, it appeared of theoretical as well as of practical interest to investigate whether the properties of pH gradient decay observed in IFPA applied to BEF, particularly since deficiency of neutral Ampholine components or specific chemical instability of neutral Ampholine had been postulated as some of the possible causes of pH gradient instability (3). Is it possible to improve the flexibility of electrofocusing by forming pH gradients in buffers? Ampholine preparations provide a very limited number of pH ranges and relatively steep slopes of pH gradient. The possibility of producing stable pH gradients with buffers suggested the capability of generating a very wide variety of pH gradients of any desirable slope, tailored to the needs of any fractionation problem, by use of acidic, neutral, and basic buffers as “pH gradient modifiers”. The original pH gradients in BEF were very flat. They barely covered more than 1 pH unit in the linear range. Although such flat pH gradients may be desirable in some problems requiring maximal resolution, steeper gradients may be required in others.2 pH Gradient “tailoring” may use: (a) for increasing the pH range covered by the pH gradient, a basic or neutral buffer addition. In the representative cases shown in (Figs. 1 and 2), these shifts are of the order of l- 2 pH units per 15 to 50% increase in the number of buffer constituents. (b) For increasing the slope of the linear segment of the pH gradient, addition of neutral and acidic compounds (Fig. 2), not that of basic and acidic compounds alone (at least in the cases tested). The magnitude of the effect is considerable. The slope increases by a factor of 5-6 upon a 15% addition of neutral and acidic buffer components. (c) For vertically displacing a pH gradient along the pH scale while maintaining a constant slope, the addition of basic buffer components, not that of acidic ones is indicated (Figs. 1 and 2). In each case, a 15 to 50% addition in the number of buffer components caused a pH shift covering 2-3 pH units. Are the characteristics of pH gradient instability the same as observed in IFPA? The previous study (1) showed that amphoteric buffer pH gradients, like the pH gradients produced by Ampholine, exhibited decay. This decay was also shown to involve progressive acidification * It does not appear reasonable to attempt making very wide ranges of pH gradients, such as a gradient covering the entire pH scale, by use of buffer mixtures. It appears easier. in this case, to make multicomponent mixtures of carrier ampholytes synthetically (15) or to use commercial wide-range ampholyte mixtures.
SHORT
COMMUNICATIONS
293
of the gel with time, starting at the anodic end. This report also demonstrates a progressive cathodic drift of protein zones in analogy to the Ampholine gradient (3). These data exclude the hypothesis that Amphohne instability during electrofocusing or deficiency of neutral Ampholine components is a possible cause of pH gradient instability. They suggest that the mechanism of electrofocusing conducted with buffers may be similar to that carried out with Ampholine. Practical advantages of pH gradients formed in BEF: The finding that pH gradients formed in BEF exhibit most of the qualities of Ampholine pH gradients, with some advantage of Ampholine with regard to gradient linearity, promises to be of practical value if these gradients are reproducible and relatively stable with time. The previous study (1) had already shown stability with amphoteric buffers. This report confirms stability for the case in which amphoteric buffers and nonamphoteric amines were mixed. It also shows that buffer pH gradients are sufficiently reproducible, especially with regard to the extent of the pH range. This should allow one to carry out protein fractionation in BEF in the same fashion as in IFPA. Staining of proteins in electrofocusing on polyacrylamide gels containing Ampholine has suffered from the fact that, to date, applicable protein procedures also stained the carrier ampholytes. Therefore, it was necessary, in IFPA, to diffuse the carrier ampholytes out of the gels under conditions of protein fixation before staining could be conducted. Alternatively, a stained Ampholine background had to be removed by a separate destaining procedure. Thus, in electrofocusing in Ampholine pH gradients (IFPA), one had the choice of either using a no-background stain with relatively poor sensitivity (10) or a background procedure (11) which was sensitive but required lengthy destaining. BEF is free of such dilemma. It is compatible with the sensitive no-background protein stains used in PAGE (12,13).3 Ampholine also interfered with protein staining and detection of the reference boundary in two-dimensional IFPA-PAGE gels (14). This difficulty is apt to disappear with the application of BEF to two-dimensional gel electrophoresis. It is concluded, from the present study, that stable natural pH gradients formed with buffers provide advantages of flexibility in electrofocusing. They exhibit a sufficient degree of reproducibility to be useful for protein fractionation. They are compatible with fast, no-background staining procedures. The instability of pH gradients in BEF mimics that previously observed in IFPA with regard to rate, progressive acidification, and 3 While this manuscript no-background staining Ampholine appeared.
was in press, a report procedure for isoelectric
[Ref. (16). focusing
procedure B] of a sensitive, in pH gradients formed by
294
SHORT COMMUNICATIONS
cathodic drift of protein zones. The method promises advantages in twodimensional electrophoresis. REFERENCES 1. Nguyen, Y. N., and Chrambach, A. (1976) Anal. Biochem. 74, 145. H. (l%l)Acra Chem. Scud. 15, 325, 456. 3. Baumann, G., and Chrambach, A. (1975) in Progress in Isoelectric Focusing and Isotachophoresis. (Righetti, P. G., ed.), pp. 13-23, Elsevier, Amsterdam. 4. Everaerts, F. M., and Verheggen, Th. P. E. M. (1975) in Progress in Isoelectric Focusing and Isotachophoresis (Righetti. P. G., ed.), pp. 309-326, Elsevier, Amsterdam. 5. Brown, R. K., Lull, J. M., Lowenkron, S., Bagshaw, J. C., and Vinogradov, S. N. (1976) Anal. Biochem. 71, 325. 6. Chidakel,B. E., Nguyen, Y. N., andchrambach, A. (1977)Anal.Biochem. 77,216-225. 7. Salokangas, A., Talmadge, K., Bechtel, E.. Eppenberger, U.. and Chrambach, A. Eur. J. Biochem., in press. 8. Chrambach, A., Jovin, T. M., Svendsen, P. J., and Rodbard, D., in Methods of Protein Separation: A Modern Survey (Catsimpoolas, N., ed.), Vol. 2, pp. 27-144, Plenum Press, New York. 9. Doerr, P., and Chrambach, A. (1971) Anal. Biochem. 42, %. 10. Reisner, A. H., Nemes, P., and Bucholtz, C. (1975)Anal. Biochem. 64, 509. 11. Vesterberg, 0. (1971) Biochim. Biophys. Acta 243, 345. 12. Diezel, W., Kopperschlaeger, G., and Hoffman, E. (1972) Anal. Biochem. 48, 617. 13. Chrambach, A., Reisfeld, R. A., Wyckoff, M., and Zaccari, J. (1967)Anal. Biochem. 20, 150. 14. O’FarrelI, P. H. (1975) J. Biol. Chem. 250, 4007. 15. Vinogradov. S. N., Lowenkron, S., Andomian, M. R., and Bagshaw, J. C. (1973) 2. svensson,
Biochem.
Biophys.
Res. Commun.
54, 50.
16. Vesterberg, O., and Hansen, L. (1976) in Electrofocusing and Electrophoresis (Radola, B. J., and Graesslin, D., eds.) Walter de Gruyter, New York, in press. NGA Y. NGUYEN ANNELI ANDREAS Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20014, and 4Hormone Laboratory, Dept. of Gynecology University Clinic CH 4004 Basel, Switzerland Received May 20. 1976; accepted October 21, 1976
SALOKANGAS~ CHRAMBACH
BIOCHEMISTRY
78,
287-294 (1977)
Electrofocusing in Natural pH Gradients by Buffers: Gradient Modification
Formed
Natural pH gradients formed in buffers (1) can be shifted in slope or in parallel along the pH scale by addition or substitution of buffers to obtain, for each particular fractionation problem, a gradient in which the species of interest occupies the center position. Similar to the natural pH gradients formed in Ampholine (2). the gradients formed in buffer decay concomitantly with a cathodic drift and progressive acidification of the pH gradient from the anodic end (3). Reproducibility of the pH gradient in buffer electrofocusing was found to be adequate. The stability of pH gradients with time is not affected by addition of 10 amine (basic) buffers to the standard mixture of IO amphoteric buffers.
Buffer electrofocusing (BEF) has recently been introduced (1) as an alternative to isoelectric focusing in pH gradients formed by Ampholine (2). In both cases, the pH gradient is formed “naturally” (2), i.e., by the electric field rather than by use of a gradient maker. While, at this time, Ampholine pH gradients made of 50-500 amphoteric constituents (43) excel in linearity of pH gradient over those made from as few as 10 buffers, the buffer gradients appear to have advantages in temporal stability when made at high (0.1 M) concentration, in two-dimensional electrofocusing-PAGE experiments, and in the ease of staining in addition to economic advantages. In principle, they also appear to have advantages in flexibility of gradient design to suit the particular requirements of a fractionation. This report will provide evidence for this concept. Another theoretically important property of buffer pH gradients was that they could be made, although with imperfect linearity and possibly with slight deviations from monotonic pH variation [Fig. 9 of Ref. (6)], from nonamphoteric buffers (1). It appeared of interest to us to investigate whether improved temporal stability or linearity can be obtained for pH gradients formed by a mixture of amphoteric and nonamphoteric buffers. The initial study (1) has shown that the BEF gradients decay in a similar fashion to pH gradients established by Ampholine, with progressive acidification of the gradient during gradient decay, thus ruling out Ampholine-specific reactions as the possible causes of gradient instability. This study will show further similarity between Ampholine and buffer gradients in the cathodic drift of protein zones. MATERIALS
AND METHODS
1. Proteins. A preparation of calf ovarian cyclic AMP-dependent protein kinase was obtained as previously described (7). 287 Copyright 0 lY77 by Academic Pres,, Inc. All right5 of reproduction in any form reserved.
ISSN tXW3.2697
288
SHORT COMMUNICATIONS
BUFFER MIXTURE NONE BASIC
0 SLICE NUMBER
10
20
30
40
50
TIME lhoursl
FIG. 1. Left panel: pH gradients formed in BEF by a mixture of amphoteric buffers prior to and subsequent to the addition of basic nonamphoteric buffers. Gel concentration = 10% T, 2% Csis; buffer concentration = 0.1 M; catholyte = 0.2 N KOH; anolyte = 0.2 N H,SO,; current = 1 mA per tube; Duration of BEF = 48 hr. Right panel: time curve of the initial rate of decrease of current at constant voltage (200 V) per 6-mm i.d. gel under the conditions listed above.
2. Gel B&U mixtures. (A) The amphoteric (standard) buffer mixture was prepared as previously described (1). The standard gel buffer was modified by: (B) addition, in equal proportions, of the mixture of 10 nonamphoteric amine (basic) buffers prepared as described (1); (C) addition, to a final concentration of 0.1 M, of pyridine (pK, = .5..50), picoline (pK, = 6.21), histidine (pKZ = 6.35), and Bis-(2-hydroxyethyl)-imino-tris(hydroxymethyl) methane (Bistris; pK, = 6.88); and (D) addition of 0.1 M histidine, Bistris, lactic acid (pK = 3.84), and propionic acid (pK = 4.86).’ 3. BEF in Gel Buffers. BEF was carried out in gel buffers A, B, C, and D in polyacrylamide gel of 10% T, 2% CBis. In BEF of the kinase preparation, a gel concentration of 5% T and 15% CDATD was used, and preelectrofocusing (20-hr duration) preceded application of the proteins. Polymerization was carried out at 0-4°C in the Pyrex PAGE apparatus as described (8). Conditions of BEF were similar to those used previously in IFPA (3,9). Duration of BEF ranged from 16 to 186 hr. At various intervals, gel tubes were withdrawn from the apparatus and gels were sliced and allowed to diffuse into 0.5 ml of 0.02 M KC1 overnight in vucuo over NaOH to adsorb CO, prior to pH analysis (3). RESULTS
1. Upward parallel shift of pH gradients in BEF along the pH scale by addition of 10 basic buffers to the standard amphoteric gel buffer mixture. A pH gradient was formed in BEF. A mixture of the standard assortment of amphoteric buffers and 10 basic buffers was used as the gel buf1 Abbreviations: %T, acrylamide + crosslinking agent (g)/lOO ml; Bis. N.N’-methylenebis-acrylamide; %C, crosslinking agent/acrylamide + crosslinking agent (g); DATD, N,N’diallyltartardiamide; pK,. pK of amino group at 0°C.
289
SHORT COMMUNICATIONS
FIG. 2. pH Gradients formed in BEF of a mixture of amphoteric buffers prior to and subsequent to the addition of either basic and neutral or neutral and acidic components. Gel concentration = 10% T, 2% Csis. The conditions and procedure of electrofocusing are listed in Fig. I.
fer. Electrofocusing was carried out for 48 hr. The addition of the basic buffers (a) extended the pH range covered by the “linear” pH gradient from (4.5-5.5) to (6.5-8.5); (b) caused the pH gradient to shift at the cathodic end from 6.5 to 9.5, while, at the anodic end, the pH remained constant at 2.4-3.2; (c) shifted the linear segment of the pH gradient upward by 2-3 pH units; (d) did not improve the linearity of the pH gradient; and (e) caused the current to decrease, as a function of time, at a slower rate than observed with the standard buffers alone (Fig. 1). The addition did not prevent, however, the attainment of a very low final current at the steady-state, suitable for BEF. 2. Similar parallel shift of pH gradients in BEF by addition of t+lvo basic and two neutral buffers to the standard arnphoteric gel buffer
10
20
30
40
50
60
SLICENUMBER
FIG. 3. pH Gradients formed with a mixture of amphoteric and acidic buffers as a function of time of electrofocusing. Conditions of experiment as in Fig. 1. Duration of BEF = 12. 17, 20, 24. 30. and 58 hr, respectively.
SHORT COMMUNICATIONS
290
-.
36
68
92
112
20
20
20
20
16
48
72
92
With Pmtoin
FIG. 4. Cathodic drift of kinase protein bands: gel concentration = 5% T, 15% CDATD; buffer concentration = 0.05 M; duration of BEF = 36, 68, 92, and 112 hr. Conditions of BEF as described in Fig. 1.
291
SHORT COMMUNICATIONS
PH
I
I
I
I
I
10
20
30
40
50
60
SLICE NUMBER
FIG. 5. pH Gradients formed in BEF repetitively with a mixture of amphoteric buffers under a single set of conditions. Duration of BEF = 48 hr; buffer concentration = 0.1 M; current = 1 mA per tube; gel concentration = 5% T, 15% CDATD.Other conditions as in Fig. I.
mixture. The addition of two basic and two neutral buffers to the standard amphoteric buffer mixture (a) extended the pH gradient in the linear range from (4.5-5.5) to (5.8-7.5); (b) shifted the pH gradient at the basic end from 6.5 to 7.8 and maintained it at the acidic end at pH 2.4-2.2; (c) shifted the linear part of the pH gradient by 1.5 to 2 pH units upward; and (d) rendered the pH gradient more nonlinear (Fig. 2). 3. Increase in the slope of pH gradients in BEF by addition of two neutral and/or two acidic buffers to the standard amphoteric gel buffer mixture. The addition of two neutral (histidine and Bistris) and two acidic (propionic and lactic acids) compounds to the standard gel buffer mixture (a) extended the linear range of the pH gradient from (4.5-5.5) to (3.0-7.5); (b) reduced the linearity of the pH gradient; and (c) shifted the pH gradient at the basic end from 6.4 to 7.8 and maintained it at the acidic end at pH 2.4-2.2 (Fig. 2). The addition of two acidic buffers (propionic and lactic acids) alone to the standard buffer mixture had no effect on the pH gradient. 4. pH Gradient instability, progressive acidi$cation, and cathodic drift of protein in BEF. BEF was conducted in gel buffer D for durations up to 112 hr (Figs. 3 and 4) and resulted in (a) instability of the pH gradient with time as previously observed (1); (b) progressive acidification with time starting at the anodic end (Fig. 3); and (c) a progressive cathodic migration of proteins constituting the kinase preparation (Fig. 4). 5. Reproducibility of pH gradients in BEF. BEF was repetitively carried out, at different times, in the standard buffer mixture using a single duration of electrofocusing and otherwise identical conditions. The pH gradients appeared reproducible within 1 pH unit, both with regard to the displacement along the pH axis and the slope of the pH gradient. The extent of the pH range was constant (Fig. 5).
292
SHORT COMMUNICATIONS
DISCUSSION
pH Gradients formed in BEF (1) promised, in addition to economy, increased flexibility in the choice of both the pH range and the slope of pH gradient. BEF also promised advantages of gradient stability, reproducibility, and applicability of fast no-background protein-staining procedures. Also, it appeared of theoretical as well as of practical interest to investigate whether the properties of pH gradient decay observed in IFPA applied to BEF, particularly since deficiency of neutral Ampholine components or specific chemical instability of neutral Ampholine had been postulated as some of the possible causes of pH gradient instability (3). Is it possible to improve the flexibility of electrofocusing by forming pH gradients in buffers? Ampholine preparations provide a very limited number of pH ranges and relatively steep slopes of pH gradient. The possibility of producing stable pH gradients with buffers suggested the capability of generating a very wide variety of pH gradients of any desirable slope, tailored to the needs of any fractionation problem, by use of acidic, neutral, and basic buffers as “pH gradient modifiers”. The original pH gradients in BEF were very flat. They barely covered more than 1 pH unit in the linear range. Although such flat pH gradients may be desirable in some problems requiring maximal resolution, steeper gradients may be required in others.2 pH Gradient “tailoring” may use: (a) for increasing the pH range covered by the pH gradient, a basic or neutral buffer addition. In the representative cases shown in (Figs. 1 and 2), these shifts are of the order of l- 2 pH units per 15 to 50% increase in the number of buffer constituents. (b) For increasing the slope of the linear segment of the pH gradient, addition of neutral and acidic compounds (Fig. 2), not that of basic and acidic compounds alone (at least in the cases tested). The magnitude of the effect is considerable. The slope increases by a factor of 5-6 upon a 15% addition of neutral and acidic buffer components. (c) For vertically displacing a pH gradient along the pH scale while maintaining a constant slope, the addition of basic buffer components, not that of acidic ones is indicated (Figs. 1 and 2). In each case, a 15 to 50% addition in the number of buffer components caused a pH shift covering 2-3 pH units. Are the characteristics of pH gradient instability the same as observed in IFPA? The previous study (1) showed that amphoteric buffer pH gradients, like the pH gradients produced by Ampholine, exhibited decay. This decay was also shown to involve progressive acidification * It does not appear reasonable to attempt making very wide ranges of pH gradients, such as a gradient covering the entire pH scale, by use of buffer mixtures. It appears easier. in this case, to make multicomponent mixtures of carrier ampholytes synthetically (15) or to use commercial wide-range ampholyte mixtures.
SHORT
COMMUNICATIONS
293
of the gel with time, starting at the anodic end. This report also demonstrates a progressive cathodic drift of protein zones in analogy to the Ampholine gradient (3). These data exclude the hypothesis that Amphohne instability during electrofocusing or deficiency of neutral Ampholine components is a possible cause of pH gradient instability. They suggest that the mechanism of electrofocusing conducted with buffers may be similar to that carried out with Ampholine. Practical advantages of pH gradients formed in BEF: The finding that pH gradients formed in BEF exhibit most of the qualities of Ampholine pH gradients, with some advantage of Ampholine with regard to gradient linearity, promises to be of practical value if these gradients are reproducible and relatively stable with time. The previous study (1) had already shown stability with amphoteric buffers. This report confirms stability for the case in which amphoteric buffers and nonamphoteric amines were mixed. It also shows that buffer pH gradients are sufficiently reproducible, especially with regard to the extent of the pH range. This should allow one to carry out protein fractionation in BEF in the same fashion as in IFPA. Staining of proteins in electrofocusing on polyacrylamide gels containing Ampholine has suffered from the fact that, to date, applicable protein procedures also stained the carrier ampholytes. Therefore, it was necessary, in IFPA, to diffuse the carrier ampholytes out of the gels under conditions of protein fixation before staining could be conducted. Alternatively, a stained Ampholine background had to be removed by a separate destaining procedure. Thus, in electrofocusing in Ampholine pH gradients (IFPA), one had the choice of either using a no-background stain with relatively poor sensitivity (10) or a background procedure (11) which was sensitive but required lengthy destaining. BEF is free of such dilemma. It is compatible with the sensitive no-background protein stains used in PAGE (12,13).3 Ampholine also interfered with protein staining and detection of the reference boundary in two-dimensional IFPA-PAGE gels (14). This difficulty is apt to disappear with the application of BEF to two-dimensional gel electrophoresis. It is concluded, from the present study, that stable natural pH gradients formed with buffers provide advantages of flexibility in electrofocusing. They exhibit a sufficient degree of reproducibility to be useful for protein fractionation. They are compatible with fast, no-background staining procedures. The instability of pH gradients in BEF mimics that previously observed in IFPA with regard to rate, progressive acidification, and 3 While this manuscript no-background staining Ampholine appeared.
was in press, a report procedure for isoelectric
[Ref. (16). focusing
procedure B] of a sensitive, in pH gradients formed by
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SHORT COMMUNICATIONS
cathodic drift of protein zones. The method promises advantages in twodimensional electrophoresis. REFERENCES 1. Nguyen, Y. N., and Chrambach, A. (1976) Anal. Biochem. 74, 145. H. (l%l)Acra Chem. Scud. 15, 325, 456. 3. Baumann, G., and Chrambach, A. (1975) in Progress in Isoelectric Focusing and Isotachophoresis. (Righetti, P. G., ed.), pp. 13-23, Elsevier, Amsterdam. 4. Everaerts, F. M., and Verheggen, Th. P. E. M. (1975) in Progress in Isoelectric Focusing and Isotachophoresis (Righetti. P. G., ed.), pp. 309-326, Elsevier, Amsterdam. 5. Brown, R. K., Lull, J. M., Lowenkron, S., Bagshaw, J. C., and Vinogradov, S. N. (1976) Anal. Biochem. 71, 325. 6. Chidakel,B. E., Nguyen, Y. N., andchrambach, A. (1977)Anal.Biochem. 77,216-225. 7. Salokangas, A., Talmadge, K., Bechtel, E.. Eppenberger, U.. and Chrambach, A. Eur. J. Biochem., in press. 8. Chrambach, A., Jovin, T. M., Svendsen, P. J., and Rodbard, D., in Methods of Protein Separation: A Modern Survey (Catsimpoolas, N., ed.), Vol. 2, pp. 27-144, Plenum Press, New York. 9. Doerr, P., and Chrambach, A. (1971) Anal. Biochem. 42, %. 10. Reisner, A. H., Nemes, P., and Bucholtz, C. (1975)Anal. Biochem. 64, 509. 11. Vesterberg, 0. (1971) Biochim. Biophys. Acta 243, 345. 12. Diezel, W., Kopperschlaeger, G., and Hoffman, E. (1972) Anal. Biochem. 48, 617. 13. Chrambach, A., Reisfeld, R. A., Wyckoff, M., and Zaccari, J. (1967)Anal. Biochem. 20, 150. 14. O’FarrelI, P. H. (1975) J. Biol. Chem. 250, 4007. 15. Vinogradov. S. N., Lowenkron, S., Andomian, M. R., and Bagshaw, J. C. (1973) 2. svensson,
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16. Vesterberg, O., and Hansen, L. (1976) in Electrofocusing and Electrophoresis (Radola, B. J., and Graesslin, D., eds.) Walter de Gruyter, New York, in press. NGA Y. NGUYEN ANNELI ANDREAS Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20014, and 4Hormone Laboratory, Dept. of Gynecology University Clinic CH 4004 Basel, Switzerland Received May 20. 1976; accepted October 21, 1976
SALOKANGAS~ CHRAMBACH
