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Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing
Journal of Biochemical and Biophysical Methods, 6 (1982) 219-227
219
Elsevier Biomedical Press
Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing Pier Giorgio Righetti, Gabriela Tudor and Elisabetta Gianazza Department of Biochemistry, University of Milan, Via Celoria 2, Milan 20133, Italy (Received 27 April 1982) (Accepted 30 April 1982)
Summary When hydrophobic samples, or membrane proteins, are disaggregated in buffers containing detergents (e.g. Nonidet P-40), urea and 2-mercaptoethanol, and applied at the cathodic end of a gel cylinder or slab for isoelectric separation, as routinely performed for two-dimensional techniques, a severe disturbance of the alkaline region of the pH gradient ensues. This phenomenon has been attributed to high protein loads, which supposedly overcome the buffering power of isoelectric carrier ampholytes. On the contrary, in the present study it has been found that this suppression of the alkaline end of the pH gradient is due to 2-mercaptocthanol, which is a buffer with pK 9.5. This compound ionizes at the basic gel end and is driven ¢lectrophorctically along the pH gradient, sweeping away, along its path, the focused carrier ampholytes. Key words: isoelectric focusing; two-dimensional techniques; 2-mercaptoethanol.
Introduction High resolution, two-dimensional (2D) gel electrophoresis procedures have now become standard tools for the analysis of complex protein mixtures [1]. By combining isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulphate (SDS) gel electrophoresis in the second dimension, this procedure (IEF-SDS or ISO-DALT) [2] can resolve over 1000 proteins with isoelectric points (pI) in the pH range 4-7. However, when the pH gradient is extended to higher pH, the few slightly basic proteins which enter the gel are not well resolved and the pH gradient cannot be further extended to include very basic proteins [3]. This unpaired resolution at the cathodic gel end in the IEF dimension has been attributed to the cathodic drift [4,5], which results in a progressive flattening of the pH gradient in the neutral gel region with concomitant loss of proteins and carder ampholytes into the cathodic electrode reservoir. The cathodic drift, which is, among other parameters, a function of the time of IEF analysis and voltage applied, markedly reduces the reproducibility of spot positions in 2D runs, thus complicating the interpretation of serial maps 0165-022X/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
I
220 obtained from tissue proteins and body fluids under different physiological or pathological conditions. Recently, it has been suggested [6] that this flattening of pH gradient at the cathodic gel end is directly proportional to the amount of protein applied in the IEF dimension, indicating that proteins themselves could be responsible for the cathodic drift. We have noticed, however, that in the vast majority of 2D runs the sample is usually denatured in a lysis buffer containing 9.5 M urea, 2% Nonidet P-40 (NP-40) and 5% 2-mercaptoethanol (2-ME) and applied directly at the cathodic end of the gel cylinder used for the IEF run [ 1,2,7-11]. It is our contention that the cathodic pH gradient flattening is not related to protein load but to the presence of 2-ME in the sample solution. Simple ways to obviate this noxious phenomenon will be presented.
Materials and Methods
Polyacrylamide gels for IEF were prepared according to Refs 4 and 12 to contain 5% T, 4% C, 1.6% Ampholine (LKB Produkter, Bromma, Sweden) pH 3.5-10, 0.2% Ampholine pH 3.5-5, 0.2% Ampholine pH 8-10, 8 M urea, 2% NP-40 and 1 mg/ml each of Asp, Lys and Arg. The gels were polymerized either as rods (4 mm diam., 90 mm h) or as thin slabs (0.5 X 110 X 110 mm) [13]. The former were run in a vertical apparatus, with the cathode uppermost. After 30 min prefocusing at 250 V, the samples were applied and the run continued for a total of 5000 V. h. The gel slabs were run 2 h at 7 W. After 30 min prefocusing, the samples were applied 2 mm from the electrodic strips on Paratex pads that were removed half an hour before the completion of the run. For the pH gradient readings, segments 5 mm long were eluted in 300/xl of 10 mM KCI. No correction was applied to compensate for the presence of urea. For -SH determination, 50 #1 of the above eluates were diluted with 1 ml of phosphate buffer (200 mEq/l, pH 7.6) and reacted with 20 #1 of 10 mM 5,5'-dithiobis(2-nitrobenzoate) (DTNB) solution [14], pH 7..4412nm w a s read after 2 min with a Cary 219 spectrophotometer (Varian, Palo Alto, Calif.). The protein sample was a mixture of heme-free globin chains, prepared by the acid-acetone procedure [15]. Refractile lines of focused carrier ampholytes in gel slabs were observed and photographed after [16].
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Fig. 1 shows that increasing amounts of protein (between 0.5 and 1.5 mg of globin chains from HbA~ per gel, dissolved in 8 M urea, 2% NP-40, but in the absence of 2 ME) fail to modify the pH gradient course in IEF. No dose-dependent pH shift is seen - either in the alkaline portion of the gradient or at pH close to the protein's pI, even though the range of concentrations used is 1 to 2 orders of magnitude higher than the usual loads under such experimental conditions. However, adding a thioi to the sample solution does modify the shape of the pH gradient. In the experiment of Fig. 2, increasing amounts of 2-ME were applied, as in Fig. 1, above
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Fig. I. Effect of protein load on pH gradients in IEF. 5% T, 4% C gels were cast to contain 8 M urea, 2% NP-40, I mg/ml each of Asp, Lys, Arg and the following Ampholines: !.6% pH 3.5-10, 0.2% pH 3.5-5 and 0.2% pH 8-10. IEF was run in vertical gel tubes according to Ref. 12, for a total of 5000 V.h at 10°C. Anolyte: 20 mM H3PO4; catholyte: 20 mM NaOH. The cathode was uppermost. Sample: 10 mg/ml of human globin chains dissolved in 8 M urea, 2% NP-40 but in the absence of 2-ME. 50, 100 and 150 pl were applied to duplicate gel tubes at the cathodic gel end. Two control gel tubes were loaded with 100 and 150 #1, respectively, of 8 M urea and 2% NP-40. After IEF, 5 mm long gel segments were added with 300 ~1 of 10 mM KCi and the pH read immediately at room temperature.
the cathodic end of the gel rods run in a vertical apparatus. Between pH 4 and 6.8 all the gradients are superimposable, while they diverge above pH 7. The highest pH measured is 10 in the control, 9 in presence of 2.5 #1 of 2 ME, 8.3 with 5/~1 and 7.6 with 10 #!. A pH plateau is evident at the highest concentration (centered around pH 7.4) while a change of slope is apparent with the lower concentrations. In the experiment of Fig. 3, 2-ME was applied both at the anodic and at the cathodic side on a gel slab. Fig. 3A shows the pattern of the focused carrier
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Fig. 2. Effect of 2-ME on pH gradients in IEF. All experimental conditions as in Fig. 1 except that the gels were loaded at the cathodic end with a solution containing 8 M urea, 2% NP-40 and 5% 2-ME but no protein. 50, 100 and 200/~1 of this mixture were applied to duplicate gel tubes, corresponding to 2.5/tl, 5 t~l and 10 pl, respectively of pure, undiluted 2-ME. Notice the devastating effects of the thioi compound on pH gradients.
ampholytes visible as refractive lines under side illumination. No distortion is present in the acidic half of the gel in either track (anodic or cathodic 2-ME); on the contrary the lines at the basic edge are shifted towards the cathode for the width of the cathodic 2-ME application zone. Parallel is the effect on the pH gradient (Fig. 3B): identical courses in the acidic region, plateauing in the alkaline when 2-ME is applied at the cathodic side. The same figure plots the distribution in the gel of 2~ME when run from either side. This is much wider for cathodic than for anodic 2-ME: in the former the thiol is detectable over more than 50% of the slab, down to p H 7.0 where the two pH gradients begin to diverge, while in the latter case 2-ME is distributed over 2.5 cm of gel length, i.e. barely one half the length covered by cathodic 2-ME.
223 O n the basis of the A412nm profile of 2 - M E at the c a t h o d i c gel end, we have c a l c u l a t e d its m o l a r i t y d i s t r i b u t i o n along the p H axis (e412n m of 3 - c a r b o x y l a t o - 4 n i t r o t h i o p h e n o l a t e = 13600) a n d its respective buffering p o w e r (Fig. 4). I n t e g r a t i o n o f the a r e a u n d e r the solid circles gives a 2 - M E recovery of ca. 70% as c o m p a r e d with the a m o u n t a p p l i e d to the gel. However, since a b o v e p H 8.3 the a m o u n t of thiol c o m p o u n d , in the assay mixture, was close to a l : l m o l a r ratio with D T N B , which c o u l d lead to an u n d e r e s t i m a t i o n of 2-ME, we have e x t r a p o l a t e d this line to the
Fig. 3. Distribution of 2-ME at the anodic and cathodic ends of gel slabs. All conditions as in Fig. I, except that the gel was polymerized as a thin slab (0.5 X 110X I l0 mm). 7.5 ~l of pure 2-ME were applied soaked in tldn strips of Paratex pads simultaneously at the cathodic and anodic gel ends, over a 3 cm gel width. A. After IEF, the gel slab was photographed against a white cloth with shallow side illumination. Notice the strong distortion of the refractile lines of Ampholine when 2-ME is applied at the cathode,
224
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while anodic 2-ME loading leaves carrier ampholytes totally unperturbed. B. After IEF the slab tracks containing the cathodic and anodic 2-ME samples were cut in 5 ram slices, added with 300 ~l of l0 raM KCI and subjected to pH measurements. 50/~! aliquots of these ehiates were transferred to I ral of 200 raequiv./l phosphate buffer, pH 7.6 and reacted with DTNB (see Materials and Methods). cathodic gel end, assuming a distribution proportional to 2-ME titration curve. In this last case, ca. 90~ recovery of applied 2-ME could be estimated. It can be seen (Fig. 4) that 2-ME has a good buffering capacity (/3, Ref. 17) down to ca. p H 8.
Discussion For all practical purposes, 2-ME is a buffer with a p K 9.5 [18]. Thus, its use in samples applied at the cathodic gel end is simply disastrous to the I E F process. 2-ME ionizes and is driven electrophoretically down the gel tube, until it loses its charge (below p H 8 its charge is too low to ensure a meaningful electrophoretic transport). Thence it is still transferred down the gel tube by diffusional mass transport, so that amounts of it can still be found even at p H 7. Thus, the distribution profile of cathodic 2-ME in Fig. 3B represents probably an equilibrium between electrophoretic and diffusional mass transport of this buffering ion, while it can be assumed that the anodic distribution of 2-ME is simply due to diffusion, since no ionization of -SH groups takes place at this low pH. When running electrophoretic titration curves, we had already hinted at this possible strong interference of 2-ME on p H gradients and protein mobilities (see Fig. 1 in Ref. 19)
225
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Fig. 4. Distribution profile and buffering power (/~) of cathodic 2-ME. From the A412,m data of 2-ME in Fig. 3, the molarity distribution of 2-ME along the pH gradient has been calculated. The corresponding buffering capacity (fl), given a pK of 9.5, has also been plotted. Above pH 8.3, the data have been extrapolated as it was found that DTNB was not any longer in excess in the assay mixture.
but a direct effect had not been previously demonstrated, indeed had been completely overlooked by users of 2D techniques. That proteins, contrary to Ref. 6, could not be responsible for the large suppression of pH gradients in the alkaline regions is also well known from the literature: according to Ref. 20 even l ~ protein concentration (i.e. loads l0 times higher than the highest sample amount we have used) is unable to overcome the buffering capacity of carder ampholytes and thus to modify pH slopes in IEF. On the contrary, cathodic 2-ME can elicit such an effect. According to Davies [21], Gelsema et al. [22] and Fredriksson [23] 2% Ampholine in the pH range 8-9.5 has a buffering capacity of 3-6 /~equiv./ml. In the same pH region, the amount of 2-ME we have used has a buffering power of 0.3-1.5 /~equiv./ml. According to Ref. 24, when a species reaches at least 10~ of the buffering power of surrounding Ampholine, it will alter the pH gradient in IEF. This is in fact what happens by applying 2-ME at the cathode. The net result of this is that alkaline carrier ampholytes are driven away from their pI position and lost in the cathodic chamber. Why this should happen is a more elusive question to answer. According to Ref. 25, distortion of focused zones (and thus pH gradients) happens each time a second pH gradient, perpendicular to the current-generated pH gradient,
226
is produced in the gel matrix. This can be easily understood in a gel slab, due to lateral diffusion of 2-ME, but is not so readily apparent in gel tubes. The remedy to this common malpractice is very simple: 2-ME-containing samples should not be applied at the cathodic, but at the anodic gel end. Alternatively, dithiothreitol should be used instead of 2-ME, since it is effective at concentrations 100 times lower than those commonly employed with 2-ME.
Simplified description of the method and its applications It has been found that 2-mercaptoethanol, commonly added to denatured protein samples, for reduction of -S-S- bridges, has disastrous effects on the alkaline portion of the pH gradient in isoelectric focusing. The basic region of the pH gradient cannot extend above pH 8, thus impairing the resolution of basic proteins. This is not due to the amount of protein loaded, however high it might be, but to the fact that 2-mercaptoethanol is a buffer, with a pK of 9.5. The remedy to it is very simple: samples containing the sulphydryl compound should not be applied at the cathodic, but at the anodic gel end, where 2-mercaptoethanol has no charge or no buffering power. Alternatively, dithiothreitol should be used. By this modification, reproducible pH gradients extending up to pH 10 are routinely obtained. The importance of these findings, for two-dimansional separations, cannot be overemphasized.
Acknowledgements This work was supported in part by grants from Consiglio Nazionale delle Ricerche (CNR) and Ministero della Pubblica Istrtmione (MPI, Rome).
References 10'Farrell, P.M. (1975) J. Biol. Chem. 250, 4007-4021 2 Anderson, N.G. and Anderson, N.L. (1978) Anal. Biochem. 85, 331-340 30'Farreli, P.M. and O'Farrell, P.Z. (1977) in Methods in Cell Biology (Stein, G., Stein, J. and Kleinsmith, L.J., eds.), Vol. 16, pp. 407-420. Academic Press New York 4 Righetti, P.G. and Drysdale, J.W. (1974) J. Chromatogr. 98, 271-321 5 Righetti, P.G. and Drysdale, J.W. (1976) Isoelectric Focusing, pp. 525-526. North Holland/American Elsevier, Amsterdam, New York 6 Cantrell, S.J., Babitch, J.A. and Tones, S. (1981) Anal. Biochem. 116, 168-173 7 Alexander, A., Cullen, B., Emigholz, K., Norgard, M.V. and Monahan, J.J. (1980) Anal. Biochem. 103, 176-183 8 Jltckle, H. (1979) Anal. Biochem. 98, 81-84 9 Voris, B.P. and Young, D.A. (1980) Anal. Biochem. 104, 478-484 10 Vlasuk, G.P. and Walz, F.G. Jr. (1980) Anal. Biochem. 105, 112-120 I1 Dean, B. (1979) Anal. Biochem. 99, 105-111 12 Righetti, P.G. and Drysdale, J.W. (1971) Biochim. Biophys. Acta 236, 17-28 13 Gianazza, E., Chillemi, F., Gelfi, C. and Righetti, P.G. (1979) J. Biochem. Biophys. Methods 1, 237-251 14 Ellman, G.L. (1975) Arch. Biochem. Biophys. 82, 70-76 15 Cle88, J.B., Naughton, M.A. and Weatherall, J.D. (1966) J. Mol. Biol. 19, 91-108 16 Rishetti, P.G., Pagani, ~!. and Giann,,J. E. (1975) J. Chromatogr. 109, 341-356 17 Long, C., ed. (1961) Biochemist's Handbook, Section I, pp. 27-28. Van Nostrand Co., Princeton, N.J.
227 18 Sober, H.A., Harte, R.A. and Sober, E.K. (1970) Handbook of Biochemistry, 2nd Edn., J. 223. Chemical Rubber Co., Cleveland, Ohio 19 Righetti, P.G., Kr/shnamoorthy, R., Lapoumerou]ie, C. and Labie, D. (1979) J. Chromatogr. 177, 219-225 20 Vesterberg, O. (1973) Ann. N.Y. Acad. Sci. 209, 23-32 21 Davies, H. (19"/0) Prot. Biol. Fluids 17, 389-396 22 Gelsema, W.J., De Ligny, C.L. and Van der Veen, N.G. (1979) J. Chromatogr. 1"/3, 33-41 23 Fredriksson, S. (1977) J. Chromatogr. 135, 441-446 24 Freddksson, S. (1977) in Electrofocusing and Isotachophoresis (Radola, BJ. and Graesslin, D., eels.), pp. 71-83. de Gruyter, Berlin 25 Jonsson, M. (1980) Electrophoresis !, 141-149
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Elsevier Biomedical Press
Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing Pier Giorgio Righetti, Gabriela Tudor and Elisabetta Gianazza Department of Biochemistry, University of Milan, Via Celoria 2, Milan 20133, Italy (Received 27 April 1982) (Accepted 30 April 1982)
Summary When hydrophobic samples, or membrane proteins, are disaggregated in buffers containing detergents (e.g. Nonidet P-40), urea and 2-mercaptoethanol, and applied at the cathodic end of a gel cylinder or slab for isoelectric separation, as routinely performed for two-dimensional techniques, a severe disturbance of the alkaline region of the pH gradient ensues. This phenomenon has been attributed to high protein loads, which supposedly overcome the buffering power of isoelectric carrier ampholytes. On the contrary, in the present study it has been found that this suppression of the alkaline end of the pH gradient is due to 2-mercaptocthanol, which is a buffer with pK 9.5. This compound ionizes at the basic gel end and is driven ¢lectrophorctically along the pH gradient, sweeping away, along its path, the focused carrier ampholytes. Key words: isoelectric focusing; two-dimensional techniques; 2-mercaptoethanol.
Introduction High resolution, two-dimensional (2D) gel electrophoresis procedures have now become standard tools for the analysis of complex protein mixtures [1]. By combining isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulphate (SDS) gel electrophoresis in the second dimension, this procedure (IEF-SDS or ISO-DALT) [2] can resolve over 1000 proteins with isoelectric points (pI) in the pH range 4-7. However, when the pH gradient is extended to higher pH, the few slightly basic proteins which enter the gel are not well resolved and the pH gradient cannot be further extended to include very basic proteins [3]. This unpaired resolution at the cathodic gel end in the IEF dimension has been attributed to the cathodic drift [4,5], which results in a progressive flattening of the pH gradient in the neutral gel region with concomitant loss of proteins and carder ampholytes into the cathodic electrode reservoir. The cathodic drift, which is, among other parameters, a function of the time of IEF analysis and voltage applied, markedly reduces the reproducibility of spot positions in 2D runs, thus complicating the interpretation of serial maps 0165-022X/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
I
220 obtained from tissue proteins and body fluids under different physiological or pathological conditions. Recently, it has been suggested [6] that this flattening of pH gradient at the cathodic gel end is directly proportional to the amount of protein applied in the IEF dimension, indicating that proteins themselves could be responsible for the cathodic drift. We have noticed, however, that in the vast majority of 2D runs the sample is usually denatured in a lysis buffer containing 9.5 M urea, 2% Nonidet P-40 (NP-40) and 5% 2-mercaptoethanol (2-ME) and applied directly at the cathodic end of the gel cylinder used for the IEF run [ 1,2,7-11]. It is our contention that the cathodic pH gradient flattening is not related to protein load but to the presence of 2-ME in the sample solution. Simple ways to obviate this noxious phenomenon will be presented.
Materials and Methods
Polyacrylamide gels for IEF were prepared according to Refs 4 and 12 to contain 5% T, 4% C, 1.6% Ampholine (LKB Produkter, Bromma, Sweden) pH 3.5-10, 0.2% Ampholine pH 3.5-5, 0.2% Ampholine pH 8-10, 8 M urea, 2% NP-40 and 1 mg/ml each of Asp, Lys and Arg. The gels were polymerized either as rods (4 mm diam., 90 mm h) or as thin slabs (0.5 X 110 X 110 mm) [13]. The former were run in a vertical apparatus, with the cathode uppermost. After 30 min prefocusing at 250 V, the samples were applied and the run continued for a total of 5000 V. h. The gel slabs were run 2 h at 7 W. After 30 min prefocusing, the samples were applied 2 mm from the electrodic strips on Paratex pads that were removed half an hour before the completion of the run. For the pH gradient readings, segments 5 mm long were eluted in 300/xl of 10 mM KCI. No correction was applied to compensate for the presence of urea. For -SH determination, 50 #1 of the above eluates were diluted with 1 ml of phosphate buffer (200 mEq/l, pH 7.6) and reacted with 20 #1 of 10 mM 5,5'-dithiobis(2-nitrobenzoate) (DTNB) solution [14], pH 7..4412nm w a s read after 2 min with a Cary 219 spectrophotometer (Varian, Palo Alto, Calif.). The protein sample was a mixture of heme-free globin chains, prepared by the acid-acetone procedure [15]. Refractile lines of focused carrier ampholytes in gel slabs were observed and photographed after [16].
R ~
Fig. 1 shows that increasing amounts of protein (between 0.5 and 1.5 mg of globin chains from HbA~ per gel, dissolved in 8 M urea, 2% NP-40, but in the absence of 2 ME) fail to modify the pH gradient course in IEF. No dose-dependent pH shift is seen - either in the alkaline portion of the gradient or at pH close to the protein's pI, even though the range of concentrations used is 1 to 2 orders of magnitude higher than the usual loads under such experimental conditions. However, adding a thioi to the sample solution does modify the shape of the pH gradient. In the experiment of Fig. 2, increasing amounts of 2-ME were applied, as in Fig. 1, above
22t 10.0 0.5 mg 1.0 mg 1.5 mg
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Fig. I. Effect of protein load on pH gradients in IEF. 5% T, 4% C gels were cast to contain 8 M urea, 2% NP-40, I mg/ml each of Asp, Lys, Arg and the following Ampholines: !.6% pH 3.5-10, 0.2% pH 3.5-5 and 0.2% pH 8-10. IEF was run in vertical gel tubes according to Ref. 12, for a total of 5000 V.h at 10°C. Anolyte: 20 mM H3PO4; catholyte: 20 mM NaOH. The cathode was uppermost. Sample: 10 mg/ml of human globin chains dissolved in 8 M urea, 2% NP-40 but in the absence of 2-ME. 50, 100 and 150 pl were applied to duplicate gel tubes at the cathodic gel end. Two control gel tubes were loaded with 100 and 150 #1, respectively, of 8 M urea and 2% NP-40. After IEF, 5 mm long gel segments were added with 300 ~1 of 10 mM KCi and the pH read immediately at room temperature.
the cathodic end of the gel rods run in a vertical apparatus. Between pH 4 and 6.8 all the gradients are superimposable, while they diverge above pH 7. The highest pH measured is 10 in the control, 9 in presence of 2.5 #1 of 2 ME, 8.3 with 5/~1 and 7.6 with 10 #!. A pH plateau is evident at the highest concentration (centered around pH 7.4) while a change of slope is apparent with the lower concentrations. In the experiment of Fig. 3, 2-ME was applied both at the anodic and at the cathodic side on a gel slab. Fig. 3A shows the pattern of the focused carrier
222 10.0-
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Fig. 2. Effect of 2-ME on pH gradients in IEF. All experimental conditions as in Fig. 1 except that the gels were loaded at the cathodic end with a solution containing 8 M urea, 2% NP-40 and 5% 2-ME but no protein. 50, 100 and 200/~1 of this mixture were applied to duplicate gel tubes, corresponding to 2.5/tl, 5 t~l and 10 pl, respectively of pure, undiluted 2-ME. Notice the devastating effects of the thioi compound on pH gradients.
ampholytes visible as refractive lines under side illumination. No distortion is present in the acidic half of the gel in either track (anodic or cathodic 2-ME); on the contrary the lines at the basic edge are shifted towards the cathode for the width of the cathodic 2-ME application zone. Parallel is the effect on the pH gradient (Fig. 3B): identical courses in the acidic region, plateauing in the alkaline when 2-ME is applied at the cathodic side. The same figure plots the distribution in the gel of 2~ME when run from either side. This is much wider for cathodic than for anodic 2-ME: in the former the thiol is detectable over more than 50% of the slab, down to p H 7.0 where the two pH gradients begin to diverge, while in the latter case 2-ME is distributed over 2.5 cm of gel length, i.e. barely one half the length covered by cathodic 2-ME.
223 O n the basis of the A412nm profile of 2 - M E at the c a t h o d i c gel end, we have c a l c u l a t e d its m o l a r i t y d i s t r i b u t i o n along the p H axis (e412n m of 3 - c a r b o x y l a t o - 4 n i t r o t h i o p h e n o l a t e = 13600) a n d its respective buffering p o w e r (Fig. 4). I n t e g r a t i o n o f the a r e a u n d e r the solid circles gives a 2 - M E recovery of ca. 70% as c o m p a r e d with the a m o u n t a p p l i e d to the gel. However, since a b o v e p H 8.3 the a m o u n t of thiol c o m p o u n d , in the assay mixture, was close to a l : l m o l a r ratio with D T N B , which c o u l d lead to an u n d e r e s t i m a t i o n of 2-ME, we have e x t r a p o l a t e d this line to the
Fig. 3. Distribution of 2-ME at the anodic and cathodic ends of gel slabs. All conditions as in Fig. I, except that the gel was polymerized as a thin slab (0.5 X 110X I l0 mm). 7.5 ~l of pure 2-ME were applied soaked in tldn strips of Paratex pads simultaneously at the cathodic and anodic gel ends, over a 3 cm gel width. A. After IEF, the gel slab was photographed against a white cloth with shallow side illumination. Notice the strong distortion of the refractile lines of Ampholine when 2-ME is applied at the cathode,
224
10.0 .,•
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I 15
while anodic 2-ME loading leaves carrier ampholytes totally unperturbed. B. After IEF the slab tracks containing the cathodic and anodic 2-ME samples were cut in 5 ram slices, added with 300 ~l of l0 raM KCI and subjected to pH measurements. 50/~! aliquots of these ehiates were transferred to I ral of 200 raequiv./l phosphate buffer, pH 7.6 and reacted with DTNB (see Materials and Methods). cathodic gel end, assuming a distribution proportional to 2-ME titration curve. In this last case, ca. 90~ recovery of applied 2-ME could be estimated. It can be seen (Fig. 4) that 2-ME has a good buffering capacity (/3, Ref. 17) down to ca. p H 8.
Discussion For all practical purposes, 2-ME is a buffer with a p K 9.5 [18]. Thus, its use in samples applied at the cathodic gel end is simply disastrous to the I E F process. 2-ME ionizes and is driven electrophoretically down the gel tube, until it loses its charge (below p H 8 its charge is too low to ensure a meaningful electrophoretic transport). Thence it is still transferred down the gel tube by diffusional mass transport, so that amounts of it can still be found even at p H 7. Thus, the distribution profile of cathodic 2-ME in Fig. 3B represents probably an equilibrium between electrophoretic and diffusional mass transport of this buffering ion, while it can be assumed that the anodic distribution of 2-ME is simply due to diffusion, since no ionization of -SH groups takes place at this low pH. When running electrophoretic titration curves, we had already hinted at this possible strong interference of 2-ME on p H gradients and protein mobilities (see Fig. 1 in Ref. 19)
225
2 ~J r
.\ \
\
-
1.5
-
1.0 C)
o
U L.
t~a
•
1
I 8.5
I
I 8.0
I
I 7.5
pH
Fig. 4. Distribution profile and buffering power (/~) of cathodic 2-ME. From the A412,m data of 2-ME in Fig. 3, the molarity distribution of 2-ME along the pH gradient has been calculated. The corresponding buffering capacity (fl), given a pK of 9.5, has also been plotted. Above pH 8.3, the data have been extrapolated as it was found that DTNB was not any longer in excess in the assay mixture.
but a direct effect had not been previously demonstrated, indeed had been completely overlooked by users of 2D techniques. That proteins, contrary to Ref. 6, could not be responsible for the large suppression of pH gradients in the alkaline regions is also well known from the literature: according to Ref. 20 even l ~ protein concentration (i.e. loads l0 times higher than the highest sample amount we have used) is unable to overcome the buffering capacity of carder ampholytes and thus to modify pH slopes in IEF. On the contrary, cathodic 2-ME can elicit such an effect. According to Davies [21], Gelsema et al. [22] and Fredriksson [23] 2% Ampholine in the pH range 8-9.5 has a buffering capacity of 3-6 /~equiv./ml. In the same pH region, the amount of 2-ME we have used has a buffering power of 0.3-1.5 /~equiv./ml. According to Ref. 24, when a species reaches at least 10~ of the buffering power of surrounding Ampholine, it will alter the pH gradient in IEF. This is in fact what happens by applying 2-ME at the cathode. The net result of this is that alkaline carrier ampholytes are driven away from their pI position and lost in the cathodic chamber. Why this should happen is a more elusive question to answer. According to Ref. 25, distortion of focused zones (and thus pH gradients) happens each time a second pH gradient, perpendicular to the current-generated pH gradient,
226
is produced in the gel matrix. This can be easily understood in a gel slab, due to lateral diffusion of 2-ME, but is not so readily apparent in gel tubes. The remedy to this common malpractice is very simple: 2-ME-containing samples should not be applied at the cathodic, but at the anodic gel end. Alternatively, dithiothreitol should be used instead of 2-ME, since it is effective at concentrations 100 times lower than those commonly employed with 2-ME.
Simplified description of the method and its applications It has been found that 2-mercaptoethanol, commonly added to denatured protein samples, for reduction of -S-S- bridges, has disastrous effects on the alkaline portion of the pH gradient in isoelectric focusing. The basic region of the pH gradient cannot extend above pH 8, thus impairing the resolution of basic proteins. This is not due to the amount of protein loaded, however high it might be, but to the fact that 2-mercaptoethanol is a buffer, with a pK of 9.5. The remedy to it is very simple: samples containing the sulphydryl compound should not be applied at the cathodic, but at the anodic gel end, where 2-mercaptoethanol has no charge or no buffering power. Alternatively, dithiothreitol should be used. By this modification, reproducible pH gradients extending up to pH 10 are routinely obtained. The importance of these findings, for two-dimansional separations, cannot be overemphasized.
Acknowledgements This work was supported in part by grants from Consiglio Nazionale delle Ricerche (CNR) and Ministero della Pubblica Istrtmione (MPI, Rome).
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