ANALYTICAL

BIOCHEMISTRY

An Anodic

88,

186- 195 (1978)

Drift of pH Gradients in Isoelectric on Polyacrylamide Gel

NGA Y. NGUYEN,

ALICE

G. MCCORMICK,'

Focusing

AND ANDREAS CHRAMBACH

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda. Maryland 20014 Received November 30, 1977; accepted February 17, 1978 pH gradients in isoelectric focusing on polyacrylamide gel (IFPA) are known to be unstable as a function of time and voltage. Under conditions generally used in IFPA, pH gradient decay is accompanied by migration of carrier ampholytes and isoelectric protein zones into the cathode chamber and progressive acidification of the gel starting from the anodic end. A first case of anodic drift of the pH gradient with progressive alkalinization of the gel and migration of isoelectric protein zones and the carrier ampholytes into the anode chamber has been found. It occurs under conditions of IFPA (carrier ampholyte pl range, 6-8) when glycine is the anolyte and arginine is the catholyte (contained in the upper buffer reservoir). The direction of the progressive displacement of pH gradient is reversed to cathodic when (i) the catholyte is positioned below the gel (in the lower buffer reservoir): (ii) tubes are coated by linear polyacrylamide; and (iii) arginine is replaced by lysine. A cathodic drift ensues when arginine is used as the catholyte and IFPA is conducted on a horizontal gel slab; mechanical stress on the gel gives rise to a reversal in the direction of the drift.

pH gradients in isoelectric focusing on polyacrylamide gel (IFPA) are unstable in proportion to time and voltage (1). The instability involves a progressive drift of pH gradient, carrier ampholytes, and protein zones into the cathode chamber (2) in all but one of the reported case? (3,4). The cathodic drift also occurs when Ampholine is replaced by buffers (5) or when strongly acidic and basic electrolytes are replaced by buffer electrolytes (6). Even under conditions where pH gradient stabilization is achieved, the residual drift is in the cathodic direction (7,8). In rare cases, a temporary anodic drift has been observed shortly after electrofocusing had started; soon thereafter, it was superseded by a cathodic drift (7). This investigation concerns the first case in which the drift of the pH ’ Guest worker. * Since the fractionations indicating bidirectional drift of pH gradients and protein zones (4) were carried out in 8 M urea, involved large protein loads derived from acetic acid lyophilization, and were restricted to the pH range of 3 to 10 and gels of 10% T, 3% CBLS,they are not easily comparable to other work on the pH gradient instability in IFPA. 0003-2697/78/0881-0186$02.00/O Copyright 8 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

186

ANODIC

DRIFT

IN IFPA

187

gradients and protein patterns has been consistently in the opposite direction from the one commonly observed. The relevance of such a case for the electrofocusing mechanisms will be discussed. MATERIALS

AND METHODS

(i) Materials. Hemoglobin was a fresh hemolyzate (approximately 50 mg/ml) provided by Ms. Minna Feld (Clinical Pathology Department, Clinical Center, NIH). Cyanocobalamin (Calbiochem No. 6791, Lot No. 700102), arginine, and glycine (Sigma No. A-5006 and G-7126) were reagent grade. Ampholine (pZ range, 6-8) of LKB Produkter AB (Bromma, Sweden) (Catalogue No. 1809-131) was used. (ii) Isoelectric focusing on cylindrical polyacrylamide gels. IFPA was carried out at 0 to 4°C in gels of 1.7-ml volume, 6-mm internal diameter, 5% 1% Ampholine, using the all-Pyrex tube apparatus and the T, 15% Gm, procedures described previously (2,9,10). Gel tubes were cleaned by reacting them for several days with methanolic KOH and rinsing to neutrality. Where indicated under Results, tubes were coated with linear polyacrylamide by immersion into 1% Gelamide 250 (American Cyanamid Co., Stamford, Connecticut, or Polysciences, Warrington, Pennsylvania), draining, and drying at room temperature for several days. Electrofocusing was conducted in gels containing, in addition to Ampholine (~1 range, 6-8). 0.01 M arginine (or lysine) and glycine. Gel tubes were supported by nylon mesh except in the experiments described under Sections (i, Fig. 1A) and (v) of Results. The catholyte was 0.01 M arginine (pZ,,., = 10.7) or lysine (PZ,,., = 9.5) in the experiments reported under Section (ii) of Results, and the anolyte was 0.01 M glycine (pl,,., = 6.2). Steady-state IFPA was carried out at 400 V. At the time intervals stated under Results, single gels were withdrawn, photographed, and subjected to automated pH gradient analysis (11). Electroosmosis was tested by conducting IFPA with a sample of cyanocobalamin (12) not exceeding 150 ~1 and containing 25% sucrose and 1% Ampholine (pZ range, 8-9.5). (iii) ZFPA on horizontal gel slabs. The procedures described under.Section (ii) were applied to a horizontal gel slab (Multiphor apparatus, LKB) of 2-mm thickness, as described in the instruction manual (No. I-2117-EOl). Polymerization proceeded at room temperature. The apparatus was cooled to 0 to 4°C. Samples of hemoglobin and cyanocobalamin were absorbed into strips of filter paper (Whatman No. l), 15 x 10 mm and 5 x 10 mm, respectively, and applied by placing the strips onto the gel surface at a distance of 0.5 cm from the cathodic end. Catholyte and anolyte were 0.6 M arginine and glycine, respectively; they were applied on four filter paper strips (LKB No. 94-92-3572) that were equal to the length of the gel and

NGUYEN,

188

MCCORMICK,

AND CHRAMBACH

stacked on top of another and saturated with the electrolyte Anolyte and catholyte strips were renewed daily.

solutions.

RESULTS

(i) Anodic drift of pH gradients. When IFPA was carried out in gels containing Ampholine (pZ range, 6-8) between 0.01 M arginine (PHwc = 10.3) and glycine (PH250c = 6.0) as catholyte and anolyte, respectively, the pH gradients became progressively more alkaline with time. The isoelectric protein zones were concurrently displaced on the gels toward the anode (anodic drift) (Figs. IA and 1B). (ii) Reversal of the anodic direction of the pH gradient drift by lowering the pH of the catholyte. The experiment described under Section (i) was repeated, using 0.01 M lysine QJH,,~~ = 9.5) in lieu of arginine (pH250C = 10.3) as the catholyte. The drift of pH gradients and concurrent displacement of the isoelectric protein zone was cathodic (Fig. 2). (iii) The effect of coating the gel tubes on the direction of the pHgradient

PH

6

6

i 4’

0

I 0.5

I 1.0 RELATIVE

1 0.5

I 0

I 1.0

GEL LENGTH

f (6hlS)

(26 hrs)

FIG. 1. Anodic drift. IFPA (Ampholine pl range, 6-8,5% T, 15% CoaTD,0-4°C). Catholyte: 0.01 M arginine (pHzsDC= 10.3); and anolyte: 0.01 M glycine (pHzsDc = 6.0). Load: 20 ~1 of 50 mgiml hemoglobin and 25 ~1 of cyanocobalamin (approximately 2 mg/ml). Voltage: 400 V/5 cm (A); 100 V/5 cm (15-18 hr) and 400 V/5 cm thereafter (B). (A) No nylon mesh gel support. (B) With nylon mesh gel support.

ANODIC

DRIFT IN IFPA

Relative

189

Gel Length

(20 hd FIG. 2. Catholyte pH effect. Conditions are the same as those described for the results shown in Fig. lB, except that 0.01 M lysine (pH,,-c = 9.5) was the catholyte.

drift. The experiment described under Section (i) was carried out using tubes coated with 1% Gelamide 250 (linear polyacrylamide). The direction of pH gradient and protein zone displacement was cathodic (Fig. 3). (iv) The effect of polarity of the electric$eld on the direction of the pH gradient drift. The experiment described under Section (i) was repeated with the catholyte in the lower buffer reservoir and the anolyte in the upper buffer reservoir. Reversal of the positions of catholyte and anolyte relative to the gel reversed the anodic direction of the drift of the pH gradient and displacement of protein zones to the cathodic direction (Fig. 4). Polarity reversal also reversed the distribution of hydration along the pH gradient gel. Under the conditions of anodic drift, gel swelling occurred at the anodic gel terminus, while swelling at the cathodic end of the gel coincided with a cathodic drift (Fig. 5). (v) Intermittent cathodic and anodic direction of the pH gradient drijlt within experiments. Changes in the direction of pH gradient drifts have been observed during single experiments, such as in systems using either lysine-glycine or histidine-threonine as the catholyte-anolyte pairs. In each case, pH gradients were stable or moved slightly cathodically, until sudden and rapid gel alkalinization set in, accompanied by observable gel swelling and wall separation (7). Similarly, an anodic drift arose at

190

NGUYEN,

MCCORMICK,

AND CHRAMBACH

---

PH

7hrs 23

1

"

. . . 24 ooo 26 ..a 27 AA.4 26 . . . 46 000 46 . . . 50 944 56

6

I

0

RELATIVE

GEL LENGTH

FIG. 3. Tube coating effect. Conditions are the same as those given for the results shown in Fig. lB, except that tubes were coated with linear polyacrylamide (Gelamide 250).

both edges of a horizontal slab (see below) subjected to mechanical stress through periodic removal of gel strips for purposes of pH analysis (Fig. 6). (vi) The effect of replacement of vertical gel tubes by a horizontal gel slab on the direction of the drift of the pH gradient. The experiment described under Section (i) was repeated using a horizontal gel slab. The direction of displacement of pH gradient and protein zones was initially toward the cathode but became anodic with time (Fig. 7). in IFPA. Under the conditions of (vii) Displacement of cyanocobalamin the experiments described under Sections (i), (iv), and (vi), cyanacobalamin diffused out from the sample application zones but did not significantly enter into the gel (Fig. 8). DISCUSSION

The purpose of this study differed from previous ones dealing with pH gradient instability. Whereas the earlier work aimed at overcoming the instability (3,6-8), this study aimed at inducing it. The nature of the induced instability was, however, unique: It involved a drift of pH gradients

ANODIC

DRIFT

191

IN IFPA

-~~

16 hrs 19

I

I

6

. . . 24 000 27 . . . 43

PH

I 1.0 Relative

FIG. 4. Effect of the polarity of the electric same as those described for the results shown buffer reservoir (B).

I 0 Gel Length

field relative to the gel. Conditions are the in Fig. 1B (A). The catholyte is in the lower

and protein positions toward the anode, rather than toward the cathode, as had been universally observed. It was hoped that by a study of the “anodic drift” our understanding of the mechanism of isoelectric focusing could be advanced, in view of the following considerations. It was shown previously (2) that natural pH gradients arise and decay by the action of the electric field and that carrier ampholyte distributions coincident with the pH gradients are changing continuously during gel electrofocusing, with no clear possibility to recognize a steady-state (“equilibrium”) (1). It appears therefore that pH gradient formation, “stabilization,” and decay are subject to uniform mechanisms and are equally deserving of attention. The presently known mechanisms contributing to pH gradient decay have recently been reviewed by Rilbe (13). Only the latest facts need to be considered here. (i) pH gradient stabilization. Replacement of strongly acidic and basic anolyte and catholyte by the “terminal” constituents of the carrier ampholytes has stabilized pH gradients, at least in the pZ range of 6 to 8 (7). This result is compatible with any mechanism by which terminal constituents are lost into the adjacent electrolyte reservoirs, by field strength effects and zone e.g., by electroosmosis “amplified”

192

NGUYEN,

MCCORMICK,

AND CHRAMBACH

lo.A ------~ ‘%% PH 9-

1

--....._..,

-.-.._ \,, ---....., --.--- .-........_._,,,,_ --....

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RELATIVE

I 0.5 GEL LENGTH

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pH I’:\& _._.,_ ~-..-.....----.-F_.._.__....._ 1 RELATIVE

0.5 GEL LENGTH

1.O

GEL (92hrsl

FIG. 5. Correlation of gel swelling with the direction of the pH gradient shift. (A) conditions are the same as those given for the resuits illustrated in Fig. 4A; (B) conditions are the same as those given for the results illustrated in Fig. 4B.

expansion at constant concentration (13), or by a redistribution across a phase boundary which necessarily gives rise to a pH and conductivity gradient (14). Any form of mass transport into a reservoir of identical buffer composition should be balanced by a reverse mass transport, maintaining the original composition of the terminal constituent. (ii) pH gradient drift reversaf. Under otherwise identical conditions of IFPA, an arginine catholyte gave rise to an anodic drift, and a lysine catholyte gave rise to a cathodic drift of the pH gradient. This result does not appear compatible with the electroosmotic hypothesis (13), according to which a cathodic drift is produced by carboxylate charges on the gel, whereas an anodic drift would be generated by fixed positive amine charges on the gel. It does not appear possible to devise a mechanism by which the particular catholyte exchange made could lead to a charge reversal from fixed carboxylate to ammonium charges on the gel. (iii) Stationary cyanocobalamin position. Lundahl and Hjerten have previously failed to detect electroosmosis in IFPA measured by the dis-

193

ANODIC DRIFT IN IFPA

RELATIVE

GEL LENGTH

FIG. 6. Anodic drift of pH gradients on a gel slab, localized in areas subjected to mechanical stress, with concurrent cathodic drift of pH gradients localized in areas nor subjected to stress (A) 73 hr; and (B) 163 hr of electrofocusing. Other conditions are the same as those described in the legend to Fig. 7. pH gradients in those parts of the gel slab on which they occurred are depicted. The solid lines indicate pH gradients at the center of the gel slab not subjected to stress. The open symbols illustrate positions on the gel subjected to stress through excision and removal of gel strips for pH analysis. The open circles in (A) and (B) correspond to identical positions on the gel. The hemoglobin zone serves as a reference for a pI = 7 zone across the gel. Empty spots on the gel are due to erasure on the photographs of cyanocobalamin spots irrelevant for the purposes of this illustration.

i

1 r-7 . ---.. . 000

I 26 50 42 18 hrs

-

DH

5’ 0 RELATIVE

I 0.5 GEL LENGTH

L I 1.0

FIG. 7. pH gradient drift on the horizontal gel slab as a function of the time of IFPA. Conditions are the same as those given for the results illustrated in Fig. 1A. except a horizontal Multiphor apparatus was used (2-mm gel thickness, 12.5 x 20.0 cm).

194

NGUYEN,

MCCORMICK,

AND CHRAMBACH

TIME (hrs)

42

75

FIG. 8. Constancy of position of cyanocobalamin in IFPA. Conditions are the same as those given in the legend to Fig. 7, except that cathodically and anodically applied spots of cyanocobalamin are shown as a function of the time of IFPA. The areas of application of cyanocobalamin are shown by dotted line on the left margin: the extent of diffusion spreading is shown by the solid lines.

placement of DNP-ethanolamine from its initial position (14). These authors even failed to detect electroosmotic flow when carboxylate charges were increased in the gel by partial amidolysis of the monomers (S. Hjerten, personal communication). This result is fully confirmed by the failure of cyanocobalamin to move away from its starting position during IFPA. The time-dependent zone spreading of cyanocobalamin was symmetrical in the cathodic and anodic directions and occurred to the same extent at the anodic and cathodic ends. This zone spreading is therefore presumed to be due to diffusion. (vi) Polarity effect. The reversal in the direction of the drift of pH

ANODIC DRIFT IN IFPA

195

gradients as a function of the geometric position of catholyte and anolyte relative to the gel will be termed polarity effect. Since the polarity effect is remedied by tube coating, it appears to be due to wall separation of the gel, induced most likely by the hydration changes along the pH gradient gel. The observed hydration changes accompanying the changes in constituent concentrations along the gel and the progressive acidification or alkalinization phenomena (Fig. 5) support an electroosmotic mechanism only in the case of cathodic drift (13). Even in the case of cathodic drift, however, gel swelling may be an epiphenomenon or consequence of the mechanisms of pH gradient decay rather than their cause. (v) Reversal of the direction of the pH gradient drijt within experiments. The observed changes in the direction of pH gradient decay within experiments have been accompanied in all cases by hydration and wall separation phenomena. They are therefore considered secondary phenomena, which, like gel swelling itself, give us no information with regard to the mechanisms leading to pH-dependent gel swelling. REFERENCES 1. Chrambach, A., and Nguyen, N. Y. (1976) in Electrofocusing and Isotachophoresis (Radola, B. .I., and Graesslin, D., eds.), pp. 51-58, de Gruyter, Berlin-New York. 2. Baumann, G., and Chrambach, A. (1975) in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P. G., ed.). pp. 13-23, Elsevier, Excerpta Medica, North-Holland, Amsterdam. 3. Chrambach, A., Doerr, P., Finlayson, G. R., Miles, L. E. M. Sherins, R., and Rodbard, D. (1973) Ann. N. Y. Acad. Sci. 209, 44-64. 4. Miles. L. E. M., Simmons, J. E., and Chrambach. A. (1972) Anal. Biochem. 49, 109- 117. 5. Nguyen, N. Y., and Chrambach, A. (1976) Anal. Biochem. 74, 145-153. 6. Nguyen, N. Y., and Chrambach, A. (1977) Anal. Biochem. 79, 462-469. 7. Nguyen, N. Y., and Chrambach, A. (1977) Anal. Biochem. 82, 226-235. 8. Nguyen, N. Y., and Chrambach, A. (1977) Anal. Biochem. 82, 54-62. 9. Chrambach, A., Jovin. T. M., Svendsen, P. .I., and Rodbard, D. (1976) in Methods of Protein Separation (Catsimpoolas. N., ed.), pp. 27-144, Plenum, New York. 10 . Doerr, P., and Chrambach, A. (1971) Anal. Biochem. 42, 96-107. 11. Chidakel, B. E.. Nguyen, N. Y.. and Chrambach, A. (1976) Anal. Biochem. 77, 216-225. 12. Ghosh, S., and Moss, D. B. (1974) Anal. Biochem. 62, 365-370. 13. Rilbe, H. (1976) in Electrofocusing and Isotachophoresis (Radola, B. J., and Graesslin, D., eds.), pp. 35-50, de Gruyter, Berlin-New York. 14. Lundahl, P., and Hjerten, S. (1973) Ann. N. Y. Acad. Sci. 209, 94-111.

An anodic drift of pH gradients in isoelectric focusing on polyacrylamide gel.

ANALYTICAL BIOCHEMISTRY An Anodic 88, 186- 195 (1978) Drift of pH Gradients in Isoelectric on Polyacrylamide Gel NGA Y. NGUYEN, ALICE G. MCCOR...
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