fractionation of carrier ampholytes in multicompartment electrolyzers with isoelectric membranes

1930 A. Bossi and P. G. Righetti Electrophoresis 1995, 16, 1930-1934

Alessandra Bossi Pier Giorgio Righetti

Fractionation of carrier ampholytes in multicompartment electrolyzers with isoelectric membranes

Department of Cell Biology, University of Calabria, Arcavacata di Rende (Cosenza)

Multicompartment electrolyzers with isoelectric membranes can be successfully utilized for the preparation of carrier ampholytes of narrow pH ranges from commercially available wider pH intervals. An example is given on the preparation of a 0.6 pH unit (pH 6.7-7.3) range from a standard pH 6-8 interval. A 10% Ampholine solution is focused in an electrolyzer equipped with the following isoelectric membranes: pl6.0, 6.7, 7.3 and 8.0. The narrow pH 6.7-7.3 cut can be efficiently utilized for base-line separation and quantitation of hemoglobin A from its glycated form, Hb A,c. This analysis is impor- tant for the screening and follow up of diabetic patients. The advantage of multicompartment electrolyzers are: (i) precision in the preparation of narrow pH cuts, due to the presence of membranes with defined plvalues; (ii) ability to perform in both small- and large-scale operations and (iii) absence of contaminants leaching from granulated supports, as typical of some previous techniques.

1 Introduction

Production of narrow-range carrier ampholytes (CA) is gaining importance, in view of the increasing use of isoelectric focusing (IEF) in capillary electrophoretic proce- dures. Unfortunately, a consistent number of papers published to date in the field (for a review, see [l]) has failed to prove the high resolution typically obtained in gel slab techniques. This might be due to the high elec- troosmotic flow typically existing on the inner capillary surface, due to silanol ionization. Even in coated capilla- ries, however, resolution is not high enough to guar- antee acceptance in, e.g., the clinical chemistry field, where clear-cut and reproducible results are needed for precise diagnosis. Just as an example, not a single paper so far published in capillary IEF has been able to offer base-line resolution between adult hemoglobin (Hb A) and its glycated form (Hb AJ. Quantitation is thus difficult and diagnosis of diabetic conditions and its remis- sion is therefore unreliable.

In the past, the problem of increased resolution in IEF had been solved by commercially-available narrow CA ranges, encompassing as little as 0.5 pH units (as opposed to the standard 2 pH unit interval routinely available). Although such narrow pH ranges had proven valuable in routine clinical analysis [2], their production has been mostly discontinued. In the early days, several methods were proposed for preparing narrow pH cuts from wide pH range CAs. Gianazza et al. [3] suggested a preparative apparatus based on the continuous flow technique of Fawcett [4]. This method seemed to offer advantages over multi-compartment electrolyzers, as described by Rilbe [5] and Vesterberg [6], by eliminating problems of osmotic pressure and membrane polarization and minimizing anodic oxidation of CAs. In principle, CAs

~~

Correspondence: Prof. P. G . Righetti, L.I.T.A., Via Fratelli Cervi 93, Segrate 20090 (Milano), Italy (TellFax: +02-2642-3364)

Nonstandard abbreviations: CA, carrier ampholyte; CZE, capillary zone electrophoresis; Hb, hemoglobin; Hb A,,, glycated hemoglobin; Hb Fa,, acetylated fetal hemoglobin

Keywords: Carrier ampholytes / Isoelectric focusing / Isoelectric membranes / Immobilized pH gradients / Multicompartment electrolyzer

could also be fractionated in narrow ranges by using the zone convection apparatus of Valmet [7]. By this technique, this author separated Ampholine encompassing the pH 3.5-10 range into a number of fractions, each covering about 0.2 of a pH unit, although Charlionet et al. [8] claimed that the minimum pH interval that could be successfully used in IEF was a span of 0.3 pH units. Also attractive, for preparation of narrow-range CA cuts, could be the flow-through systems described by Just [9] and by Righetti and Hjerttn [lo]: since the CAs are syn- thesized via a gradient of chemicals filling the length of a capillary tubing along the length of a capillary tubing, fractionation of its content gives automatically narrow pH cuts. Other chambers that can be exploited for this purpose are the polyethylene coil tubing of Macko and Stegemann [Il l and the thin-layer, continuous free flow apparatus of Just et al. [12]. In principle, any apparatus used for preparative IEF can be utilized for preparation of narrow pH ranges; thus, Rilbe’s density gradient col- umns [13] or Radola’s granulated flat beds [14, 151 could be advantageously utilized for this task. Otavsky et al. [16] have used horizontal troughs filled with a mixture of Sephadex and Pevikon C870 (plastic beads made of polyvinyl chloride and polyvinyl acetate) for the same purpose: after IEF, the desired CA pH range could be recovered simply by centrifuging the cake through sin- tered glass.

Ideally, however, equipment which does not utilize capillary systems as anticonvective media is of interest, since the fractionated CAs do not have to be separated from the inert support (usually a granulated gel, which might also leach out contaminants into the CA buffers). From this point of view, our multicompartment electrolyzer with isoelectric membranes [17, 181 would appear to be ideally suited for this task. In the latter technique (devel- oped for purifying proteins to homogeneity), the com- pound to be purified is always kept in a liquid vein and it is trapped into a chamber delimited by two membranes having pls encompasisng the p l value of the protein being purified. Thus, by a continuous titration process, all other impurities, either nonisoelectric or having different p l values, are forced to leave the chamber, in which the protein of interest will ultimately be present as the sole species, characterized by being

0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1995 0173-0835/95/1010-1930 $5.00+.25/0

Electrophoresis 1995, 16, 1930-1934 Production of narrow-range carrier ampholytes 193 1

isoelectric and isoionic as well. This purification procedure was successfully applied to a number of proteins, including eglin C [17], monoclonal antibodies against the gp-41 of the AIDS virus [18], recombinant human growth hormone [19], the epidermal growth factor receptor [20, 211, recombinant superoxide dismutase [22] and interleukin 6 [23] and glucoamylase [24]. For the first time, this process is applied to preparation of narrow CA pH ranges.

2 Materials and methods

2.1 Materials

Acrylamide, N,N-methylenebisacrylamide (Bis), N, N, - N,W-tetramethylethylenediamine (TEMED) and ammo- nium persulfate (APS) were from Bio-Rad (Hercules, CA, USA). The following Immobiline species: pK 3.1, pK 6.2 and pK 7.0 and Ampholine and Pharmalyte CAs pH 6-8 were from Pharmacia-LKB Biotechnologies (Uppsala, Sweden).

2.2 Biologicals

Normal human adult hemoglobin and its glycated deriv- ative (Hb AJ were prepared by lysis of red blood cells. The two species were purified by small-scale preparative immobilized pH 6.8-7.8 gradients according to Gelfi and Righetti [25]. Umbilical cord blood was taken at delivery and processed as adult Hb. In order to prevent auto- oxidation, the isolated fractions were gassed with carbon monoxide for 30 s and stored at 4°C.

Table 1. Physico-chemical parameters of the set of four isoelectric membranes used in CA fractionation

p l Buffering ion“’ Titranta) p Power b M ) (mM) (mequiv. L-’ pH-’)

6.0 pK 6.2 (20) pK 3.1 (13.3) 10.95 6.7 pK 7.0 (20) pK 3.1 (14.3) 10.23 7.3 pK 7.0 (20) pK 3.1 ( 7.4) 10.23 8.0 pK 8.5 (25) pK 3.1 (20.0) 10.4

a) The first value refers to the pK of the different Immobilines; the value in parenthesis is their respective molarity.

achieved at 150 V constant (over an 8 cm electrode dis- tance) in only 3 to 4 h. The anolyte was 20 mM acetic acid (pH 3.32, conductivity: 213 pmhos) and the catholyte 20 mM Tris (pH 10.3; conductivity 34 pmhos). The instrument was thermostatted at 10°C.

2.4 Analytical CA gels

Isoelectric focusing in both pH 6-8 and narrow cut pH 6.7-7.3 CA was performed in 5 O/oT, 4O/oC polyacrylamide matrices cast onto a GelBond PAG support. “Empty” gels were prepared, washed three times in distilled water, dried and reswollen in the appropriate Ampholine pH range. Dilute acetic acid (20 mM) and Tris (20 mM) were used as anolyte and catholyte, respectively. The samples (ca. 30 pL, from 20 to 40 pg protein) were applied in surface basins at the anodic side. Focusing was started at 10°C at 500 V for the first hour and then at 1600 V for additional 3 h for the 2 pH unit ranges, but at 2500 V for the narrow pH ranges. Staining was with Coomassie Bril- liant Blue R-250 in presence of Cu2+ [27].

3 Results 2.3 Preparation of isoelectric Immobiline membranes

Preparation of narrow pH cuts from pH 6-8 Ampholine was achieved with the Iso-PrIME apparatus (Hoefer Sci., San Francisco), consisting of a multichamber electrolyzer to be assembled with isoelectric, buffering membranes. Four isoelectric membranes were made with the following p l values: 6.0, 6.7, 7.3 and 8.0. All membranes contained 20 mM buffering ion and the pK 3.1 Immobi- line as counter-ion, for titrating to the respective pH values (except for the pl8.0 membrane, which contained 25 mM buffering ion; for their composition and properties, see Table 1). Calculation of the various amounts of titrants was performed with the program of Giaffreda et al. [26] (available from Hoefer Sci.). All membranes were cast as a 1O%T, 5%C matrix in the form of disks of 4.7 cm diameter and a thickness of ca 1 mm. Note that the membranes are supported by glass fiber filters (see [17] for a detailed description of their properties). After washing the membranes three times in distilled water, the multicompartment apparatus was assembled and 22.5 mL total of Ampholine pH 6-8 were loaded (7.5 mL per chamber), diluted to 10% (from a 40% stock solution). Only the content of anolyte and catholyte chambers is recycled from 200 mL reservoirs. No reservoirs have been connected to the other three electrolyzer chambers since the amount to be processed was ade- quate for our purposes. Focusing of the pH 6-8 Ampho- line (and of a pH 6-8 Pharmalyte) solutions has been

Figure 1 shows the assembly of the multicompartment electrolyzer with the p l values of the isoelectric membranes. Since we were interested in the analysis and separation of Hb A from its glycated form (Hb A,=), we have assembled the apparatus in such a way as to collect in the central chamber the desired narrow pH range cut (pH 6.7-7.3 interval). This interval should be ideal for Hb A/Hb A,, separation, since the former has a p1 of 6.97. It should be noted, additionally, that, by decreasing the pH interval from 2 to 0.6 pH units, one should expect an increment of resolution (all other conditions being identical) proportional to the ratio of the two pH gradient widths. The progress in focusing of the CAs in the three chambers is shown in Fig. 2. After 3 h, a pla- teau in the current decline is reached (the power supply was operated in the constant voltage mode), indicating the attainment of isoelectric conditions. As a precaution, the focusing process was continued for an additional hour.

One way to ascertain if fractionation in narrow ranges has been achieved is to measure pH and conductivity of the starting and fractionated material. Table 2 gives theses values for two different experiments, regarding the fractionation of pH 6-8 Ampholine and pH 6-8 Pharmalyte (although nominally identical pH ranges, they should contain quite different amphoteric species, due to the different synthetic principles) [2]. As shown

1932 A. Bossi and P. G. Righetti Electrophoresis 1995, 16, 1930-1934

PI: 6.0 6.7 7.3 8.0 Figure 1. Scheme of the assembly of the multicompartment electrolyzer. The pZ values and the position of the isoelectric membranes are indicated by vertical arrows. Chamber numbering from left to right: chamber No. 1, anodic compartment (pH 3.3); 2-4, sample collection chambers; No. 5, cathodic compartment (pH 10.3).

24 T

10% Ampholine pH 6-8 h Figure 3. Focusing of Hb A and its glycated form, Hb AlC and of umbilical cord blood Hbs in pH 6-8 (upper panel) and narrow pH 6.7-7.3 (lower panel) Pharmalyte ranges. Note the much increased resolution in the last case. Both gels were 5%T, 4%c, containing either 2% or 3 % Ampholine in the pH 6-8 or 6.7-7.3, respectively. The photographic enlargement is identical in both pictures.

b

I 0 40 80 120 160 200 240

Time (min)

Figure 2. Progress of focusing free CAs in the multicompartment electrolyzer. The three central chambers were filled with 7 mL each of 10% Ampholine pH 6-8. The system was operated at 150 V constant, at room temperature, for a period of 3 h. The plateauing of the current at 4 h of operation suggests attainment of steady-state conditions.

Table 2. pH and conductivities of unfractionated and fractionated

Typeb) PH Conductivity CAs")

(mmho)

Ampholine pH 6-8 pH 6.0-6.7 pH 6.7-1.3 pH 7.3-8.0

Pharrnalyte pH 6-8 pH 6.0-6.7 pH 6.7-7.3 nH 7.3-8.0

7.2 6.6 7.1 7.8

7.0 6.6 7.1 7.8

0.725 0.154 0.187 0.243

0.690 0.153 0.240 0.295 r-- - - -

a) pH and conductivity refer to 10% w/v solutions at 25OC. b) The 3 narrow pH ranges refer to either Ampholine or Pharmalyte

in Table 2, for all 3 narrow pH cuts the nominal pH value falls in between the extremes of each narrow pH range and all conductivities are markedly decreased as compared to the starting 2 pH unit material. Although the conductivities of the narrow pH cuts are substan-

commercial carrier ampholytes.

tially different between Ampholine and Pharmalyte (being higher for the latter) the nominal average pH of each fraction is essentially identical (as it should be, since both compounds have been fractionated by using the same isoelectric membranes).

Figure 3 shows the separation of Hb A from its glycated form and the fractionation of the three main compo- nents of cord blood (Hb F, Hb A and Hb FJ in two Ampholine ranges: the commercially available 2 pH unit cut (pH 6-8) and the narrow pH 6.7-7.3 range obtained by fractionation in the multicompartment electrolyzer. It can be appreciated that, whereas in the first case the separation is minimal (less than 1 mm free space between the two bands), rendering quantitation quite difficult, better separation was obtained in the narrow pH cut.

4 Discussion

4.1 Generation of narrow pH ranges

For difficult IEF separations, especially for samples of clinical-chemical interest, one can resort to two variants of IEF. In one case (as adopted in the present report), ultranarrow pH cuts are prepared, so as to progressively improve the separation. This approach was adopted for, e.g., screening for polymorphism of a number of serum proteins, such as a,-antitrypsin, phosphoglucomutase, apolipoproteins, group-specific component, transferrin, protein C (for a review see [28]). In fact, in most cases,

Electrophoresis 1995, 16, 1930-1934 Production of narrow-range carrier ampholytes 1933

the problem of creating narrow pH ranges was simply solved by resorting to immobilized pH gradients, which allow a full engineering of the width and shape of the pH gradient. In a second approach, one has to resort to nonlinear pH gradients. In this last case, one still utilizes the standard 2 pH unit cuts, but flattened (thus rendered nonlinear) in a given portion of the pH interval, so as to maximize separation in that region. This approach, first reported by Brown et al. [29], consists of adding substan- tial amounts (10-40 mM for good species, up to 500 mM for poor CAs) of an amphoteric buffer, having a p l in the pH region to be flattened, to the standard Ampholine pH range. This ampholyte, thus called “separator”, has the ability of flattening the portion of the pH gradient in which it is isoelectric (and its surrounding), thus greatly increasing the separation power. Cossu et al. [30] have reported this approach for a clear-cut separation of fetal (Hb F), adult and acetylated fetal hemoglobins in the screening of cord blood for B-thalassemias. Since the separation between Hb A and Hb Fa, was poor, the pH 6-8 Ampholine was supplemented with an equimolar mixture of 0.2 M P-Ala and 0.2 M 6-aminocaproic acid, with an impressive improvement of separation. In another instance, Beccaria et al. [3 11 utilized P-Ala or the dipeptide His-Gly for improved separation of Hb A from its glycated form.

4.2 Preparation of narrow-range CAs

The present approach, for preparing narrow-range CAs, offers some interesting advantages. First of all, the narrow pH cuts can be prepared with high precision, since only those CA fractions trapped between two isoelectric membranes can be harvested. This is in contrast with all other focusing techniques for CA fractionation, where only approximate pH cuts can be isolated, since no initial and terminal pH boundaries can be identified during the fractionation. Secondly, both small- and large- scale fractionations can be performed with the same apparatus, according to the adopted procedure. In the absence of recycling and external reservoirs, only the content of each chamber of the electrolyzer will be fractionated (ca. 7 mL volume). However, if larger quantities are required, the apparatus can be operated while connected to reservoirs, in the recycling mode, and then large quantities can be processed in a single run. Addi- tionally, with the present methodology, one can prepare precisely the pH cuts needed to maximize the separation of any biological sample, as required by the A p l of any set of neighboring proteins under analysis. This cannot be done with the narrow pH ranges commercially available. For example, we noticed in the last catalogue of Pharmacia (18-1101-95), that indeed a few, 1 pH unit spans are still available. While they might work to solve some analytical problems, they of course cannot be used to optimize any other one.

In our specific case, while we could prepare a pH 6.7-7.3 gradient, centered on the p l (7.0) of Hb A, and thus optimal for pulling apart from this major component the minor, glycated Hb, the best range commercially available is a Pharmalyte pH 6.7-7.7, which presumably has been optimized to cover all acidic and basic Hb variants, but is still not the best range for Hb A/Hb A,, separa-

tion. In this last case, in fact, the separation is rendered most difficult by two adverse conditions: (i) the minute Apl between Hb A (p16.97) and Hb A,, (pf 6.92); (ii) the large difference in concentration between the two species (concentration ratio HbA/Hb A,, of 30:l in normal adults). Finally, with the Immobiline technology here adopted, one could prepare ultranarrow CA ranges, e.g. encompassing only 0.1 or 0.2 pH units. According to Hjertkn (personal communication) such very narrow cuts could be utilized as isoelectric buffers in capillary zone (thus not in the focusing mode) electrophoresis for per- forming zonal separations at very high voltage gradients, while minimizing Joule heating. As an extra bonus, the focusing technique here adopted allows harvesting of ultrapure CA cuts. Our membranes produce no leacha- bles and our carrier-free technique allows recovery of uncontiminated fractions. Conversely, in gel techniques (either continuous or granulated), there is always the risk of impurities leaching out from the gel phase. While charged impurities will most probably be transported in the eletrodic reservoirs, neutral ones will remain in the CA solution recovered.

4.3 Conclusions

Fractionation of carrier ampholytes in narrow and ultranarrow pH cuts seems to be gaining momentum, as shown in the present report, and as additionally reported, recently, by Schlicht et al. [32]. The latter authors have successfully fractionated up to 20% w/v crude (as well as prefractionated) carrier ampholytes of defined pH ranges. They have presented a capillary- cooled electrolyzer in which the various multicompart- ments are delimited either by polyester nets or by “neutral” ultrathin polyacrylamide (3 O/oT, 4 %C) membranes, supported by a net. In their system, narrow CA pH ranges could be isolated with improved performance in analytical runs. In our system, one might object that the CAs have a much too high buffering power, so that they might overcome the B power of our isoelectric membranes. In reality, this objection does not hold: first of all, we have also set our membranes so as to have a high S power (averaging 10-11 mequiv. L-’ pH-’; see Table 1); secondly, even if the CAs had a higher B power than our membranes, once they are focused between a set of membranes, they cannot, by definition, leave the chamber by electrophoretic transport. They, might, however, move by diffusion; the latter event, however, is counteracted by the highly sieving membranes (10 %T) and by their intrinsic fi power (it is to be expected that the small amount of material diffusing at any given time will be less concentrated than the Immobilines in the membranes). Our data, in fact, confirm this remarkable reproducibility of the narrow pH cuts harvested from the electrolyzer. The precise buffering power of the various CAs is a bit more difficult to evaluate: different reports give such values (for 1% solutions) ranging from 1 to 5 mequiv. L-’ pH-’ (for a review, see. pp. 66-68 in [2]).

P. G.R. gratefully acknowledges support from the Radius in Biotechnology (ESA, Paris, France) and from Progetto Stra- tegico CNR, Comitato di Chimica (Roma, Italy).

Received February 21, 1995

1934 A. Bossi and P. G. Righetti

5 References

[31

Righetti, P. G., Gelfi, C., J. Cap. Elec. 1994, I, 27-35. Righetti, P. G., Isoelectric Focusing: Theory, Methodology and Appli- cations, Elsevier, Amsterdam 1983. Gianazza, E., Pagani, M., Luzzana, M., Righetti, P. G., J. Chroma- togr. 1975, 109, 357-364. Fawcett, J. C., Ann. N . Y. Acad. Sci. 1973, 209, 112-126. Rilbe, H., Prot. Biol. Fluids 1970, 17, 369-382. Vesterberg, O., Acta Chem. Scand. 1969, 23, 2653-2666. Valmet, E . , Sci. Tools 1969, 16, 8-13. Charlionet, R., Morcamp, C., Sesboue, R., Martin, J. P., J. Chro- matogc 1981, 205, 355-366. Just, W. W., Anal. Biochem. 1980, 102, 134-144. Righetti, P. G., Hjertin, S . , J . Biochem. Biophys. Methods 1981, 5,

Macko, V., Stegemann, H., Anal. Biochem. 1970, 37, 186-190. Just, W. W., Leon-V, J. O., Werner, G., Anal. Biochem. 1975, 67, 590-601. Vesterberg, O., Wadstrom, T., Vesterberg, K., Svennson, H., Malm- gren, B., Biochim. Biophys. Acta 1967, 133, 435-445. Radola, B. J., Biochim. Biophys. Acta 1973, 295, 412-428. Radola, B. J., in: Arbuthnott, J. P., Beeley, J. A. (Eds.), Isoelectric Focusing, Buttenvorths, London 1975, pp. 182-197. Otavsky, W. I., Bell, T., Saravis, C., Drysdale, J. W., Anal. Biochem.

Righetti, P. G., Wenisch, E., Faupel, M., J. Chromatogr. 1989, 475,

Righetti, P. G., Wenisch, E., Jungbauer, A,, Katinger, H., Faupel, M., J. Chromatogr. 1990, 500, 681-696.

25 9-272.

1977, 78, 302-307.

293-309.

Electrophoresis 1995, 16, 1930-1934

[19] Ettori, C., Righetti, P. G., Chiesa, C., Frigerio, F., Galli, G.,

[20] Wenisch, E., Righetti, P. G., Weber, W., Electrophoresis 1992, 13,

[21] Weber, W., Wenisch, E., Gunther, N., Marnitz, U., Betzels, C., Righetti, P. G., J. Chromatogr. A 1994, 679, 181-189.

[22] Wenisch, E., Vorauer, K., Jungbauer, A,, Katinger, H., Righetti, P. G., Electrophoresis 1994, 15, 647-653.

[23] Breton, J., Avanzi, N., Valsasina, B., Sgarella, L., La Fiura, A., Breme, U., Orsini, G., Wenisch, E., Righetti, P. G., J. Chromatogr.

1241 Wenisch, E., Schneider, P., Hansen, S. A. Rezzonico, R., Righetti,

[25] Gelfi, C., Righetti, P. G., J. Biochem. Biophys. Methods 1983, 8,

[26] Giaffreda, E., Tonani, C., Righetti, P. G., J. Chromatogr. 1993, 630,

[27] Righetti, P. G., Drysdale, J. W., J. Chromatogr. 1974, 98, 271-321. [28] Righetti, P. G.: Immobilized p H Gradients: Theory and Methodo-

logy. Elsevier, Amsterdam 1990. [29] Brown, R. K., Caspers, M. L., Lull, J. M., Vinogradow, S. N., Fel-

genhauer, K., Nekic, M., J. Chromatogr. 1977, 131, 223-232. [30] Cossu, G., Manca, M., Gavina, P. M., Bullitta, R., Bianchi-Bosisio,

A,, Gianazza, E., Righetti, P. G., Am. J. Haematol. 1982, 13,

1311 Beccaria, L., Chiumello, G., Gianazza, E., Luppis, B., Righetti, P. G., Am. J. Haematol. 1978, 4, 367-374.

[32] Schlicht, C., Dettke, H., Radola, B. J., Electrophoresis Forum '94 Technical University of Munich, October 24-26, 1994, Abstract No. 15.

Grandi, G., J. Biotechnol. 1992, 25, 307-318.

6 68-673.

A 1995, 706, 463-468.

P. G., J. Biochem. Biophys. Methods 1993, 27, 199-213.

156-17 1.

3 13-327.

149-157.

fractionation of carrier ampholytes in multicompartment electrolyzers with isoelectric membranes

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