AtNALYTICALBIOCHEMISTRY71, 325-332 (1976)

Analysis of Carrier Ampholytes by Ion Exchange Chromatography I RAY K. BROWN, JUNE M. LULL, STEVEN LOWENKRON, JOSEPH C. BAGSHAW, AND SERGE N . VINOGRADOV

Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201 Received March 10, 1975; accepted September 10, 1975 Commercially available and synthetic wide-range and short-range ampholytes used in the isoelectric focusing of proteins were analyzed by ion-exchange chromatography. A pH gradient over the pH range 3.8 to 11.0 was used to elute the ampholytes from a column of a sulfonated polystyrene resin. The widerange ampholytes were resolved into some 60 to 70 ninhydrin-positive components. The recovery obtained with the method was quantitative, Analysis of the cathodic and anodic fractions of wide-range ampholytes focused concurrently with whale myoglobin on a polyacrylamide gel, suggested that no simple relationship exists between the pI of the ampholyte components and the pH at which they are eluted from the ion-exchange column.

Isoelectric focusing in stabilized pH gradients is one of the most powerful techniques for the isolation and characterization of biological macromolecules. The artificial pH gradients can be formed by mixtures of synthetic ampholytes possessing closely-spaced isoelectric points covering the required range of pH (1,2). The commercially available carrier ampholytes are complex mixtures of low molecular weight polyamine polycarboxylic acids (3). It is evident that the formation of reproducible pH gradients is of paramount importance in the successful focusing of proteins and other macromolecules and that it is related to the reproducibility in the synthesis of the ampholytes. Control of the latter requires a reliable method for the quantitative analysis of the ampholyte mixtures. The commercially available ampholytes are known to be complex mixtures from dye staining patterns (4,5) and refractive index variations (6) observed upon isoelectric focusing in polyacrylamide and Sephadex gels. There has also been an appreciable variation in the estimates of the number of individual components in commercially available ampholytes (1,3-5). A qualitative method based on the so-called "caramelization pattern" has been developed by Felgenhauer and Pak (7). It allowed, for the first time, direct visual observation of a number of 1 Portions of this paper were presented at the Spring, 1975 meeting of the Federation for Experimental Biology (Fed. Proc. 34, 685 (1975)). 325 Copyright © 1976byAcademicPress, Inc. All rights of reproductionin any form reserved.

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ampholyte components and the estimation of their relative concentrations over defined p H ranges. It also has permitted a comparison to be made of patterns produced with different batches of commercially available ampholytes and with ampholytes prepared by Vinogradov et al. (8) by the copolymerization of acrylic acid and oligoethyleneamines. We describe below a method for the quantitative analysis of synthetic ampholytes by ion-exchange chromatography on a column of a sulfonated polystyrene resin employing a pH and ionic strength gradient. EXPERIMENTAL PROCEDURE Ampholytes Several lots of wide-range (pH 3-10) and short-range, acidic (pH 4-6) and basic (pH 8-9.5) ampholytes were purchased from LKB. Widerange ampholytes were also prepared by the copolymerization of acrylic acid with hexamethylene heptamine (HEHA) as described previously by Vinogradov et al. (8). Chromatographic Analysis All analyses were performed on a Technicon Amino Acid Analyzer. The following buffers were used to prepare the elution gradient. (A) Citrate buffer (pH 3.80) containing 14.71 g Na3 citrate.2H20, 25.0 ml of 2.00 N N a O H , 5 ml thiodiglycol, titrated to pH 3.80 with 6 N HC1, and diluted to 1 liter; (B) pH 5.00 citrate buffer containing 14.71 g Na3 citrate" 2HzO, 25.0 ml of 2.00 N N a O H , 35.07 g NaCl, titrated to p H 5.00 with 6 N HC1 and diluted to 1 liter; (C) pH 7.50 phosphate buffer prepared by mixing a 0.05 M Na2HPO4 solution 0.75 M in NaCI with a 0.05 M NazHPO4 solution 0.70 M in NaC1 until a pH of 7.50 is reached; (D) pH 9.4 borate buffer prepared by titration of a solution 0.05 M in H 3 B Q and 0.8 M in NaC1 with a 0.2 M N a O H solution 0.6 M in NaC1 to pH 9.4; (E) alkaline phosphate buffer prepared by mixing 900 ml of a solution 0.05 M in Na3PO4 and 0.65 M in NaCI with 100 ml of a solution 0.05 M in Na2HPO4 and 0.70 M in NaC1; (F) 0.2 N N a O H . To each liter of buffer were added 10 ml of Brij 35 solution (100 g plus 200 ml H20) and to each buffer except (F), 100/zl of octanoic acid. A nine-chamber gradient maker was filled as follows: Chamber 1, 55 ml pH 3.8 buffer and 20 ml pH 5.0 buffer; chamber 2, 15 ml pH 3.8 buffer and 60 m l p H 5.0 buffer; chamber 3, 75 ml o f p H 5.0 buffer; chamber 4, 20 ml pH 5.0 buffer and 55 ml pH 7.5 buffer; chamber 5, 75 ml o f p H 7.5 buffer; chamber 6, 35 ml pH 7.5 buffer and 40 ml pH 9.4 buffer; chamber 7, 75 ml of pH 9.4 buffer; chamber 8, 75 ml of alkaline phosphate buffer; chamber 9, 75 ml of 0.2 Y N a O H . Approximately 6 mg of the ampholyte mixture (calculated as dry weight) in 500/xl o f p H 3.8 citrate buffer was placed on a 0.5 x 135 cm column of

ANALYSIS OF CARRIER A M P H O L Y T E S

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sulfonated polystyrene ion-exchange resin (Aminex A-6, Bio-Rad) maintained at 60°C and previously equilibrated with the p H 3.80 buffer. The buffers from the gradient maker were pumped through the system at a pressure of 180-200 psi and flow rates of about 30 ml/hr. The effluent was sampled continuously, mixed with 2.88 vol ninhydrin reagent (40 g ninhydrin, 3 g hydrindantin, 5.3 liters of methyl cellosolve, 4 liters of water, 700 ml of 4 M sodium acetate buffer, p H 5.5), and the absorbance was measured at 550 nm. A stream-splitting arrangement was used to collect a portion of the column effluent in 3-ml fractions. The p H of the fractions was measured with a glass electrode and a Radiometer PHM4 pH meter.

Color Yield and Recovery Two types of experiments were performed to determine the recovery of ninhydrin-positive components during the analysis. In one, the color yield of a sample injected directly into the analyzer was compared with the total color yield calculated from the chromatogram. In a typical experiment, 5 ml of a 1:50,000 dilution of a 40% (w/~v)ampholyte was fed directly into the analyzer, and 15/~1 of the 40% (w/v) ampholyte diluted in 0.5 ml of p H 3.8 citrate buffer was applied to the ion-exchange column and eluted with the pH gradient described above. In another experiment, the weight of the ion-exchange resin was determined before and after equilibration with ampholyte and its elution. One-hundred milligrams of Aminex A-6 resin was placed on a fritted glass funnel and washed with 10 ml of citrate buffer pH 2.70 (same as (A) except titrated to p H 2.7 with 6 N HC1), followed by 10 ml of 0.2 N NaOH. The resin was washed with water and dried to constant weight at 160°C. The resin was next washed with 10 ml of. 1 N HC1 and 5 ml of H E H A wide-range ampholyte mixture (10% (w/v) in H20) adjusted to p H 2.5 with 0.1 N HC1. The ampholyte mixture was followed by 10 ml of pH 2.7 citrate buffer, 10 ml of 0.2 N N a O H , and 10 ml of water. The ion-exchange resin was then dried to constant weight.

Isoelectric Focusing on Polyacrylamide Gels The following experiment was performed in order to obtain some information about the correspondence between the pI's of the ampholyte components and their position in the elution profiles. Sperm whale myoglobin (Mann) was focused in three identical 3 mm x 10 cm polyacrylamide gels using the H E H A wide range ampholyte. Gels were crosslinked with methylene bis acrylamide (6% T, 2.5% C) and contained 2% (w/v) ampholyte. Focusing was carried out at 4°C for 18 hr in an analytical apparatus (Medical Research Apparatus) using 0.01 M H3PO4 and 0.02 M N a O H as anolyte and catholyte, respectively. A pulsed power supply (Ortec) was used at 150 V, 100 pulses/sec and 0.1/xFD. At the end of the run the myoglobin band (< 1 mm) was cut out of the gel. The anodic

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and cathodic portions of the three gels were combined and extracted separately in a volume of water equal to the volume of the gel for 2 days at room temperature. Approximately 1 ml of the supernatant was applied to the column and eluted as described earlier. RESULTS Three lots of commercial wide-range ampholytes (LKB lots No. 16, 17, and 52) were analyzed by the foregoing method. The results of the analyses of LKB ampholines lots No. 17 and 52 are shown in Fig. 1, A and B, respectively. Approximately 62 peaks are seen in both mixtures over the pH range 4 to 11. Most of the components are eluted over the pH range 4-7. The results of analyses of LKB pH 8-9.5 (lot No. 3) ampholine and LKB pH 4 - 6 ampholine (lot No. 7) are shown in Fig. 2, A and B, respectively. The acidic ampholyte consisted of about 35 components, while the basic ampholyte consisted of about 52 components. Most of the acid short-range ampholyte components are eluted in the first 230 ml while only a very small proportion of the components of the basic ampholytes are eluted in this volume. The broad peak at about 470 ml observed in most of the chromatograms, but which is most prominent in the chromatograms of the short-range ampholytes (Fig. 2), is a baseline artifact due probably to the elution of ammonia. The reproducibility of the elution volumes of the most prominent peaks in the chromatograms was ___3ml. The ninhydrin color yields of the ampholytes were measured in the i

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ANALYSIS OF CARRIER AMPHOLYTES

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various buffers used to form the gradient. They varied _+5% from the average. The color yield was slightly lower in the alkaline buffers. The recoveries obtained with commercial ampholytes as well as with ampholytes synthesized by us were usually quite good. In the first type of recovery experiment, two difficulties interfered with accurate interpretation of the data. The ampholyte mixtures were of such high complexity that some of the components were incompletely resolved, making estimation of the baseline difficult. The baseline used in the calculations was obtained from a run in which the ampholyte was omitted. Another difficulty was the fact that the system does not analyze all of the column eluate; of the 675 ml passing through the column, only 590 ml are analyzed. After the appropriate corrections were introduced, the recoveries of ninhydrin were calculated to be 97-101%. In the second type of recovery experiment, the gain in weight of the resin after the application of a large excess of ampholyte was found to be only 0.2 mg. This result is within the error of weighing and would represent 0.04% of the added ampholyte; hence, no ampholyte appeared to be irreversibly bound to the ionexchange resin. These experiments suggested that all of the material placed on the ion-exchange column is eluted. The column used for more than 25 analyses of ampholytes was also employed intermittently for amino acid analyses. No deleterious effects upon the resolution or sharpness of the amino acid peaks have been observed so far. This is a further indication that no accumulation of nonelutable material occurred on the ion-exchange column.

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The results of the analyses of the cathodic and anodic portions of wide-range H E H A ampholyte focused on a polyacrylamide gel concurrently with myoglobin are shown in Fig. 3. The wide-range H E H A ampholyte, consisted of some 70 components (Fig. 3C). The anodic (Fig. 3A) and cathodic (Fig. 3B) portions consisted of about 42 and 27 components, respectively. Some components, between the vertical arrows, appear in both fractions. DISCUSSION

The results of the analyses of commercial ampholytes shown in Figs. 1 and 2 provide compelling evidence for the view (Haglund, 1971; Catsimpoolas, 1973) that they are a complex mixture of closely related polyamine polycarboxylic acids. Comparison of the three wide-range LKB ampholines revealed a great deal of similarity in their compositions. Peaks corresponding to the major components in the chromatogram of lot No. 17 ampholyte can be easily located in the chromatogram of lot No. 52 ampholytes. They appear to be slightly shifted in position. The areas of these major peaks were approximately proportional in the two chromatograms. The peaks occurring at 110, 244,358,486, 528 and 544 ml in the pattern of lot No. 52 (Fig. 1A) possessed areas that were 43, 40, 48, 36, 39, 47, and 42%, respectively, of the areas under the corresponding peaks observed in the lot No. 17 pattern (Fig. 1B). Two major peaks seen in lot No. 52 were not found in lot No. 17; these occurred at 25 ml and at 305 i

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Analysis of carrier ampholytes by ion exchange chromatography.

AtNALYTICALBIOCHEMISTRY71, 325-332 (1976) Analysis of Carrier Ampholytes by Ion Exchange Chromatography I RAY K. BROWN, JUNE M. LULL, STEVEN LOWENKRO...
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