High resolution gel chromatography of proteins

High resolution gel chromatography of proteins

ANALYTICAL BIOCHEMISTRY 124, 134-138 (1982) High Resolution Gel Chromatography of Proteins MALTERUTSCHMANN,LOTHARKUEHN,' BURKHARDTDAHLMANN,AND ...

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ANALYTICAL

BIOCHEMISTRY

124,

134-138 (1982)

High Resolution

Gel Chromatography

of Proteins

MALTERUTSCHMANN,LOTHARKUEHN,' BURKHARDTDAHLMANN,AND HANS REINAUER Biochemical Department, Diabetes Forschungsinstitut

an der Universit& Diisseldorf; D-4 Diisseldorf

Germany

Received December 31, 1981 The protein fractionation pattern on three different gels-Sephadex G-75 supertine, Ultrogel AcA 54, and Bio-Gel P-100 minus 400 mesh-has been compared. Analysis of the column eluates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated B&Gel P-100 to be a gel with very high separation efficiency, allowing complete resolution of proteins differing in M, by as little as 5000. The method is simple and inexpensive and allows the investigator to perform protein fractionations with resolution similar to that possible by highperformance liquid chromatography, but at a preparative scale and with little risk of protein denaturation.

Gel-permeation chromatography has been widely used in biochemical research and molecular weight estimation of macromolecular compounds (1). Often, a concentrated salt medium is required for solubility and/or activity of the desired sample component, and its purification may largely depend on the efficiency of gel-filtration techniques. High-performance liquid chromatography (HPLC),’ which has recently been introduced for the fractionation of proteins (2), gives excellent resolution and requires short times of analysis. This method, however, is confined to analytical (microgram) separations, precluding studies which involve larger (milligram) sample sizes. With the aim to achieve gel chromatographic separation of proteins similar to that possible under HPLC conditions but applying milligram quantities of sample, we have examined the chromatographic behavior of a protein mixture on three different gels of

similar fractionation range, namely, Sephadex G-75 superfine, Ultrogel AcA 54, and Bio-Gel P-100 minus 400 mesh. The results show that proteins with molecular weights between 13,000 and 68,000, the range tested, could be resolved with different efficiencies. Among the three gels, Bio-Gel P-100 was found to have an optimal resolving power. As judged by polyacrylamide gel electrophoresis, fractionation of the protein mixture on this gel resulted in a complete resolution of all components, including carbonic anhydrase and chymotrypsinogen A, two proteins differing in molecular weight by as little as 5000.

’ Address correspondence to Dr. Lothar Kuehn, Biochemical Department, Diabetes Forschungsinstitut, Auf ‘m Hennekamp 65 D-4 Dusseldorf 1, Germany. 2 Abbreviations used; HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate. 0003-2697/82/l Copyright A,, rider

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134

MATERIALS

AND METHODS

Proteins for gel chromatography were purchased from the following sources: bovine serum albumin, ovalbumin, chymotrypsinogen A, and ribonuclease A from Pharmacia, Uppsala, Sweden; carbonic anhydrase from Sigma, Mtinchen, Germany; Dextran Blue 2000 from Pharmacia. Low molecular weight marker proteins for SDS-polyacrylamide gel electrophoresis were purchased from BioRad Laboratories, Mtinchen, Germany. Chemicals for polyacrylamide gel electro-

HIGH

RESOLUTION

GEL CHROMATOGRAPHY TABLE

REPRODUCIBILITY

OF V, VALUES

OF PROTEINS

1 FRACTIONATED

ON DIFFERENT

Reproducibility” Protein Bovine serum albumin Ovalbumin Carbonic anhydrase Chymotrypsinogen A Ribonuclease A

135

OF PROTEINS

Molecular weight

Sephadex G-75 superfine

68,000 43,000 30,000 25,000 13,700

186 + 1.5 (4) 218 + 2.5 (4) 243 + 2.0 (4) 256 + 1.5 (4) 304 + 2.0 (4) r = 0.999’

GEL MEDIA

I’. (ml); means + SEM Utrogel AcA 54

176 f 206 f 239 f 260 + 298 + r =

1.25 (5) 2.75 (5) 1.75 (5) 1.75 (5) 2.5 (5) 0.996*

Bio-Gel P- 100 -400 mesh 91 f 111+2 139 + 155 t 187 + r =

2

(6) (6) 1.75 (6) 2.5 (6) 2.25 (6) o.995b

a Figures in parentheses indicate the number of fractionations. b Correlation factor calculated from linear regression analysis (see text).

phoresis were purchased from Serva Feinbiochemica, Heidelberg, Germany. The gel-permeation materials used were Sephadex G-75 superfine (fractionation range 3000-70,000 daltons, Pharmacia), Ultrogel AcA 54 (fractionation range 5000-70,000 daltons, LKB Producter AB, Bromma, Sweden), and Bio-Gel P-100 minus 400 mesh (fractionation range 5000100,000 daltons, Bio-Rad Laboratories). The fractionation ranges quoted are those obtained with globular proteins. Fractionation was carried out at 4°C in glass columns (20 X 900 mm, Multichrom CR, Reichelt Chemie, Heidelberg, Germany), having a bed volume of 283 ml and fitted with flow adapter heads as well as three-way valves for sample application. Sephadex G-75 and Ultrogel AcA 54 columns were run at hydrostatic pressure according to the directions supplied by the manufacturer, yielding flow rates of about 10 ml/h. Mariotte-type buffer reservoirs were used to ensure constant flow rates. Bio-Gel P-100 columns were developed under a constant nitrogen pressure of 3 bar, giving flow rates of about 3 ml/h. The eluant used throughout was 0.05 M TrisHCl buffer solution, pH 8.5, containing 1 M KCl. Protein in the eluate was monitored at 280 nm with an LKB Uvicord detection unit. SDS-polyacrylamide gel electrophoresis of the column eluates was carried out on 5

to 23% linear gradient slab gels as described by Chua (3). Prior to electrophoresis, protein in the respective eluate fraction was concentrated by mixing a 1-ml aliquot with 0.1 ml of a solution of 50% (w/v) trichloroacetic acid. After centrifugation at 23,OOOg, the supernatant was discarded and the pellet was dissolved in 0.3 ml of electrophoresis sample buffer (3), containing 6 M urea. Ten microliters of the sample was applied to individual gel slots. All other experimental details were as given in the legends to figures. RESULTS AND DISCUSSION

In gel-permeation chromatography, proteins are separated by size, with the larger molecules eluting ahead of the smaller ones. Table 1 shows that this assumption is fulfilled for the protein mixture fractionated on each of the three gels. In multiple fractionations, proteins are reproducibly eluted at the same position. With each gel, a linear relationship is obtained when the log of the molecular weight of the proteins is plotted against the elution volume of the respective peak fraction. Linear regression analysis of compiled data for each set of columns yields correlation coefficients in excess of 0.99. However, distinct differences between the resolving power of the three gel media became apparent when the chromatograms are

136

RUTSCHMANN

ET AL.

compared (Fig. l A-C). Thus, on Sephadex G-75, the peaks corresponding to carbonic anhydrase and chymotrypsinogen A are poorly resolved, with the two proteins essentially coeluting (Fig. 1A, peaks 3 and 4). A different fractionation pattern is observed

0

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after chromatography on AcA 54 columns in that all components are eluted as distinct peaks (Fig. 1B). Still, effluent peaks are not separated at the baseline, suggesting incomplete resolution into individual components. Finally, with Bio-Gel P-100 columns (Fig.

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FIG. 1. Elution profile of different proteins on (A) Sephadex G-75 superfine, and (C) B&Gel P-100 minus 400 mesh. Samples (1 ml) containing 1 mg of proteins were applied: peak 1, bovine serum albumin; peak 2, ovalbumin; peak peak 4, chymotrypsinogen A; and peak 5, ribonuclease A. Fractions (2.5 ml) were indicate the position of the proteins in the eluate; V, is the exclusion volume of

(B) Ultrogel AcA 54, each of the following 3, carbonic anhydrase; collected. The numbers the column.

HIGH RESOLUTION

GEL CHROMATOGRAPHY

OF PROTEINS

137

FIG. 2. SDS-poiyacrylamide gradient gel electrophoresis of column eluates from (A) Sephadex G-75 superfine, (B) Ultrogel AcA 54, and (C) Bio-Gel P-100 minus 400 mesh. Starting with the peak fraction of the V, peak in Fig. 1, every second fraction of the eluates was analyzed. Preparation of samples for gel electrophoresis was as described under Materials and Methods. Electrophoresis lasted for 16 to 20 h at a constant 25 mA. Gels were stained with 0.2% (w/v) Serva Blue R in ethanol/acetic acid/water (91219) and destained. The numbers on the horizontal bars indicate the proteins from the column eluates in Fig. 1A-C, respectively. Std: low molecular weight SDS standard protein mixture with (top to bottom) phosphorylase B (M. 94,000), bovine serum albumin (M, 68,000), ovalbumin (M, 43,000), carbonic anhydrase (M, 30,000), soybean trypsin inhibitor (M, 21,000), and lysozyme (M, 14,300).

lC), both resolution into individual components and separation of peaks at the baseline are achieved. The different efficiency of the three gel media is best illustrated by results obtained when column eluates are analyzed on SDSpolyacrylamide gradient gels. The finding that carbonic anhydrase and chymotrypsin-

ogen A are coeluted from Sephadex G-75 is confirmed by this analysis (Fig. 2A). While resolution of the two components is much improved upon fractionation on AcA 54 columns, SDS gels still reveal their incomplete separation (Fig. 2B), while analysis of Bio-Gel P- 100 column fractions shows no partial overlap of any two components.

138

RUTSCHMANN

The signi~cance of this result is underling by the fact that the peak 3 and 4 proteins differ in their M, by as little as 5000. While the results shown here are from experiments performed with 1 mg of each of the f&e proteins, up to 5 mg per protein component could be fractionated on Bio-Gel P-100, without loss of resolution (not shown). It should be noted that, due to the different physical properties of the gels employed, columns could not be eluted under identical conditions. Thus, optimal resolution was achieved when Sephadex G-75 or AcA 54 columns were eluted at hydrostatic pressure with flow rates of about 10 ml/h. Flow rates different from this value ensued a rapid decrease in separation efficiency. On the other hand, the fine-particle-sized Bio-Gel P-l 00 showed a high flow resistance, and only at 3 bar, the maximum pressure possible with this type of chromatographic equipment, a flow rate of about 3 ml/h was obtained. Nevertheless, the results clearly demonstrate that protein separations on B&Gel P- 100 columns are superior to those obtained with Sephadex G-75 or AcA 54 columns. In another study involving a biological sample, the resolving power of Bio-Gel P100 could be demonstrated (4). A proteolytic activity present in extracts of rat skeletal muscle, which during all previous attempts at purification had behaved as a single protein species, was eluted from Bio-Gel columns as two distinct activities with an M, of 25,000 and 18,000, respectively. For the different resolution of proteins achieved on the three gels, band-broadening effects could be a major cause. Band broadening and thus separation efficiency are often expressed as a function of the number of theoretical plates N according to Iv = 5.54( v,/w3

ill

where V, is the elution volume of a peak and Wo.5 is the width of the peak at half its height (5). Calculation of N, however, shows Sephadex G-75 to have the highest and Bio-

ET AL.

Gel P-100 to have the lowest number of theoretical plates. Thus, on the basis of N, the efficiency of separation should have been in the inverse order of that observed in the present study. An alternative explanation for the poor resolution of chymotrypsinogen A and carbonic anhydrase might be a relatively high content of aromatic amino acids of the latter, a property known to retard proteins on dextran-type gels such as Sephadex (6) but not on polyacrylamide gels of the Bio-Gel P series. Furthermore, the high rigidity of BioGel P- 100, when compared with the two other gels, may well have contributed to the results obtained in this study. In conclusion, we have shown that, under appropriate conditions, gel chromatography on Bio-Gel P- 100 minus 400 mesh allows separation of proteins with excellent resolution. Although this type of protein fractionation is more time consuming than are HPLC techniques, it has several obvious advantages: it is simple and economical, the risk of protein denaturation is minimized, and preparative quantities of sample can be processed. ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (SFB 113), Bonn, by the Ministerium fur Wiisenschaft und Forschung des Landes Nordrhein-Westfalen, Dusseldorf, and by the Ministerium fur Jugend, Familie und Gesundheit, Bonn, Germany. The expert technical assistance of Mrs. R. Schwitte is gratefully acknowledged.

REFERENCES 1. Determann, H. (1968) Gel Chromatography, p. 4, Springer, New York. 2. Rubinstein, M. (1979) Anal. Biochem. 98, 1-7. 3. Chua, N. H. (1980) in Methods in Enz~ology (San Pietro, A., ed.), Vol. 69, pp. 434-446, Academic Press, New York. 4. Rutschmann, M. (198 1) Masters thesis, University of Dusseldorf, Germany. 5. Schifreen, R. S., Hanna, D. A., Bowers, L. D., and Carr, P. W. (1977) Anal. Chem. 49,1929-1939. 6. Gelotte, B. (1960) J. Chromatogr. 3, 330-341.