Biochemistry and Molecular Biology Education 28 (2000) 203}206

Chromatofocusing

Tomaz' MakovecInstitute of Biochemistry, Medical Faculty, University of Ljubljana, 1000 Ljubljana, Vrazov trg 2, Slovenia

Received 6 November 1999; accepted 20 January 2000

Abstract

A simple laboratory procedure is described for demonstrating the separation of proteins according to their isoelectric point. Itrequires a minimum of equipment and can be completed in 2 h laboratory class. ( 2000 IUBMB. Published by Elsevier Science Ltd.All rights reserved.

1. Introduction

Whether the goal is high-resolution separations on theanalytical scale or the preparative scale, isolation ofbiologically active macromolecules often includes theexploitation of molecular charge properties. Thus, ion-exchange chromatography is frequently used due to itsversatility and simplicity. Similarly, electrophoretic fo-cusing procedures, although generally more labor inten-sive and cumbersome with larger sample volumes, allowmacromolecular separations based upon di!erences innet surface charge. Indeed, isoelectric focusing, as is oftenreferred to, is perhaps the most discriminating high-resolution protein separation method available. Between1977 and 1981, Sluyterman and his colleagues o!ereda theoretical basis and practical means of realizing themost favorable attributes of both ion-exchangechromatography and isoelectric focusing in a singlechromatofocusing procedure [1}3].

During chromatofocusing, proteins are separated asa result of the isocratic formation of `internala pH gradi-ents on anion-exchange columns. This is achieved byemploying bu!ering system that maximize the bu!eringpower, and using strongly bu!ering ion exchangers. Inorder to minimize ionic strength, polymeric bu!ers calledpolyampholytes (commercial name: Polybu!er 96 andPolybu!er 74) are used, together with an anion ex-changer that has a very broad titration over the pH range2}9. The polyampholytes have both positive and nega-tive charges, and contribute very little to overall ionicstrength. Consequently, they can be used at relativelyhigh concentration in order to control the pH on the

E-mail address: tmakovec@ibmi.mf.uni-lj (T Makovec).

column very closely. Proteins are eluted according to theirisoelectric points, starting at high pH. The operating pro-cedure is to start by equilibrating the column at a pH highenough to bind the protein of interest, in polyampholytebu!er, then to apply to the column the polyampholyteadjusted to a pH lower than the protein's pI. A pHgradient develops on the column and a steady drop in pHeluting from the column is attained. One of the greatbene"ts of chromatofocusing is the ease of operation. Nogradient forming devices or mixers are required. The pHgradient is formed with a single eluent, isocratically. ThepH gradient slope is a function of the column bed volume,bu!er capacity of the matrix and chromatofocusing bu!erconcentration as well as the initial and "nal adjusted pH ofthe stationary and mobile phase, respectively. The proteinsshould be eluted close to their isoelectric points. In idealconditions, resolution of proteins di!ering in pI by as littleas 0.05 units is possible on a chromatofocusing column.The anion exchanger used (Polybu!er exchanger 94) haspolyethylene imine substitutients; the charged iminogroups interact with each other to create a continuum ofpK

!values. Chromatofocusing is successful with proteins

that are stable and soluble at their isoelectric point, as theyare eluted in a bu!er close to their pI. It is necessary to beable to remove the polyampholytes followingchromatofocusing before any further treatments. Someexamples of using chromatofocusing in puri"cation pro-cedures of proteins are listed in Refs. [4}7].

2. Experimental

Reagents required include Polybu!er exchanger 94(PBE 94); Polybu!er 74 (PB 74); bu!er A: 20 mM

1470-8175/00/$20.00 ( 2000 IUBMB. Published by Elsevier Science Ltd. All rights reserved.PII: S 0 3 0 7 - 4 4 1 2 ( 0 0 ) 0 0 0 2 0 - 0

Tris/HCl pH 8.5, 0.9% NaCl, 0.5 mM EDTA; bu!er B:20 mM Tris/HCl pH 8.5, 0.5 mM EDTA; bu!er C: 1 MTris/HCl pH 8.5; bu!er D: polybu!er 74 diluted 1 : 10with water, pH adjusted to 5.5 with 2 M NaOH; cyto-chrome c, 10 mg/ml in bu!er B.

All operations are carried out at room temperature.Erythrocytes were prepared from 100 ll of bovine blood,washed twice and then haemolyzed by incubation for5 min in 1 ml of bu!er B. The supernatant after centrifu-gation at 10,000 g for 5 min serves as a source of haemo-globin.

A column (U"0.5 cm) is "lled with 3}4 ml PBE 94,and the gel washed with 1 bed volume of bu!er C and2 bed volumes of bu!er B. The column is now ready foruse, and 50 ll of cytochrome c is added to the exposedtop of the gel. When all the sample has just entered thecolumn, 1 ml of bu!er D is added and the eluent collecteduntil the gel is exposed (the #ow of the eluent stopsautomatically at this point). Collect fractions in the samemanner until red material is eluted. The content of cyto-chrome c in the fractions is determined by measuring theabsorbance at 410 nm.

A 100 ll sample containing haemoglobin is added tothe top of another column and the procedure repeated inthe same manner as for cytochrome c. The pH of eachfraction is also determined. The content of haemoglobinis determined by measuring the absorbance at 410 nm.The pH of the fraction with the highest absorbance at410 nm approximately equals the isoelectric point ofhaemoglobin.

Fifty microliters of cytochrome c is mixed with 50 ll ofhaemoglobin and the resulting solution is added to thetop of the third column and the procedure is repeated.

3. Results and discussion

The laboratory exercise reported here demonstratesusefulness of chromatofocusing for the puri"cation andcharacterization of proteins. The use of coloured proteinssuch as cytochrome c and haemoglobin allows the possi-bility of following the chromatographic process by directobservation. In all experiments students have to predictthe results.

The "rst group of students examine the binding ofcytochrome c to the column. Cytochrome c (IEP"10.5),under the conditions used, does not bind to the columnand elutes in the void volume (Fig. 1a).

The second group of students examine the binding ofhaemoglobin to the column. Haemoglobin (IEP"7.1) atpH 8.5 binds to the column and during elution moves

cFig. 1. Chromatofocusing of (a) cytochrome c, (b) human haemoglobin,and (c) cytochrome c and haemoglobin on PBE 94; pH (j) and absorb-ance at 410 nm (r) were measured for each fraction.

204 T. Makovec / Biochemistry and Molecular Biology Education 28 (2000) 203}206

Fig. 2. Separation of haemoglobins A, E, F and S with MONO P HR5/20 column. pH (L) and absorbance at 280 nm (***) were mea-sured in each fraction.

down as a sharp red band. The pH of fraction withhighest absorbance was 7.1$0.1 (Fig. 1b).

The third group of students apply the mixture of cyto-chrome c and haemoglobin to the column which allowsthe separation of these two haem proteins. Both proteinsseparate immediately after beginning of the elution intotwo well-resolved red bands. The identity of both bandsis determined according to the results obtained or totheir spectroscopic properties in the range 500}600 nm(see Fig. 1c).

4. Problem

The possibility of detecting structural di!erences be-tween normal and abnormal haemoglobins is of signi"-cant clinical value. Since the discovery of HbS in 1949, atleast 350 other Hb variants have been described [8], andto date, electrophoresis has been the standard method forHb analysis. However, the technique has some limita-tions including di$culties in quantitating and recoveringthe separated haemoglobins. The introduction ofchromatofocusing column with high-resolution capabili-ties (MONO P, Pharmacia) allows very e$cient analyti-cal and preparative separations of human haemoglobins.Fig. 2 shows the well-resolved separation of di!erenthaemoglobin analogs, A,E,F and S, with MonoP HR5/20chromatofocusing column. Determine the identity of par-ticular haemoglobin variant in Fig. 2 (designated there asa, b, c and d) given that the mutations are: HbS:b6 GluPVal; HbE: b26 GluPLys; and HbF:b143 HisPSer. HbA is the normal adult haemoglobin.

Solution: The isoelectric points decrease from a to d.Peak a corresponds to HbE because HbE contains acidicamino acid substituted with the basic one. HbS containsacidic amino acid substituted with the amino acid with-out charge, so HbS elutes after HbE (b in Fig. 2). HbFcontains basic amino acid substituted with amino acidwithout charge and therefore elutes after HbA so theidentity is: peak a"HbE, peak b"HbS, peak c"HbAand peak d"HbF.

5. Notes

The fourth group of students performs the separationof cytochrome c and blue dextran on a Sephadex G100Super"ne column. Four groups of 3}4 students easilycomplete the whole experiment in a 2 h laboratory ses-sion.

Regeneration of PBE 94 can be done in the column,without repacking. The gel should be washed (2}3 bedvolumes) with 1 M NaCl to remove bound substances.Strongly bound proteins can be removed by washingwith 0.1 M HCl. If HCl is used, the gel must be re-equilibrated to a higher pH as soon as possible afterwashing. The gel is then ready for re-equilibration withstart bu!er at the desired pH.

Instead of PBE 94 other less expensive anion ex-changers can be used (DEAE-cellulose, DEAE-Sephacel,etc.). Unfortunately, a linear pH gradient with low ionicstrength, required for e$cient separation of proteins, isobtained only with expensive polybu!er PB 74. Forexample (see methods), elution of the PBE 94 with 20mM MES bu!er pH 5.5 results in total loss of resolvingpower of the column and haemoglobin and cytochromec elute together from the column.

Acknowledgements

I thank Spela Podkoritnik Mokorel for checking theEnglish.

References

[1] L.A. AE, Sluyterman, O. Elgersma, Chromatofocusing: isoelectricfocusing on ion-exchange columns I. General principles,J Chromatog. 150 (1978) 17}30.

[2] L.A. AE, Sluyterman, O. Elgersma, Chromatofocusing: isoelectricfocusing on ion-exchange columns II. Experimental veri"cation,J. Chromatog. 150 (1978) 31}44.

[3] L.A. AE, Sluyterman, J. Wijdenes, Chromatofocusing. III.The properties of a DEAE-agarose anion exchanger and itssuitability for protein separations, J Chromatog. 206 (1981)429}440.

[4] P. Gallo, Small column chromatofocusing of cerebrospinal#uid and serum immunoglobulin G, J. Chromatog. 375 (1986)277.

T. Makovec / Biochemistry and Molecular Biology Education 28 (2000) 203}206 205

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[6] S.G. Graber, A.R. Figler, J.C. Garrison, Expression and puri"ca-tion of G-protein a subunits using baculovirus expression system,Meth. Enzymol. 237 (1994) 212}225.

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[8] P. Basset, F. Braconnier, J. Rosa, An update on electrophoretic andchromatographic methods in the diagnosis of hemoglo-binopathies, J. Chromatog. 227 (1982) 267}304.

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