Biotechnology Letters22: 927–929, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Estimation of proteins in the presence of polyethylenimine

Vandana Gupta, Sunil Nath∗ & Subhash ChandDepartment of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi110 016, India∗Author for correspondence (Fax: +91-11-6868521; E-mail: sunath@dbeb.iitd.ernet.in)

Received 13 March 2000; Revisions requested 17 March 2000; Revisions received 6 April 2000; Accepted 10 April 2000

Key words:adsorption, bioseparation, carboxymethyl-cellulose, interference, ion-exchange resin, polyethylen-imine, proteins

Abstract

Polyethylenimine (PEI) interferes strongly in all commonly employed protein estimation assays. Here, a strategyto remove this interference is developed. Polyethylenimine is selectively removed by adsorption from a mixturecontaining PEI and lysozyme using carboxymethyl-cellulose, a cation-exchange resin between an ionic strength of0.35–1 M (pH 6.0), conditions under which lysozyme remains in solution and can be accurately estimated.

Introduction

Polyelectrolytes are charged polymers that interactwith oppositely charged proteins (for example, Nath1995, Li et al. 1999). These interactions havebeen exploited for the recovery of biomolecules inbioseparation (Sternberg & Hershberger 1974, Weiret al. 1993, Dissing & Mattiasson 1994, Nathet al.1995). Polyethylenimine (PEI) is a positively chargedpolyelectrolyte (pK 9.7) with the structural formula(–CH2–NH-CH2–)n (Lukovkin et al. 1973) and hasbeen used to purify a large number of proteins (forexample, see Dissing & Mattiasson 1996). To esti-mate the protein concentration during purification, itis necessary to know the specific activity of the pro-tein of interest. However, PEI interferes strongly in allstandard methods of protein estimation and preventsaccurate quantification of the protein concentration.Previous workers have either used enzyme activity inthe supernatant as a measure of the degree of pre-cipitation or diluted the samples to decrease the con-centration of PEI below the interference level (Zhaoet al. 1990, Dissing & Mattiasson 1996). As the con-centration of PEI remaining in the supernatant afterprecipitation is unknown, this leads to erroneous es-timation of proteins. Protein was also removed froma protein-PEI mixture by trichloroacetic acid (TCA)

precipitation (Jendrisak & Burgess 1975). The degreeof protein precipitation, however, is affected by thepresence of PEI. Therefore, removal of polyethylen-imine interference is essential for accurate estimationof proteins.

Our studies are aimed at developing a strategy toeliminate this interference of PEI. It involves the batchadsorption of PEI from a sample containing PEI andprotein under conditions that prevent the binding ofthe proteins on the adsorbent. Proteins can then beestimated by any of the commonly employed methods.Another advantage of our method is that a large num-ber of samples can be processed in a relatively shortspan of time.

Materials and methods

Materials

Polyethylenimine (number average molecular weight-60000), BSA, lysozyme, Coomassie Brilliant BlueG-250, bicinchonic acid (BCA) reagent, andcarboxymethyl-cellulose were from Sigma. All otherchemicals were of reagent grade.

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Fig. 1. Absorption of polyethylenimine (PEI) expressed as equiv-alent concentration of BSA as a function of PEI concentration bydifferent protein assays. (N) Folin–Lowry test at 660 nm; ( )bicinchonic acid (BCA) test at 562 nm; (�) Bradford test at 595 nm.

Protein and PEI estimation

PEI estimation by the Bradford, Folin–Lowry andBCA (bicinchonic acid) (Smithet al. 1986) methodswas accomplished following the standard protocols.Absorbance of PEI in these assays was converted intothe equivalent BSA concentration from the standardcurve of BSA.

Adsorption studies

Carboxymethyl-cellulose was equilibrated in sodium-succinate buffer (0.1 M, pH 6.0) for 24 h. NaCl (4M)was added to the protein, PEI or their mixtures to varythe ionic strength. Solutions containing protein (0.01–1 mg ml−1) and/or PEI (0.01–0.5 mg ml−1) and NaClwere mixed with 0.2 g carboxymethyl-cellulose (dryweight) per ml solution and vortexed for 30 s followedby centrifugation at 1100g for 5 min. The degree ofadsorption was measured by estimating the residualconcentration of protein and PEI in the supernatant bymodified Bradford method (Löffler & Kunzeh 1989).

Fig. 2. Adsorption of lysozyme and PEI on CMC as a func-tion of ionic strength at pH 6.0. (�) lysozyme (0.5 mg ml−1);(M) lysozyme (1.0 mg ml−1); ( ) PEI (0.1 mg ml−1); ( ) PEI(0.5 mg ml−1); (♦) lysozyme (0.5 mg ml−1)+ PEI (0.1 mg ml−1);(#) lysozyme (1.0 mg ml−1) + PEI (0.1 mg ml−1); (�) lysozyme(0.5 mg ml−1) + PEI (0.5 mg ml−1); (N) lysozyme (1.0 mg ml−1)+ PEI (0.5 mg ml−1).

Results and discussion

Interference of polyethylenimine in protein estimation

The interference by PEI in the commonly employedprotein assays is shown in Figure 1. Conversion of thedata of PEI absorbance to equivalent concentrationsof BSA shows a significant interference of the poly-ethylenimine in all these assays. PEI demonstrates themaximum sensitivity for the Bradford assay followedby Folin–Lowry and BCA methods. This interferenceexists even at very low concentrations of PEI, elim-inating the possibility of dilution of PEI-containingsamples below their interference levels. The results(Figure 1) show that this strong interferene of PEIcan result in errors of as high as 100% in the proteinestimation.

Removal of polyethylenimine interference

From the results shown in Figure 1, selective removalof PEI from the protein samples is required to per-mit an accurate estimation of protein concentration.A batch adsorption strategy to remove the PEI wasdesigned, using the carboxymethyl-cellulose as thecation-exchange adsorbent. The results of adsorptionof PEI and lysozyme on carboxymethyl-cellulose as afunction of ionic strength are shown in Figure 2. PEI,a highly charged polyelectrolyte, adsorbs stronglyon carboxymethyl-cellulose; 100% binding of PEI isachieved with 1 M salt concentration (as shown bythe bold circles in Figure 2). Lysozyme, on the otherhand, remains in the solution at this high ionic strength(open triangles in Figure 2). Further, the adsorptionof PEI and lysozyme in a mixture also shows sim-ilar segregated behaviour with salt concentration as

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shown individually by them at least at concentrationsof ∼1 mg ml−1. The data of Figure 2 demonstratesthat operation at greater than 0.5 M NaCl concen-trations leads to a selective removal of PEI fromdilute PEI-lysozyme mixtures and hence to removalof interference by PEI in protein estimation.

Conclusion

A strategy to remove polyethylenimine interference inprotein estimation has been developed. It has beenshown that above 0.5 M NaCl concentrations, thepolyethylenimine gets adsorbed to the carboxymethyl-cellulose resin, while the model protein lysozyme re-mains in the supernatant, thereby permitting accuratequantitation of proteins. The strategy should find ap-plication in a variety of protein-polymer precipitationand partitioning processes in bioseparation.

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