Biochemical Engineering Journal 29 (2006) 23–26

Eliminating nucleic acids contaminants by hydrogen peroxide-inducedfree radicals during the preparation of proteins

Qing Yang a,∗, Jianqiang Xu a, Xuhong Qian b,∗∗, Kun Zhang a, Xuyu Lei a

a Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116024, PR Chinab National Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, PR China

Received 4 October 2004; accepted 8 February 2005

Abstract

Elimination of high order nucleic acids to reduce the systematic viscosity is an important step in the separation and purification ofproteins/enzymes. In this research, hydroxyl radicals produced by H2O2 was applied for the degradation of genomic nucleic acids from boththe leaf of maize and recombinant Escherichia coli cells. One percent agarose gel electrophoresis analysis showed that all genomic DNA wasdegraded by the exposure under 10 mM H2O2 in the presence of Fe(II) for 5 min to produce 70 bp fragments. To prolong the exposure time willruw©

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esult in much smaller nucleotides fragments. Compared with hydrogen peroxide, other peroxides showed lower DNA degradation activitynder the same conditions. Both SDS-PAGE analysis and western blotting assay showed that exposure at lower concentration (<10 mM)ithin 20 min, H2O2 did not impair proteins of interest, but >20 min exposure could cause damages to proteins.2006 Published by Elsevier B.V.

eywords: Degradation; Free radical; Genomic DNA; Hydrogen peroxide; Intracellular preparation

. Introduction

Elimination of high order nucleic acids to reduce theystematic viscosity is an important step in the separationnd purification of proteins/enzymes [1]. As to intracellu-ar preparations, high order nucleic acids can give rise to anncrease of viscosity interfering with the followed ultrafiltra-ion operation, and can bind to the separation media or pro-eins thus impairing the separation efficiency. Some organ-sms contain sufficient nuclease activity to eliminate thisroblem but, otherwise, the nucleic acids must be removedy precipitation or degraded by the addition of exogenousucleases. Ammonium sulphate precipitation can be effec-ive in removing nucleic acids; however, it will remove someroteins at the same time. Nucleic acids bind efficiently to

∗ Corresponding author. Tel.: +86 4118 4709 687;ax: +86 4118 2300 288.∗∗ Corresponding author. Tel.: +86 4118 3673 466;ax: +86 4118 3673 488.

E-mail addresses: qingyang@dlut.edu.cn (Q. Yang),

anion exchangers (DEAE- and Q-) but sometimes tracesof nucleic acids can escape and be mixed with the pro-tein of interest. Various more specific precipitants, such aspolyethyleneimine [2], the cationic detergent cetyltrimethylammonium bromide [3], streptomycin sulphate [4] and pro-tamine sulphate [5] have been used. All of these are expensiveand possibly toxic, and also they may complex undesirablywith certain enzymes resulting in a loss of purity. There-fore, it is important to make sure that this kind of reagentscan be removed easily in the purification procedure. Nucleicacid degrading enzymes are the often choice early in thepurification process and to be sure to be removed in thelater stages. However, these enzymes are very expensive, sonew alternatives for nucleic acids degradation are of greatpotential.

H2O2 can cleave DNA through releasing hydroxyl radi-cals by Fenton reaction [6]. Under an irradiation of ultravioletlight or in the presence of metal ions or metal iron–EDTAcomplexes, the generated hydroxyl radical from hydrogenperoxide abstracts the hydrogen atoms on the deoxyribosemoieties and splits DNA chain into pieces without nucleotide

hqian@dlut.edu.cn (X. Qian). specificity after a series of consecutive reactions [7,8]. As it

369-703X/$ – see front matter © 2006 Published by Elsevier B.V.oi:10.1016/j.bej.2005.02.022

24 Q. Yang et al. / Biochemical Engineering Journal 29 (2006) 23–26

produces only water, there is no contamination left in the reac-tion system. Thus, it may have much potential in many fields.Aside from the wide application in sterilization, the mostimportant contribution of this reaction is in DNA footprinting[9,10]. The others include secondary structure analysis [11]and covalent linkage to intercalating agents [12]. In 2001,Zhang et al. applied this reaction in the preparation of nucleicacids for microarray hybridization [13]. Recently, Konat etal. applied this reaction to have revealed a novel mechanismof oxidative genotoxicity in vivo [14] and to have found thathydrogen peroxide mediated the degradation of higher orderchromatin which is an integral component of programmedcell death [15,16].

However, there has been no concern about its potentialuse in the down-stream process of biochemical preparationsso far. The objective of this paper is to provide possibil-ity that H2O2 at an appropriate concentration may func-tion as an efficient and convenient alternative to degradegenomic DNA in the preprocess step of purification of bio-chemicals to eliminate genetic materials from transgenicvarieties.

2. Materials and methods

2.1. Total DNA extraction from the leaf of maize

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2.4. SDS-PAGE analysis and Western blotting assay

The total cellular proteins treated by hydrogen peroxidewas analyzed on 10% SDS-PAGE according to the manual ofPhastGel System (Pharmacia, Sweden). Protein bands werestained with Coomassie Brilliant Blue R-250. The proteinbands were electrophoretically transferred onto nitrocellu-lose membrane at 0.65 mA cm−2 for 2 h. The membrane wasblocked with 20 g l−1 fat-free milk and incubated with horseanti-His-Tag serum (Invitrogen, USA), diluted 1:300, fol-lowed by rabbit anti-horse IgG-HRP conjugate (Invitrogen,USA), diluted 1:500. The bound antibody was detected byusing 3,3′-diaminobenzidine and hydrogen peroxide.

3. Results and discussion

3.1. Genomic DNA degradation induced by peroxides

Twenty micrograms genomic DNA of maize in 200 �lwater solution was exposed to H2O2 at the desired con-centration. The degradation started up by the addition of1 mM Fe2+ and stopped by the addition of 10% glycerolinto the reaction mixture. After 5-min exposure at 20 ◦C, allgenomic DNA degraded at the concentration above 10 mM(Fig. 1A). The rate of this DNA degradation could be shownt0dhoawaosc

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The extraction of total DNA from the fresh leaf ofransgenic maize was performed according to the methodsescribed by Junghans and Metzlaff [17]. Resuspend theNA in TE buffer with the final concentration of 250 ng �l−1.

.2. Genomic DNA degradation

Genomic DNA (500 ng) from the leaf of maize was addedo the reaction buffer consisting of 10 mM Tris/HCl/1 mMDTA–Fe(II) (pH 7.5). The reaction was allowed to run formin, and the mixture was applied onto 1% agarose gel forlectrophoresis analysis.

.3. Intracellular nucleic acids removal

Escherichia coli BL(21)DE3 cells harboring the matureene of gloshedobin, a thrombin-like enzyme from the venomf snake, Gloydius shedaoensis [18], were grown at 37 ◦C inuria broth containing 50 mg l−1 ampicillin. The cells were

noculated into fresh medium and grown at 37 ◦C until theell density reaches 1 mg ml−1 approximately. Cells wereollected by centrifuge at 10,000 × g for 30 min. Then, theupernatant was decanted and 5 Units lysozyme in 10 mMris/HCl (pH 7.5) was added into 1 g cells at 25 ◦C for0 min. Finally, the intracellular nucleic acids were treatedith 10 mM Tris/HCl/1 mM EDTA–Fe2+ (pH 7.5) contain-

ng 10 mM hydrogen peroxide or organic peroxides. Thefficiency of nucleic acid degradation was assayed by 1%garose gel electrophoresis.

o be concentration-dependent, having lower efficiency at.1 mM and a slower degradation at 1 mM but a higher degra-ation activity at 10 mM (Fig. 1A). In case of generatingydroxyl free radicals in the presence of metal ions insteadf ultraviolet light, the experimental result showed that theddition of Cu(II), Cd(II), Co(II), Sn(II), Zn(II) or Fe(II)as much more efficient rather than that of Hg(II), Mg(II)

nd Ca(II) (Fig. 1B). Compared with hydrogen peroxide, thether organic peroxides, peracetic acid and performic acid,howed lower DNA degradation activity under the identicalonditions (Fig. 1C).

.2. Intracellular nucleic acids removal

The disruption mixture of E. coli cells was directly loadedo expose under H2O2 at the desired concentration. Theegradation started up by adding 1 mM Fe(II) and stopped byhe addition of 10% glycerol. After 5-min exposure at 20 ◦C,ll intracellular nucleic acids degraded with H2O2 concen-ration above 10 mM (Fig. 2) to give low molecular weightragments as low as 70 bp. The rate of this DNA degradationould be shown to be concentration-dependent, having lowfficiency at 0.1 mM, a slow degradation at 1 mM but a highegradation activity at 10 mM (Fig. 2).

.3. Effect on the structure of proteins

The enzyme applied here was purified from E. coli lysatesy a procedure involving Ni(II)–IDA Sepharose affinity col-mn. The purified proteins were incubated with 10 mM

Q. Yang et al. / Biochemical Engineering Journal 29 (2006) 23–26 25

Fig. 1. Degradation of genomic DNA by hydrogen peroxide. (A) One percent agarose gel electrophoresis assay for the degradation of maize genomic DNAby hydrogen peroxide at different concentrations. Two microlitres of each sample was applied. Lanes 1–5 represent the concentration of hydrogen peroxide asfollows: 100, 10, 5, 1, 0.1 mM, respectively. (B) Genomic DNA degradation by 10 mM hydrogen peroxide initiated by the addition of 1 mM metal ions. Tenmillimolars hydrogen peroxide was mixed with 200 �l solution containing 20 �g E. coli genomic DNA, then the mixture was incubated at 20 ◦C for 5 min. Twomicrolitres reaction mixture was subjected to 1% agarose gel electrophoresis analysis. Lane 1, no metal ion added; lanes 2–10, Cu(II), Hg(II), Cd(II), Co(II),Sn(II), Zn(II), Mg(II), Ca(II) and Fe(II), respectively. (C) Degradation of maize genomic DNA by 10 mM peroxides in the presence of 1 mM Fe2+. Lane 1, noaddition of peroxide; lane 2, addition of 10 mM hydrogen peroxide; lane 3, 10 mM peracetic acid; lane 4, 10 mM performic acid.

Fig. 2. Intracellular nucleic acid degradation using hydrogen peroxide. Onepercent agarose gel electrophoresis assay for the degradation of intracellu-lar nucleic acid using hydrogen peroxide at different concentrations. Twomicrolitres of each sample was applied. M represents marker ØX174-HaeIII digest; lane 1, genomic DNA; lanes 2–6 represent the concentration ofhydrogen peroxide as follows: 0.1, 1, 5, 10 and 100 mM, respectively.

hydrogen peroxide in the presence of 1 mM Fe(II) for dif-ferent time. The effect on immunology activity was ana-lyzed by both SDS-PAGE electrophoresis analysis and west-ern blotting assay. The impairment by hydrogen peroxideto protein primary structure is time-dependent (Fig. 3A).There was neither excessive band nor band position changeon SDS-PAGE gel, indicating that 20 min is the durationtime for maintaining the primary structure of gloshedobin.However, prolonging the exposure time to 60 min, the pro-tein of interest was found to be smearing on SDS-PAGE

Fig. 3. Effect on recombinant gloshedobin by exposure under hydrogen per-oxide at various time intervals. (A) The protein analyzed on 10% SDS-PAGEstained with Coomassie Brilliant Blue R-250. Fifty microlitres protein ofeach sample was applied. The intracellular enzyme was purified from E.coli lysates before exposure under 0.01 M H2O2. Lane 1, 60 min; lane 2,20 min; lane 3, blank. (B) Western blotting analysis. Lane 1, 60 min; lane 2,20 min; lane 3, blank.

gel (Fig. 3A, lane 1), suggesting the degradation of pro-teins into smaller fragments occurred. Furthermore, westernblot assay using anti-His-Tag serum proved that the corre-sponding antibody could recognize the protein of interest,suggesting no effect on immunologic activity of the proteinafter incubated with 10 mM hydrogen peroxide for 20 min(Fig. 3B).

4. Conclusion

Although the free radical cleavage agents are not suit-able for being used in vivo due to their harmful effects tothe biological systems, there are many other applications invitro. Our studies suggested that hydrogen peroxide at theconcentration lower than 10 mM for 5 min could be usedas an efficient DNA degradation agent. Elongation of expo-sure time will result in <70 bp nucleotide fragments. Theresults suggested that hydrogen peroxide could be appliedin eliminating nucleic acids during transgenic varietiesprocessing.

Acknowledgement

This study was under the auspices of 863 Project for High-TKT

R

ech (Contract number 2003AA2Z3520) and the Nationaley Project for Basic Research, the Ministry of Science andechnology of China (Contract number 2003CB114405).

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