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Electric pulse-induced precipitation of biological macromolecules in electroporation
Bioelectrochemistry and Bioenergetics 48 Ž1999. 249–254
Short communication
Electric pulse-induced precipitation of biological macromolecules in electroporation Romualdas Stapulionis
)
Institute for Biomedical Research, and Department of Biochemistry, Kaunas Medical UniÕersity, Kaunas, Lithuania Received 23 April 1998; revised 10 September 1998; accepted 5 October 1998
Abstract We found that electric discharge through solution of biological macromolecules ŽDNA, RNA and proteins. causes precipitation of significant portions of these macromolecules. This precipitation is a consequence of the interaction of biological macromolecules with the metal ions solubilized from the anode plate by the electric pulse, and occurs in both absence and presence of the cells in poration medium. Precipitated fractions of macromolecules sediments at the centrifugation speed used to pellet eukaryotic cells and does not dissolve when washed with buffer. Our data indicate a complication of the direct evaluation of electroporation efficiency based on the assumption that electroporated biological macromolecules which remain associated with the cells after several washes, are successfully electroinjected into the cytoplasm of cells. q 1999 Elsevier Science S.A. All rights reserved. Keywords: Electroporation method; Pulse-induced precipitation; Biological macromolecules; Artefact; Solubilization of electrode
1. Introduction Electroporation is a method used to introduce extracellular macromolecules into living cells by the means of electric discharge w1x. This technique has become widely recognized in cell technology as a convenient tool which allows massive microinjection of poration medium components into millions of cells simultaneously. A variety of approaches are used to determine how much of exogenous material becomes electroinjected in the cells, the easiest and simplest of which is the measurement of material which remains associated with the pulsed cells after several centrifugationrwashing cycles w2–6x. The problem with the electroporation method is that it works but many questions on how it works remain still unresolved or under discussion w7x. As a consequence, intensive research sometimes generates unexpected results which do not fit into accepted norms of the electroporation process. Among such results are reports about adverse side effects of the electric pulse on both the cells and material to be electroporated. It was shown that pulse drastically
) Tel.: q370-7-731484; fax: q370-7-796498; e-mail: [email protected]
affects such properties of the both linear and circular DNA as electrophoretic mobility, susceptibility to nucleases and biological activity w8x. A high voltage electric pulse can cause cleavage of the DNA molecule which is thought to be mediated by the pulse-generated active oxygen species w9x. There have been reports that electric pulses solubilize metals from electrodes w10,11x. These metal ions introduced into the cells are very toxic and have pronounced effects on membrane properties w12x as well as on the biochemistry of electropermeabilized cells w13x. In many cases, conflicting data in the literature can be attributed to the dissolution of electrode material w1x. In this paper we present data demonstrating a new side effect of the electric pulse, namely, that during electric pulse Žin both absence and presence of the cells. a significant amount of metal ions is released from the anode, forming an insoluble precipitate and causing a co-precipitation of the main macromolecule groups-DNA, RNA and proteins. Co-precipitated biological macromolecules resist several washes with buffer and sediment very easily at the centrifugation speed used to spin down eukaryotic cells. This finding points out a complication regarding the above mentioned determination of electroporation efficiency, since precipitated Žbut not uptaken by the cells. target macromolecules sediment together with the cells, are not
0302-4598r99r$ - see front matter q 1999 Elsevier Science S.A. All rights reserved. PII: S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 2 0 6 - 2
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R. Stapulionisr Bioelectrochemistry and Bioenergetics 48 (1999) 249–254
removed by washing, and therefore, can be mistakenly considered as being introduced into the cells.
2. Materials and methods 2.1. Materials A mixture of tritiated amino acids Žleucine, lysine, phenylalanine, proline, and tyrosine. was obtained from Amersham. Unlabelled amino acids, bovine pancreas ribonuclease A, Trypan blue, Triton X-100 were purchased from Sigma. Cell culture reagents and medium were products of GIBCO. Rabbit liver tRNA and 3 H-labelled aminoacyl-tRNA were prepared as described w14x. 2.2. Cell culture Chinese hamster ovary ŽCHO. cells were obtained from J. Pachter ŽUniversity of Connecticut Health Center.. The cells were maintained as monolayers in Dulbecco’s modified Eagle’s medium supplemented with 5% Žvolrvol. calf serum, 5% Žvolrvol. fetal bovine serum, and proline Ž40 mgrml.. Cells were cultured at 378C in air containing 5% CO 2 in Falcon flasks Ž75 cm2 .. Cells were harvested with trypsin and used for experiments in suspended state. Cells were counted in a hemocytometer and their intactness was determined by trypan blue exclusion. 2.3. Electroporation procedure Electroporation was carried out using a BTX Transfector 300 ŽBiotechnologies and Experimental Research, San Diego. which generated exponentially decaying pulse and which was equipped with stainless steel electrode plates. Gap between electrodes was 3.5 mm. In some experiments stainless steel electrode plates were replaced with the aluminium or copper plates. Electroporation was performed at 660 V cmy1 and 500 or 1000 mF, which are conditions recommended for the pulsing of eukaryotic cells w15x. For some experiments other settings were used which is indicated in the figure legends. Electroporation cuvette was kept in ice during this procedure in order to avoid possible effects of heating. Volume of pulsed solution of macromolecules in KHBS buffer Ž21 mM Hepes, pH 7.4; 137 mM KCl; 5 mM NaCl; 0.7 mM K 2 HPO4 ; 6 mM glucose. was 0.4 or 0.8 ml. In the experiments with cells, CHO cells were harvested by trypsin treatment, washed once with complete culture medium, twice with KHBS buffer, and were suspended in 0.4 or 0.8 ml of KHBS. Small volumes of the materials to be electroporated were then added to the chambers and mixed. After delivery of electric pulse cuvettes were kept on ice for an additional 10 min, precipitate or cells washed twice with ice cold HBS buffer Ž21 mM Hepes, pH 7.4; 137 mM NaCl; 5 mM KCl; 0.7 mM Na 2 HPO4 ; 6 mM glucose..
2.4. Measurement of aminoacyl-tRNA (aa-tRNA) For the experiments 3 H-labelled aa-tRNA was used. Samples containing aa-tRNA were precipitated with 10% trichloracetic acid, collected on Whatman GFrC filters, washed 5 times with 2.5% trichloracetic acid and once with ethanol:ether Ž1:1.. Filters were incubated for 30 min at 508C in 1 ml of ‘Solvable’ ŽDuPont., and radioactivity was determined in liquid scintillation counter. 2.5. Aminoacyl-tRNA synthetase assay Reaction mixture of 0.1 ml contained 250 mM Tris– acetate, pH 7.4; 5 mM ATP; 5 mM MgCl 2 ; 0.2 mM EDTA; 200 mg bovine serum albumin, 0.1 mM 3 H-labelled amino acid, and 1 mgrml rabbit liver tRNA. Small amount of aminoacyl-tRNA synthetase was added, samples were incubated for 1.5 min at 308C, precipitated with 10% trichloracetic acid, and aa-tRNA formed was determined as above.
3. Results 3.1. Electric pulse releases iron from electrode plate into poration medium We made an observation that electric discharge through KHBS buffer in the absence of cells causes appearance of a precipitate which forms in the anode region of electroporation cuvette shortly after the pulse. Electroporation from the electrochemistry point of view is an instant electrolysis process, during which electric current causes solubilization of the anodic plate made of a non-noble metal, and amount of released ions is proportional to the net electric charge passed through the sample. BTX electroporator used by us was supplied with the stainless steel electrodes, therefore, we examined poration medium subjected to the pulse for the presence of iron. As seen in Fig. 1, substantial amount of iron was found in the poration medium after the pulse. Amount of the released iron depended directly on both field strength ŽFig. 1A. and capacitance ŽFig. 1B.. All solubilized from electrodes iron Žreleased as Fe 2q and Fe 3q ions. was in precipitated form since at neutral pH free iron ions form immediately insoluble compounds with OHy and PO43y, both present in KHBS. This precipitate sediments at very low rpm’s Ž400–500 = g . and does not dissolve while washed with buffer. 3.2. Electric discharge causes precipitation of soluble macromolecules Further experiments revealed that electric pulse affects solubility of variety of biological macromolecules. When
R. Stapulionisr Bioelectrochemistry and Bioenergetics 48 (1999) 249–254
Fig. 1. Iron concentration in the electroporation cuvette after electric discharge. 0.8 ml KHBS was subjected to the electric pulse under constant electric field 660 V cmy1 ŽA. or constant capacitance 1500 mF ŽB.. Iron was determined by the o-phenanthroline method.
solutions of DNA, RNA or proteins Žwithout cells. are subjected to the pulse, significant portion of these macromolecules becomes co-precipitated with the insoluble iron compounds ŽTable 1.. Up to 30% of pulsed DNA and total RNA can be pelleted at 7000 = g in just 2 min. Aluminium electrodes cause even greater precipitation than stainless steel electrodes ŽTable 1., and similar effect was observed using copper plates Ždata not shown.. Amount of precipitated macromolecules was proportional to the both field strength and capacitance. It is unlikely that precipitation is caused by the electrolysis-generated pH-effects, since in a separate experiment acid-precipitated macromolecules easily dissolve after readdition of buffer while pulse-induced precipitate is relatively stable to the washing
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with buffer Žeach wash causes removal of around 15% of precipitated macromolecules, which is due to the insufficient centrifugation speed for the decreased in size Žduring re-suspending. aggregates, and also due to the partial dissolution of them in a fresh buffer portion.. Attempts were made to check if this precipitation of macromolecules has any relation to the pulse-released iron ions. When chelating agent EGTA, which instantly binds iron ions, was present during the pulse then neither macromolecule precipitation nor formation of the above mentioned iron precipitate was observed after the electric pulse in spite the fact that amount of released metal remained unchanged. Introduction of iron ions Žwithout electric pulse. by the addition of FeCl 3 into solution of macromolecules in KHBS simulated the effect of electric discharge. Added 0.5 mM FeCl 3 , which is approximately equal to the amount of iron generated by the pulse at 660 V cmy1 and 1200 mF ŽFig. 1A., caused precipitation of around 10% of tRNA. These observations indicate that macromolecules are precipitated not directly by the electric discharge, but by the pulse-released iron ions. Supporting such conclusion is also the fact that macromolecules must be present at the moment when the free iron ions are generated Žeither by the pulse or by the addition of FeCl 3 . for the precipitation to occur. It is because at neutral pH free iron ions form instantly insoluble compounds with inorganic medium components, and therefore, become not available for the interaction with biological macromolecules. Consequently, macromolecules added several seconds after the pulse or FeCl 3 do not become precipitated. 3.3. Macromolecule precipitation and the measurement of electroporation efficiency In many cases, described in literature, efficiency of electroporation is evaluated as the amount of porated
Table 1 Precipitation of various macromolecules due to the electric discharge Material subjected to electric discharge
Percentage of total in precipitate Ž%. Stainless steel electrodes
Aluminium electrodes
DNA Total RNA tRNA Aminoacyl-tRNA synthetase
27 31 15 6
53 55 36 18
1.12 mg of Escherichia coli chromosomal w3 Hx-DNA, 8.4 mg of E. coli total w14 Cx-RNA or 10 mg of E. coli tRNA-CCA-w14 Cx were added in 0.8 ml KHBS and subjected to electric pulse of 230 Vr0.35 mm and 1600 mF. After incubation on ice for 10 min, each probe was pelleted Ž2 min at 7000 = g ., washed twice with HBS, and precipitated with 10% trichloracetic acid. 100% was radioactivity of the initial amount of material. For the aminoacyl-tRNA synthetase experiment 20 ml of rat liver postribosomal supernatant were subjected to the pulse under conditions as above, kept on ice, and washed twice. Aminoacyl-tRNA synthetase activity was determined in well-suspended precipitate. Activity of 20 ml of supernatant without pulse was set at 100%.
R. Stapulionisr Bioelectrochemistry and Bioenergetics 48 (1999) 249–254
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Fig. 2. Association of aa-tRNA with CHO cells. Ž1. aa-tRNA and cells were subjected to the electric pulse; Ž2. aa-tRNA was pulsed alone, and the cells were added immediately after the pulse; Ž3. to the suspension of cells, containing aa-tRNA, FeCl 3 was added up to 0.5 mM final concentration Žwithout pulse.. For all points 0.4 ml KHBS with indicated amounts of w3 Hx-aa-tRNA were used. Electric pulse was set at 660 V cmy1 and 500 mF. In all series cells were kept on ice for 10 min, pelleted and washed twice with HBS Ž2 min at 500= g ., and aa-tRNA in the cell fraction was measured.
material which remains associated with the cells after several washingrcentrifugation cycles w2–6x. Our observation that the pulse causes precipitation of macromolecules and that their precipitate sediments at the rpm’s used to spin down eukaryotic cells, indicates complication of such measurement: material which was simply precipitated due to the electric discharge and became co-pelleted with the cells can be considered as successfully electroinjected into the cells, thus, causing overestimation of electroporation efficiency.
This is demonstrated by the experiment shown in Fig. 2. When electroporation is carried out in the presence of both aminoacyl-tRNA Žaa-tRNA. and CHO cells, it is very easy to interpret results as a successful electroinjection ŽFig. 2, line 1. since amount of associated with the cells Ž‘electroporated’ aa-tRNA. material is proportional to the concentration of aa-tRNA in poration cell, to the field strength and capacitance Ždata not shown., and control experiments, when cells were not pulsed, show no association of aatRNA with the cells. However, everything of mentioned is true when the solution of aa-tRNA alone is subjected to the pulse, and the cells are added after the pulse ŽFig. 2, line 2.. Moreover, introduction of the metal ions by the addition of FeCl 3 into the medium containing both the cells and aa-tRNA gives similar effect even without any electric discharge ŽFig. 2, line 3.. If pulsed material were electroinjected into the cell, it would be isolated within the cell as a soluble matter. Thus, electroinjected aa-tRNA should be protected against RNase A action and be in the soluble state. Indeed, approximately 20% of aa-tRNA associated with the CHO cells is resistant to RNase A ŽTable 2.. However, after the treatment of cells with Triton X-100 which dissolves the cell membrane, most of the resistant to RNase A aa-tRNA sediments with the detergent insoluble fraction of cells, indicating that precipitation of macromolecules occurs during electric pulse in the presence of cells as well. Amount of aa-tRNA in X-100 supernatant which could represent soluble electroinjected aa-tRNA takes only a small fraction Žaround 4%. of the initial amount of aa-tRNA remaining associated with cells after the pulse. Moreover, this amount is comparable with that associated with the non-electroporated cells. It seems that at least within the first 30 min after the pulse there is no soluble electroporated material in the cytoplasm of the cells. Fluorescence-microscopy gives additional support for such conclusion: fluorescentlabelled tRNA was seen as ununiform clumps, most of which were associated with the cell membrane from the outer side Ždata not shown..
Table 2 Association of electroporated aminoacyl-tRNA with CHO cells after RNase A and Triton X-100 treatment Pulsing conditions
Aminoacyl-tRNArpmol Cells without RNase A treatment
y1
500 V cm ; 500 mF 700 V cmy1 ; 500 mF No pulse
0.256 0.500 0.048
Cells treated with RNase A Cells
X-100 pellet
X-100 supernatant
0.065 0.097 0.045
0.030 0.062 0.017
0.010 0.021 0.020
For electroporation at indicated conditions 213 pmol of w3 Hx-aa-tRNA were mixed with 11 = 10 6 cells in 0.8 ml KHBS, subjected to the pulse, kept on ice for 10 min, pelleted and washed twice with KHBS Ž2 min at 500= g .. One third of the cells was precipitated with 10% TCA. Remaining cells were incubated with RNase A at 278C for 10 min, washed twice with KHBS, and divided into two equal portions. One of them was immediately precipitated with TCA. The second portion was treated with 0.5% Triton X-100 for 5 min on ice, then pellet and supernatant were separated by centrifugation Ž5 min at 10 000 rpm., and each fraction precipitated with TCA. Aminoacyl-tRNA was determined as described in Section 2.
R. Stapulionisr Bioelectrochemistry and Bioenergetics 48 (1999) 249–254
4. Discussion This study reports a precipitation of nucleic acids and proteins caused by the pulse-solubilized electrode material. Earlier, there was a paper demonstrating the clotting of DNA by the electric pulse w8x, however, to our knowledge there have been no prior reports about the interaction of solubilized by the pulse metal ions with biological macromolecules. Latest review on electroporation warns about consequences of solubilization of metals from the electrode plates w1x, however, this concern focuses on toxicity and side effects of metal ions on the biochemistry of electropermeabilized cells, example of which was published recently showing pulse-induced dissolution of a substantial amount of aluminium ions which were increasing Ca2q release from L1210 cells w13x. Meanwhile, our findings indicate a complication of the often used approach for electroporation efficiency estimation w2–6x, namely, when electroporated material is considered as successfully electroinjected into the cell if it stays associated with cell pellet after several centrifugationrwashing cycles. We find that significant fraction of all tested biological macromolecules ŽDNA, RNA, proteins. becomes precipitated after the electric discharge in the absence of cells at pulsing conditions recommended for the eukaryotic cells w15x. This precipitation is a consequence of the interaction of solubilized from the anode plate metal ions with macromolecules, and it was observed using several non-noble metal electrode plates generally coming with commercial electroporators. Precipitated macromolecules sediment very easily at the rpm’s used to pellet eukaryotic cells and remain insoluble after several washings. Such precipitation occurs also in the presence of cells, therefore, electroporated cells assayed for the electroinjected material by the mentioned above approach are contaminated with the macromolecules which were precipitated and co-pelleted with the cells without having any relation to electroinjection. It seems that recently reported effects of the electric field on the susceptibility to nucleases, electrophoretic mobility and biological activity of various DNAs w8x could be explained in the terms of interaction of nucleic acid with the solubilized by the pulse metal ions. In analogy with the data in Ref. w8x we show that precipitated due to the pulse aa-tRNA is much less sensitive to the RNase A, also, it is unable to enter the gel during electrophoresis Ždata not shown.. However, these effects are lost when ‘scavenger’ of iron ions Žchelating agent EGTA. is present in the poration medium during the pulse. This fact suggests that interaction with solubilized metal ions is responsible for at least some pulse-induced properties of nucleic acids. It is too early to provide detailed view on how the precipitation of macromolecules occurs. Likely explanation would be that positively charged metal ions solubilized from the anode interact with the negatively charged residues of nucleic acids or proteins, neutralizing the repulsing
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forces between macromolecules or even creating metal ‘bridges’ between them, and in such way facilitating the aggregate formation. These events may resemble those when calcium phosphate is used for the DNA transfection. The latter procedure generates DNA aggregation and, in fact, is also based rather on the interaction of metal ion ŽCa2q . with the DNA since concentration of calcium exceeds that of phosphate by 175 times w16x. Ions solubilized from anodes of various metals ŽFe 2qrFe 3q, Al 3q and Cu2q . are capable to act in such way. There is no clear agreement about the mechanism of the electric pulse-induced macromolecule uptake by the cells. Widely accepted, at least among the non-expert users of this method, is the idea that macromolecules pass through pulse-induced pores in the cell membrane by simple diffusion w17,18x. If it is so, then overestimation of electroinjected material, determined as a probe associated with the cells after several washingrcentrifugation cycles, should be very high since we were not able to detect any soluble pulsed macromolecules in the supernatant of the disrupted by detergent cells within at least 30 min after the pulse. Other proposed mechanism is electrointernalization, when macromolecules are internalized over several hours by the means of pulse-induced endocytosis and are incorporated into membrane vesicles w1x. However, under such scenario, after the treatment of cells with detergent, electroinjected macromolecules should be found in the soluble state as well which was not the case in our experiments. Regarding the last mechanism, it is tempting to speculate that pulsed cells endocytose exogenous macromolecules, however, not in a soluble state but as precipitated due to the pulse material which becomes solubilized within the cell upon incubation in growth medium. However, the data are too preliminary to make a stronger statement. In conclusion, our study warns about the possible artefacts and suggests that at least within the 30 min after the pulse researchers should rely on more sophisticated methods for electroporation efficiency evaluation than looking for the cell-associated material.
References w1x U. Zimmermann, The effect of high intensity electric field pulses on eukaryotic cell membranes: fundamentals and applications, in: U. Zimmermann, G.A. Neil ŽEds.., Electromanipulation of Cells, CRC Press, New York Ž1996. pp. 1–106. w2x D. Kim, Y.J. Lee, C.M. Rausch, M.J. Borrelli, Electroporation of extraneous proteins into CHO cells: increased efficacy by utilizing centrifugal force and microsecond electrical pulses, Exp. Cell Res. 197 Ž1991. 207–212. w3x M. Glogauer, C.A.G. McCulloch, Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments, Exp. Cell Res. 200 Ž1992. 227–234. w4x A.K. Wilson, J. Horwitz, P. De Lanerolle, Evaluation of the electroinjection method for introducing proteins into living cells, Am. J. Physiol. 260 Ž1991. C355–C363.
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w5x D.C. Bartoletti, G.I. Harrison, J.C. Weaver, The number of molecules taken up by electroporated cells: quantitative determination, FEBS Lett. 256 Ž1989. 4–10. w6x R. Horan, R. Powell, J.M. Bird, F. Gannon, J.A. Houghton, Effects of electropermeabilization on the association of foreign DNA with pig sperm, Arch. Androl. 28 Ž1992. 105–114. w7x V.A. Klenchin, S.I. Sukharev, S.M. Serov, L.V. Chernomordik, Yu.A. Chizmadzhev, Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis, Biophys. J. 60 Ž1991. 804–811. w8x A.G. Sabelnikov, E.S. Cymbalyuk, Clotting of donor DNA during electroporation of prokaryotes, Bioelectrochem. Bioenerg. 24 Ž1990. 313–321. w9x E. Tamiya, Y. Nakajima, H. Kamioka, M. Suzuki, I. Karube, DNA cleavage based on high voltage electric pulse, FEBS Lett. 234 Ž1988. 357–361. w10x U. Zimmermann, J. Vienken, J. Halfmann, C.C. Emeis, Electrofusion: a novel hybridization technique, in: A. Mizrahi, A. Wenzel ŽEds.., Advances in Biotechnology Processes, Vol. 4, Alan R. Liss, New York Ž1985. pp. 79–150. w11x J. Teissie, Effects of electric fields and currents on living cells and their potential use in biotechnology: a survey, Bioelectrochem. Bioenerg. 20 Ž1988. 133–145.
w12x H. Gimmler, B. Treffny, M. Kowalski, U. Zimmermann, The resistance of Dunaliella acidophila against heavy metals: the importance of the Zeta potential, J. Plant Physiol. 138 Ž1991. 708–715. w13x J.W. Loomis-Husselbee, P.J. Cullen, R.F. Irvine, A.P. Dawson, Electroporation can cause artefacts due to solubilization of cations from the electrode plates, Biochem. J. 277 Ž1991. 883–885. w14x R. Stapulionis, M.P. Deutscher, A channeled tRNA cycle during mammalian protein synthesis, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 7158–7161. w15x M.N. Teruel, T. Meyer, Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells, Biophys. J. 73 Ž1997. 1785–1796. w16x J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York Ž1989.. w17x E.M. Neumann, M. Schaefer-Rider, Y. Wang, P.H. Hofschneider, Gene transfer into mouse lyoma cells by electroporation in high electric fields, EMBO J. 1 Ž1982. 841–845. w18x F. Toneguzzo, A. Keating, Stable expression of selectable genes introduced into human hematopoietic stem cells by electric fieldmediated DNA transfer, Proc. Natl. Acad. Sci. U.S.A. 83 Ž1986. 3496–3499.
Short communication
Electric pulse-induced precipitation of biological macromolecules in electroporation Romualdas Stapulionis
)
Institute for Biomedical Research, and Department of Biochemistry, Kaunas Medical UniÕersity, Kaunas, Lithuania Received 23 April 1998; revised 10 September 1998; accepted 5 October 1998
Abstract We found that electric discharge through solution of biological macromolecules ŽDNA, RNA and proteins. causes precipitation of significant portions of these macromolecules. This precipitation is a consequence of the interaction of biological macromolecules with the metal ions solubilized from the anode plate by the electric pulse, and occurs in both absence and presence of the cells in poration medium. Precipitated fractions of macromolecules sediments at the centrifugation speed used to pellet eukaryotic cells and does not dissolve when washed with buffer. Our data indicate a complication of the direct evaluation of electroporation efficiency based on the assumption that electroporated biological macromolecules which remain associated with the cells after several washes, are successfully electroinjected into the cytoplasm of cells. q 1999 Elsevier Science S.A. All rights reserved. Keywords: Electroporation method; Pulse-induced precipitation; Biological macromolecules; Artefact; Solubilization of electrode
1. Introduction Electroporation is a method used to introduce extracellular macromolecules into living cells by the means of electric discharge w1x. This technique has become widely recognized in cell technology as a convenient tool which allows massive microinjection of poration medium components into millions of cells simultaneously. A variety of approaches are used to determine how much of exogenous material becomes electroinjected in the cells, the easiest and simplest of which is the measurement of material which remains associated with the pulsed cells after several centrifugationrwashing cycles w2–6x. The problem with the electroporation method is that it works but many questions on how it works remain still unresolved or under discussion w7x. As a consequence, intensive research sometimes generates unexpected results which do not fit into accepted norms of the electroporation process. Among such results are reports about adverse side effects of the electric pulse on both the cells and material to be electroporated. It was shown that pulse drastically
) Tel.: q370-7-731484; fax: q370-7-796498; e-mail: [email protected]
affects such properties of the both linear and circular DNA as electrophoretic mobility, susceptibility to nucleases and biological activity w8x. A high voltage electric pulse can cause cleavage of the DNA molecule which is thought to be mediated by the pulse-generated active oxygen species w9x. There have been reports that electric pulses solubilize metals from electrodes w10,11x. These metal ions introduced into the cells are very toxic and have pronounced effects on membrane properties w12x as well as on the biochemistry of electropermeabilized cells w13x. In many cases, conflicting data in the literature can be attributed to the dissolution of electrode material w1x. In this paper we present data demonstrating a new side effect of the electric pulse, namely, that during electric pulse Žin both absence and presence of the cells. a significant amount of metal ions is released from the anode, forming an insoluble precipitate and causing a co-precipitation of the main macromolecule groups-DNA, RNA and proteins. Co-precipitated biological macromolecules resist several washes with buffer and sediment very easily at the centrifugation speed used to spin down eukaryotic cells. This finding points out a complication regarding the above mentioned determination of electroporation efficiency, since precipitated Žbut not uptaken by the cells. target macromolecules sediment together with the cells, are not
0302-4598r99r$ - see front matter q 1999 Elsevier Science S.A. All rights reserved. PII: S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 2 0 6 - 2
250
R. Stapulionisr Bioelectrochemistry and Bioenergetics 48 (1999) 249–254
removed by washing, and therefore, can be mistakenly considered as being introduced into the cells.
2. Materials and methods 2.1. Materials A mixture of tritiated amino acids Žleucine, lysine, phenylalanine, proline, and tyrosine. was obtained from Amersham. Unlabelled amino acids, bovine pancreas ribonuclease A, Trypan blue, Triton X-100 were purchased from Sigma. Cell culture reagents and medium were products of GIBCO. Rabbit liver tRNA and 3 H-labelled aminoacyl-tRNA were prepared as described w14x. 2.2. Cell culture Chinese hamster ovary ŽCHO. cells were obtained from J. Pachter ŽUniversity of Connecticut Health Center.. The cells were maintained as monolayers in Dulbecco’s modified Eagle’s medium supplemented with 5% Žvolrvol. calf serum, 5% Žvolrvol. fetal bovine serum, and proline Ž40 mgrml.. Cells were cultured at 378C in air containing 5% CO 2 in Falcon flasks Ž75 cm2 .. Cells were harvested with trypsin and used for experiments in suspended state. Cells were counted in a hemocytometer and their intactness was determined by trypan blue exclusion. 2.3. Electroporation procedure Electroporation was carried out using a BTX Transfector 300 ŽBiotechnologies and Experimental Research, San Diego. which generated exponentially decaying pulse and which was equipped with stainless steel electrode plates. Gap between electrodes was 3.5 mm. In some experiments stainless steel electrode plates were replaced with the aluminium or copper plates. Electroporation was performed at 660 V cmy1 and 500 or 1000 mF, which are conditions recommended for the pulsing of eukaryotic cells w15x. For some experiments other settings were used which is indicated in the figure legends. Electroporation cuvette was kept in ice during this procedure in order to avoid possible effects of heating. Volume of pulsed solution of macromolecules in KHBS buffer Ž21 mM Hepes, pH 7.4; 137 mM KCl; 5 mM NaCl; 0.7 mM K 2 HPO4 ; 6 mM glucose. was 0.4 or 0.8 ml. In the experiments with cells, CHO cells were harvested by trypsin treatment, washed once with complete culture medium, twice with KHBS buffer, and were suspended in 0.4 or 0.8 ml of KHBS. Small volumes of the materials to be electroporated were then added to the chambers and mixed. After delivery of electric pulse cuvettes were kept on ice for an additional 10 min, precipitate or cells washed twice with ice cold HBS buffer Ž21 mM Hepes, pH 7.4; 137 mM NaCl; 5 mM KCl; 0.7 mM Na 2 HPO4 ; 6 mM glucose..
2.4. Measurement of aminoacyl-tRNA (aa-tRNA) For the experiments 3 H-labelled aa-tRNA was used. Samples containing aa-tRNA were precipitated with 10% trichloracetic acid, collected on Whatman GFrC filters, washed 5 times with 2.5% trichloracetic acid and once with ethanol:ether Ž1:1.. Filters were incubated for 30 min at 508C in 1 ml of ‘Solvable’ ŽDuPont., and radioactivity was determined in liquid scintillation counter. 2.5. Aminoacyl-tRNA synthetase assay Reaction mixture of 0.1 ml contained 250 mM Tris– acetate, pH 7.4; 5 mM ATP; 5 mM MgCl 2 ; 0.2 mM EDTA; 200 mg bovine serum albumin, 0.1 mM 3 H-labelled amino acid, and 1 mgrml rabbit liver tRNA. Small amount of aminoacyl-tRNA synthetase was added, samples were incubated for 1.5 min at 308C, precipitated with 10% trichloracetic acid, and aa-tRNA formed was determined as above.
3. Results 3.1. Electric pulse releases iron from electrode plate into poration medium We made an observation that electric discharge through KHBS buffer in the absence of cells causes appearance of a precipitate which forms in the anode region of electroporation cuvette shortly after the pulse. Electroporation from the electrochemistry point of view is an instant electrolysis process, during which electric current causes solubilization of the anodic plate made of a non-noble metal, and amount of released ions is proportional to the net electric charge passed through the sample. BTX electroporator used by us was supplied with the stainless steel electrodes, therefore, we examined poration medium subjected to the pulse for the presence of iron. As seen in Fig. 1, substantial amount of iron was found in the poration medium after the pulse. Amount of the released iron depended directly on both field strength ŽFig. 1A. and capacitance ŽFig. 1B.. All solubilized from electrodes iron Žreleased as Fe 2q and Fe 3q ions. was in precipitated form since at neutral pH free iron ions form immediately insoluble compounds with OHy and PO43y, both present in KHBS. This precipitate sediments at very low rpm’s Ž400–500 = g . and does not dissolve while washed with buffer. 3.2. Electric discharge causes precipitation of soluble macromolecules Further experiments revealed that electric pulse affects solubility of variety of biological macromolecules. When
R. Stapulionisr Bioelectrochemistry and Bioenergetics 48 (1999) 249–254
Fig. 1. Iron concentration in the electroporation cuvette after electric discharge. 0.8 ml KHBS was subjected to the electric pulse under constant electric field 660 V cmy1 ŽA. or constant capacitance 1500 mF ŽB.. Iron was determined by the o-phenanthroline method.
solutions of DNA, RNA or proteins Žwithout cells. are subjected to the pulse, significant portion of these macromolecules becomes co-precipitated with the insoluble iron compounds ŽTable 1.. Up to 30% of pulsed DNA and total RNA can be pelleted at 7000 = g in just 2 min. Aluminium electrodes cause even greater precipitation than stainless steel electrodes ŽTable 1., and similar effect was observed using copper plates Ždata not shown.. Amount of precipitated macromolecules was proportional to the both field strength and capacitance. It is unlikely that precipitation is caused by the electrolysis-generated pH-effects, since in a separate experiment acid-precipitated macromolecules easily dissolve after readdition of buffer while pulse-induced precipitate is relatively stable to the washing
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with buffer Žeach wash causes removal of around 15% of precipitated macromolecules, which is due to the insufficient centrifugation speed for the decreased in size Žduring re-suspending. aggregates, and also due to the partial dissolution of them in a fresh buffer portion.. Attempts were made to check if this precipitation of macromolecules has any relation to the pulse-released iron ions. When chelating agent EGTA, which instantly binds iron ions, was present during the pulse then neither macromolecule precipitation nor formation of the above mentioned iron precipitate was observed after the electric pulse in spite the fact that amount of released metal remained unchanged. Introduction of iron ions Žwithout electric pulse. by the addition of FeCl 3 into solution of macromolecules in KHBS simulated the effect of electric discharge. Added 0.5 mM FeCl 3 , which is approximately equal to the amount of iron generated by the pulse at 660 V cmy1 and 1200 mF ŽFig. 1A., caused precipitation of around 10% of tRNA. These observations indicate that macromolecules are precipitated not directly by the electric discharge, but by the pulse-released iron ions. Supporting such conclusion is also the fact that macromolecules must be present at the moment when the free iron ions are generated Žeither by the pulse or by the addition of FeCl 3 . for the precipitation to occur. It is because at neutral pH free iron ions form instantly insoluble compounds with inorganic medium components, and therefore, become not available for the interaction with biological macromolecules. Consequently, macromolecules added several seconds after the pulse or FeCl 3 do not become precipitated. 3.3. Macromolecule precipitation and the measurement of electroporation efficiency In many cases, described in literature, efficiency of electroporation is evaluated as the amount of porated
Table 1 Precipitation of various macromolecules due to the electric discharge Material subjected to electric discharge
Percentage of total in precipitate Ž%. Stainless steel electrodes
Aluminium electrodes
DNA Total RNA tRNA Aminoacyl-tRNA synthetase
27 31 15 6
53 55 36 18
1.12 mg of Escherichia coli chromosomal w3 Hx-DNA, 8.4 mg of E. coli total w14 Cx-RNA or 10 mg of E. coli tRNA-CCA-w14 Cx were added in 0.8 ml KHBS and subjected to electric pulse of 230 Vr0.35 mm and 1600 mF. After incubation on ice for 10 min, each probe was pelleted Ž2 min at 7000 = g ., washed twice with HBS, and precipitated with 10% trichloracetic acid. 100% was radioactivity of the initial amount of material. For the aminoacyl-tRNA synthetase experiment 20 ml of rat liver postribosomal supernatant were subjected to the pulse under conditions as above, kept on ice, and washed twice. Aminoacyl-tRNA synthetase activity was determined in well-suspended precipitate. Activity of 20 ml of supernatant without pulse was set at 100%.
R. Stapulionisr Bioelectrochemistry and Bioenergetics 48 (1999) 249–254
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Fig. 2. Association of aa-tRNA with CHO cells. Ž1. aa-tRNA and cells were subjected to the electric pulse; Ž2. aa-tRNA was pulsed alone, and the cells were added immediately after the pulse; Ž3. to the suspension of cells, containing aa-tRNA, FeCl 3 was added up to 0.5 mM final concentration Žwithout pulse.. For all points 0.4 ml KHBS with indicated amounts of w3 Hx-aa-tRNA were used. Electric pulse was set at 660 V cmy1 and 500 mF. In all series cells were kept on ice for 10 min, pelleted and washed twice with HBS Ž2 min at 500= g ., and aa-tRNA in the cell fraction was measured.
material which remains associated with the cells after several washingrcentrifugation cycles w2–6x. Our observation that the pulse causes precipitation of macromolecules and that their precipitate sediments at the rpm’s used to spin down eukaryotic cells, indicates complication of such measurement: material which was simply precipitated due to the electric discharge and became co-pelleted with the cells can be considered as successfully electroinjected into the cells, thus, causing overestimation of electroporation efficiency.
This is demonstrated by the experiment shown in Fig. 2. When electroporation is carried out in the presence of both aminoacyl-tRNA Žaa-tRNA. and CHO cells, it is very easy to interpret results as a successful electroinjection ŽFig. 2, line 1. since amount of associated with the cells Ž‘electroporated’ aa-tRNA. material is proportional to the concentration of aa-tRNA in poration cell, to the field strength and capacitance Ždata not shown., and control experiments, when cells were not pulsed, show no association of aatRNA with the cells. However, everything of mentioned is true when the solution of aa-tRNA alone is subjected to the pulse, and the cells are added after the pulse ŽFig. 2, line 2.. Moreover, introduction of the metal ions by the addition of FeCl 3 into the medium containing both the cells and aa-tRNA gives similar effect even without any electric discharge ŽFig. 2, line 3.. If pulsed material were electroinjected into the cell, it would be isolated within the cell as a soluble matter. Thus, electroinjected aa-tRNA should be protected against RNase A action and be in the soluble state. Indeed, approximately 20% of aa-tRNA associated with the CHO cells is resistant to RNase A ŽTable 2.. However, after the treatment of cells with Triton X-100 which dissolves the cell membrane, most of the resistant to RNase A aa-tRNA sediments with the detergent insoluble fraction of cells, indicating that precipitation of macromolecules occurs during electric pulse in the presence of cells as well. Amount of aa-tRNA in X-100 supernatant which could represent soluble electroinjected aa-tRNA takes only a small fraction Žaround 4%. of the initial amount of aa-tRNA remaining associated with cells after the pulse. Moreover, this amount is comparable with that associated with the non-electroporated cells. It seems that at least within the first 30 min after the pulse there is no soluble electroporated material in the cytoplasm of the cells. Fluorescence-microscopy gives additional support for such conclusion: fluorescentlabelled tRNA was seen as ununiform clumps, most of which were associated with the cell membrane from the outer side Ždata not shown..
Table 2 Association of electroporated aminoacyl-tRNA with CHO cells after RNase A and Triton X-100 treatment Pulsing conditions
Aminoacyl-tRNArpmol Cells without RNase A treatment
y1
500 V cm ; 500 mF 700 V cmy1 ; 500 mF No pulse
0.256 0.500 0.048
Cells treated with RNase A Cells
X-100 pellet
X-100 supernatant
0.065 0.097 0.045
0.030 0.062 0.017
0.010 0.021 0.020
For electroporation at indicated conditions 213 pmol of w3 Hx-aa-tRNA were mixed with 11 = 10 6 cells in 0.8 ml KHBS, subjected to the pulse, kept on ice for 10 min, pelleted and washed twice with KHBS Ž2 min at 500= g .. One third of the cells was precipitated with 10% TCA. Remaining cells were incubated with RNase A at 278C for 10 min, washed twice with KHBS, and divided into two equal portions. One of them was immediately precipitated with TCA. The second portion was treated with 0.5% Triton X-100 for 5 min on ice, then pellet and supernatant were separated by centrifugation Ž5 min at 10 000 rpm., and each fraction precipitated with TCA. Aminoacyl-tRNA was determined as described in Section 2.
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4. Discussion This study reports a precipitation of nucleic acids and proteins caused by the pulse-solubilized electrode material. Earlier, there was a paper demonstrating the clotting of DNA by the electric pulse w8x, however, to our knowledge there have been no prior reports about the interaction of solubilized by the pulse metal ions with biological macromolecules. Latest review on electroporation warns about consequences of solubilization of metals from the electrode plates w1x, however, this concern focuses on toxicity and side effects of metal ions on the biochemistry of electropermeabilized cells, example of which was published recently showing pulse-induced dissolution of a substantial amount of aluminium ions which were increasing Ca2q release from L1210 cells w13x. Meanwhile, our findings indicate a complication of the often used approach for electroporation efficiency estimation w2–6x, namely, when electroporated material is considered as successfully electroinjected into the cell if it stays associated with cell pellet after several centrifugationrwashing cycles. We find that significant fraction of all tested biological macromolecules ŽDNA, RNA, proteins. becomes precipitated after the electric discharge in the absence of cells at pulsing conditions recommended for the eukaryotic cells w15x. This precipitation is a consequence of the interaction of solubilized from the anode plate metal ions with macromolecules, and it was observed using several non-noble metal electrode plates generally coming with commercial electroporators. Precipitated macromolecules sediment very easily at the rpm’s used to pellet eukaryotic cells and remain insoluble after several washings. Such precipitation occurs also in the presence of cells, therefore, electroporated cells assayed for the electroinjected material by the mentioned above approach are contaminated with the macromolecules which were precipitated and co-pelleted with the cells without having any relation to electroinjection. It seems that recently reported effects of the electric field on the susceptibility to nucleases, electrophoretic mobility and biological activity of various DNAs w8x could be explained in the terms of interaction of nucleic acid with the solubilized by the pulse metal ions. In analogy with the data in Ref. w8x we show that precipitated due to the pulse aa-tRNA is much less sensitive to the RNase A, also, it is unable to enter the gel during electrophoresis Ždata not shown.. However, these effects are lost when ‘scavenger’ of iron ions Žchelating agent EGTA. is present in the poration medium during the pulse. This fact suggests that interaction with solubilized metal ions is responsible for at least some pulse-induced properties of nucleic acids. It is too early to provide detailed view on how the precipitation of macromolecules occurs. Likely explanation would be that positively charged metal ions solubilized from the anode interact with the negatively charged residues of nucleic acids or proteins, neutralizing the repulsing
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forces between macromolecules or even creating metal ‘bridges’ between them, and in such way facilitating the aggregate formation. These events may resemble those when calcium phosphate is used for the DNA transfection. The latter procedure generates DNA aggregation and, in fact, is also based rather on the interaction of metal ion ŽCa2q . with the DNA since concentration of calcium exceeds that of phosphate by 175 times w16x. Ions solubilized from anodes of various metals ŽFe 2qrFe 3q, Al 3q and Cu2q . are capable to act in such way. There is no clear agreement about the mechanism of the electric pulse-induced macromolecule uptake by the cells. Widely accepted, at least among the non-expert users of this method, is the idea that macromolecules pass through pulse-induced pores in the cell membrane by simple diffusion w17,18x. If it is so, then overestimation of electroinjected material, determined as a probe associated with the cells after several washingrcentrifugation cycles, should be very high since we were not able to detect any soluble pulsed macromolecules in the supernatant of the disrupted by detergent cells within at least 30 min after the pulse. Other proposed mechanism is electrointernalization, when macromolecules are internalized over several hours by the means of pulse-induced endocytosis and are incorporated into membrane vesicles w1x. However, under such scenario, after the treatment of cells with detergent, electroinjected macromolecules should be found in the soluble state as well which was not the case in our experiments. Regarding the last mechanism, it is tempting to speculate that pulsed cells endocytose exogenous macromolecules, however, not in a soluble state but as precipitated due to the pulse material which becomes solubilized within the cell upon incubation in growth medium. However, the data are too preliminary to make a stronger statement. In conclusion, our study warns about the possible artefacts and suggests that at least within the 30 min after the pulse researchers should rely on more sophisticated methods for electroporation efficiency evaluation than looking for the cell-associated material.
References w1x U. Zimmermann, The effect of high intensity electric field pulses on eukaryotic cell membranes: fundamentals and applications, in: U. Zimmermann, G.A. Neil ŽEds.., Electromanipulation of Cells, CRC Press, New York Ž1996. pp. 1–106. w2x D. Kim, Y.J. Lee, C.M. Rausch, M.J. Borrelli, Electroporation of extraneous proteins into CHO cells: increased efficacy by utilizing centrifugal force and microsecond electrical pulses, Exp. Cell Res. 197 Ž1991. 207–212. w3x M. Glogauer, C.A.G. McCulloch, Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments, Exp. Cell Res. 200 Ž1992. 227–234. w4x A.K. Wilson, J. Horwitz, P. De Lanerolle, Evaluation of the electroinjection method for introducing proteins into living cells, Am. J. Physiol. 260 Ž1991. C355–C363.
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w5x D.C. Bartoletti, G.I. Harrison, J.C. Weaver, The number of molecules taken up by electroporated cells: quantitative determination, FEBS Lett. 256 Ž1989. 4–10. w6x R. Horan, R. Powell, J.M. Bird, F. Gannon, J.A. Houghton, Effects of electropermeabilization on the association of foreign DNA with pig sperm, Arch. Androl. 28 Ž1992. 105–114. w7x V.A. Klenchin, S.I. Sukharev, S.M. Serov, L.V. Chernomordik, Yu.A. Chizmadzhev, Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis, Biophys. J. 60 Ž1991. 804–811. w8x A.G. Sabelnikov, E.S. Cymbalyuk, Clotting of donor DNA during electroporation of prokaryotes, Bioelectrochem. Bioenerg. 24 Ž1990. 313–321. w9x E. Tamiya, Y. Nakajima, H. Kamioka, M. Suzuki, I. Karube, DNA cleavage based on high voltage electric pulse, FEBS Lett. 234 Ž1988. 357–361. w10x U. Zimmermann, J. Vienken, J. Halfmann, C.C. Emeis, Electrofusion: a novel hybridization technique, in: A. Mizrahi, A. Wenzel ŽEds.., Advances in Biotechnology Processes, Vol. 4, Alan R. Liss, New York Ž1985. pp. 79–150. w11x J. Teissie, Effects of electric fields and currents on living cells and their potential use in biotechnology: a survey, Bioelectrochem. Bioenerg. 20 Ž1988. 133–145.
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