cell-free extracts for the presence of phytochelatins in- ..... At 4 mM. Na,SeO,, the lower of the two .... competitor, with the overall relative ranking being Hg >.
ANALYTICAL
BIOCHEMISTRY
200, 239-243 (19%)
A Gel Electrophoresis Susan L. Abrahamson,l USDAIARS
Received
David
Plant Gene Expression
June
Assay for Phytochelatins M. Speiser,
and David
26,199l
Phytochelatins are metal-binding peptides produced by higher plants and some fungi in response to heavy metal exposure. Established methods for analyzing cell-free extracts for the presence of phytochelatins include gel-filtration chromatography and HPLC. We have developed a nondenaturing polyacrylamide gel electrophoresis assay for phytochelatins that combines a small sample size with detection via metal binding. This assay can be used for the measurement of the relative affinity of phytochelatins for a variety of metal and semimetal ions. 0 1992 Academic Press, Inc.
Trace metal contamination in the environment is increasing due to human industrial activity (l), and within the continental United States, selenium contamination in irrigation runoff waters has been linked to severe developmental problems in wildlife (2). One biological response to heavy metal stress, induction of metallothionein proteins in animals, Drosophila, Neurospora, and some yeasts, is fairly widespread and well characterized (3). These cysteine-rich small proteins are presumably involved in both the detoxification of poisonous heavy metals and metal homeostasis (4). A second type of response, found in some fungi and in higher plants, involves the synthesis of small heavy metal-complexing peptides termed phytochelatins (5-7). Also called cadystins, Cd-binding peptides, r(EC),G, or y-glutamyl metal-bindingpeptides, these small peptides are secondary metabolites derived enzymatically from glutathione (8,9). The structure of phytochelatins consists of the repeated dipeptide y-Glu-Cys attached to a carboxyterminal glycine, with a structure of (r-Glu-Cys),-Gly, where n = 2 through 11 (10,ll). 1 Current address: Department of Plant Biology, fornia, Berkeley, CA 94720. ’ To whom correspondence should be addressed. 0003-2697/92
W. Ow2
Center, Albany, California 94710
$3.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
University
of Cali-
Phytochelatins (PCS)~ have been most thoroughly studied in the fission yeast Schizosaccharomyces pombe. Passage of crude S. pombe extract through a G50 gel filtration column resolves two subsets of phytochelatin-Cd complexes: one with a mobility corresponding to a molecular mass of approximately 10,000 Da (with a lower apparent value at higher ionic strengths) and the other with a more rapid mobility corresponding to approximately 4000 Da. The two PC-Cd species contain differing peptideimetal molar ratios (13) and the highmolecular-weight (HMW) species also contains acid-labile sulfide (12). Further analysis by HPLC (14) has shown that the HMW PC-Cd complexes contain longer average chain lengths than the low-molecular-weight (LMW) PC-Cd complexes. PCs have been studied biochemically through column chromatographic methods. To assay these peptides, crude cell extracts are run over gel-filtration columns (12). If a relatively pure preparation of PCs is required, several chromatographic techniques such as ion-exchange followed by size-exclusion chromatography must be combined (11,13). A faster technique that requires less starting material is HPLC, yet this technique has the limitation that it requires acid denaturation of the PC (11,13), preventing analysis of the native PCmetal complexes. Gel electrophoresis systems (15) have been used to evaluate extracts from Cd-exposed plant tissue, but this was mainly in an effort to purify and characterize the complexes that we now know as PCs. We have applied a gel electrophoretic technique to the question of phytochelatin assessment and identification in crude samples. This method offers several advantages. The amount of sample needed is approximately 40-fold less than that needed in a standard G50 gel-filtration column assay. Several samples can be analyzed at once and so the experimental efficiency is greatly in3 Abbreviations used: PC, phytochelatin; weight; LMW, low molecular weight; PMSF, phenylmethylsulfonyl fluoride.
HMW, high molecular DEAE, diethylaminoethyl;
239
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ABRAHAMSON,
creased and direct multisample comparison within one gel by the researcher is allowed. The time required to run the samples is fairly short, the apparatus is widely available, and the assay can be performed on very crude as well as relatively pure samples. Lastly, the assays are run under nondenaturing conditions, and, thus, through the use of radioactive metal isotopes, the visualization of PCs via their bound heavy metals is allowed. MATERIALS
AND METHODS
Muteri& Chemicals were of the highest commercially available grade: media from Difco Laboratories, ultra-pure acrylamide from Bio-Rad Laboratories, and G50 and DEAE ion-exchange resins from Pharmacia. Acid-washed glass beads and miscellaneous chemicals were purchased from Sigma. ‘OVd was purchased from New England Nuclear. S. pombe strain Sp223 (h-” ade6.216 ura4.294 leu1.32) was obtained from David Levin, and Sacchuromyces cereuisiae strain DBY745 was obtained from David Botstein. Sample preparations. For gel and column assays, lysates were prepared from 200 ml of yeast cells grown in YG media to 0Dss5 of 0.4. CdC1, was added to a final concentration of 200 pM for 30 h to induce phytochelatin synthesis. Cells were harvested through centrifugation and washed with 50 mM Tris, pH 7.8. Cells were disrupted by vortexing with acid-washed glass beads in 30-s bursts followed by 1-min rests on ice in 50 mM Tris, pH 7.811 mM PMSF, followed by clearing by centrifugation at 15,000g for 5 min. The supernatant was removed and dye-binding reagent (Bio-Rad Laboratories) was used for protein determination. For isolation of phytochelatins, 4 liters of yeast was grown to ODsg5 of 0.4 and phytochelatin synthesis was induced with 200 pM CdCl,. After cell breakage, supernatants were loaded onto a 9 X l.O-cm DEAE ion-exchange column that had been equilibrated with 50 mM Tris, pH 7.8, 0.1 M KCl. The column was washed with several column beds worth of column buffer and then eluted with a continuous salt gradient of 0.1 to 0.5 M KCl. Phytochelatins eluted off at a salt concentration of 0.15 to 0.3 M KCl, determined by first running the DEAE column with a yeast lysate labeled with lmCd. The phytochelatin-containing fractions were then pooled and lyophilized. After resuspension in 50 mM Tris, pH 7.8, the eluate was loaded onto a G50 column (described in detail below) and the two phytochelatincontaining peaks were collected and pooled. These two peaks were lyophilized and dialyzed extensively against 0.5 mM Tris, pH 7.811 pM CdCl, using a Spectrum Molecularporous dialysis membrane with a d&CO of 500 Da. Gel-filtration column chromatographic assays. G50 assay columns were done according to published protocol (12). Typically, 2 mg of crude protein extract in a
SPEISER,
AND
OW
volume of 1 ml was loaded onto a 50 X 1.5-cm column equilibrated with 50 mM Tris, pH 7.8. Sample buffer was 5% glycerol, 10 mM dithiothrietol. Phytochelatins were labeled through the inclusion of 0.25 PCi ‘?Zd in the applied sample. Gel ek?ctFophoFesis. Nondenaturing gels were run according to the following protocol. Forty percent acrylamide gels were made up with 16 g acrylamide and 0.035 g bisacrylamide, 15 ml H,O and 10 ml of 3.0 M Tris, pH 8.8. Gels were polymerized by addition of 14 ~1 of N,N,N,N-tetramethylethylenediamine and 500 ~1 of 10% (NH&&O,. Samples of crude extracts containing 50 c(g of protein along with any competitor metals (total volume, 20 ~1) were mixed with 5 ~1 of sample buffer, which was 50% glycerol, 1.5 M Tris, pH 8.8, and 0.01% bromophenol blue. Twenty minutes prior to loading, 0.05 PCi “‘Cd in 5 ~1 H,O was added to each sample. Gels were run at constant current at 15 mA for 48 to 72 h. Electrode buffer (135 IXhM Tris, pH 8.0, 90 mM boric acid) recirculation was necessary due to the long run length. Upon completion of electrophoresis, the wet gel was removed, wrapped in plastic wrap, and exposed overnight to Kodak XAR film at -70°C with a Cronex intensifying screen (DuPont). Bras&a juncea czern, Plant germination andgrowth. Arabidopsis thaliana (var. Columbia), Oryza satiua (cv. Taipei 309), Nicotiana tabacum (cv. Wi38), and Lycopersicon esculentum (cv. Ruteger) seeds were surface sterilized with 0.5% sodium hypochlorite/l% sodium dodecyl sulfate, washed with sterile H,O, and then placed on sterile Murashige and Skoog (Gibco) salts medium (containing 3% sucrose, 1 pg/ml nicotinic acid, 10 pg/ml thiamine HCl, 10 pg/ml pyridoxine HCl, and 100 pg/ml myoinositol) for germination. Seedlings were grown in a Nor-Lake illuminated growth chamber and induced with CdCl, added to the media to a concentration of 100 PM for 7 days subsequent to exposure of the third set of true leaves. Extracts were prepared by washing each seedling thoroughly followed by grinding in N,(l) and then extraction with 50 mM Tris, pH 8.00 mM PMSF. The extracts were cleared by centrifugation for 10 min at 15,000g. RESULTS We were interested in examining the electrophoretic characteristics of phytochelatins to determine if polyacrylamide gel electrophoresis could be used as an assay tool for these compounds. Figure 1 shows a typical lmCd gel-filtration column chromatographic profile from a cell-free extract of S. pombe induced with 200 pM CdCl, for 30 h. The profile shows three main ‘OBCd peaks: the void volume, the HMW PC-Cd peak, and the LMW PC-Cd peak. To determine if the PCs represented in this column profile could be visualized by electrophoresis, the crude extract was electrophoresed through a
PHYTOCHELATIN
ELECTROPHORESIS
241
ASSAY
nMwLMw :I,
1m9om-
pombe were electrophoresed
g2; g izl; 4cm3ooomoo1cmo-
-I
\
010
GEL
15
20
25
30
35 40 FrmtionX
45
50
55
60
65
70
FIG. 1. Cell-free extract of S. pombe induced with 200 pM CdCI, was analyzed by gel filtration and PCs were followed by inclusion of “‘Td as a tracer. HMW PC-Cd complex and LMW PC-Cd complex are shown.
40% polyacrylamide gel (Fig. 2A, lane 3). The autoradiogram shows that the crude extract yields two main resolved ‘Yd-containing species. In addition, a poorly resolved area of lower mobility is often seen as a smear above these two bands, with its intensity dependent on the length of autoradiographic exposure and the specific cell extract. Since detection is via the labeled metal ion, the species separated are presumed to be PC-Cd complexes, and the presence of more than one is consistent with the existence of complexes with heterogeneous compositions (14). Although PCs are the main Cd-binding components produced, it was important to establish that the signals observed in the electrophoresis assay are PCs and not another Cd-binding species. PCs from the crude extract described above were purified by a combination of ion-exchange and gel-filtration chromatography as described under Materials and Methods, and purified samples were labeled with lwCd before electrophoresis (Fig. 2A). Taken together, the purified HMW PC-Cd and LMW PC-Cd peaks show the same spectrum of species as that observed in the crude extract, with the HMW samples being biased toward the lower-mobility species (lane 2) and the LMW peak containing mainly higher-mobility bands (lane 1). In comparison to the bands observed with a Cd-induced culture, an equal amount of cell-free extract from an S. pombe culture not exposed to Cd shows no signal (Fig. 2B, lane 3), nor does a cell-free extract from Cd-exposed 5’. cerevisiae, which does not produce phytochelatins (6,7) (lane 5). Finally, neither glutathione (lane 2) nor ‘@Y!d alone (lane 1) give a signal. These data indicate that signals detected in the electrophoresis assay are due to PCs. Because detection of PCs in the gel electrophoresis assay is based on binding of labeled heavy metal ions, we were interested in determining whether the assay would reflect competition between different metal ions, on the basis of the relative affinity of PCs for the different elements. Identical samples from a crude extract of S.
in the presence of lmCd and varying concentrations of unlabeled metals in the sample mix (Fig. 3A). Addition of competing metals in increasing concentrations results in a loss of the signal. CdCl, causes a loss of approximately 50% of the signal at a concentration of 0.4 mM, while a lo-fold-lower concentration restored the ‘Y!d signal. CuCl, was able to completely eliminate the signal at 0.4 mM, indicating that PCs have an affinity for Cu2+ higher than that for Cd”. This finding is consistent with previous studies on the stability of PC-Cd and PC-Cu complexes (13,14). In contrast to these two metals, ZnCl, causes only modest showed no competition at 8 mM; lower concentrations effect. Higher levels of Zn could not be tested successfully, due to interference from the electrophoresis buffer, which caused precipitation in the sample well. Figure 3B shows further application of this competition assay to other metal ions. PbCl, showed effective competition only at 4 mM, indicating that its affinity for PCs is lower than that of Cd. However, HgCl, results in a reduction of losCd binding even at 0.04 mM, the lowest concentration of any ion tested. While synthesis of PCs is induced mainly by heavy metal cations, there are two anionic inducers known to function at a relatively low level in plant tissue culture cells, SeOi- and AsO:- (16). Addition of Na,SeO, to the assay at a concentration of 40 mM results in elimination of the signal. At 4 mM Na,SeO,, the lower of the two distinct PC-Cd species is not evident. This is the only example of unequal competition of different Cd-binding species that we have observed. SeOi- has similar activity (data not shown). NaH,AsO,, the other anionic PC inducer, is unable to compete at a concentration of 40 InM, the highest tested. As expected, CaCl,, which does
A.
1
2
3
B.
1
2
3
4
5
FIG. 2. Forty percent polyacrylamide gel assay of PCs. (A) Lane 1, purified LMW PC-Cd complex; lane 2, purified HMW PC-Cd complex; lane 3, cell-free extract of S. pombe induced with 200 pM CdCl,. (B) Lane 1, sample buffer (‘YZd only); lane 2,4 mM glutathione; lane 3,50 pg cell-free extract from S. pombe not exposed to Cd; lane 4,50 pg cell-free extract from S. pombe exposed to 200 pM CdCl,; lane 5,50 pg cell-free extract from S. cereukioe exposed to 200 pM CdCI,.
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ABRAHAMSON,
SPEISER,
AND
OW
DISCUSSION
B. competitor Concenhation blrm
FIG.
3.
Gel competition assay with 50 pg of a cell-free extract of S. with 200 PM CdClz, with the following concentrations of competing metals added: (A) CdCl, (4,0.4,0.04 mM), CuCl, (4,0.4, 0.04 mM), ZnCl, (8, 4, 0.4 mM), and no competitor. (B) PbCl* (4, 0.4 mM), HgCl, (0.4,0.04 mM), Na.$eO, (40,4 mM), Na,H,A,O, (40 mM), CaCl, (4 mM), CdCl, (4 mM), and no competitor.
The gel electrophoresis assay described above can detect PC-Cd complexes on the basis of exchange binding of radiolabeled “‘Cd. Two main resolved species are routinely detected. Dependent on specific induction conditions and autoradiographic exposures, lower-moving species have also been observed as a smear above the two main species. The heterogeneity is observed even in purified PC-Cd complexes from the HMW and LMW peaks, although there does appear to be a bias toward lower-mobility species in PCs from the HMW PC-Cd peak and vice versa. The effect of sample preparation and electrophoresis on the distribution of species is not known. This heterogeneous composition is not unexpected, since PC-Cd complexes that differ in the number of PC chains, chain lengths, the number of Cd’+ ions bound, and inclusion of S2- ions will have different charge/mass ratios and different physical dimensions, therefore leading to different electrophoretic mobilities. We believe that the main utility of this assay lies in its simplicity and sensitivity; sample preparation is minimal and samples containing less than 50 pg protein from a crude extract can be measured. Signal intensity depends on the total amount of PCs present and the amount and specific activity of the radiolabeled ion. A sample containing large amounts of uncomplexed Cd, for example, would result in a less intense signal than a sample in which all of the metal was bound within PCCd complexes. This fact prevents the use of this assay as
pombe induced
not induce the synthesis of PCs, does not compete for binding, nor do MgCl, or MnCl, (data not shown). To compare the electrophoretic behavior of PCs from fission yeast with that of PCs from a variety of plant species, we tested PC synthesis by the following species by the gel electrophoresis assay: B. juncea (wild mustard), A. thdiana, 0. satiua (rice), N. tabacum (tobacco), and L. esculentum (tomato). Seedlings from each species were induced for 1 week with 100 PM CdCl, in sterile growth media prior to analysis. Equal amounts (50 pg) of protein from cell-free extracts from each of the plant species were applied to the gel in the presence of ‘OVd (Fig. 4). B. juncea has the most intense signal of any of the plant species tested, nearly as much signal as the sample from the S. pombe sample. The species with the least signal was A. thuliana, followed by 0. satiua, N. tabacum, and finally L. esculentum, which had the second-highest signal. The signal intensity in this experiment, however, does not necessarily reflect total PC content, because of possible differences in total Cd content among the samples.
FIG. 4. Cell-free extracts from the following 100 I.IM CdCl,-induced species were subjected to electrophoresis: S. pombe (induced with 200 pM CdCl,), A. thalianu, 0. satiua, N. tabacum, L. esculentum, B. juncea (uninduced), and B. juncea (induced).
PHYTOCHELATIN
GEL
ELECTROPHORESIS
a quantitative measure of PCs in samples containing varying amounts and types of metals. With this in mind, however, the assay has great utility for the analysis of large numbers of small samples for the presence of PCs. In this role, it can be used to assay mutants or samples from kinetics studies. We have utilized the gel assay to screen a large collection of metal-sensitive S. pombe mutants and have tentatively identified a mutant that lacks detectable PC production (unpublished data). The utility of the assay also extends to samples derived from plants, as shown by the similar electrophoretic properties of PCs obtained from plants and fission yeast. The ability of different metal ions to compete for binding to PCs provides a simple measure of relative affinities. The experiments described above are consistent with previous studies (14) while extending the range of ions tested. Hg2+ is shown to be an especially effective competitor, with the overall relative ranking being Hg > Cu>Cd>Pbm SeO, > Zn > AsO, = Ca. The fact that SeOg- can compete is an indication that this anion (or some in vitro derivative) can displace Cd’+ from PC-Cd complexes. The manner of this interaction is unclear. Given the size and overall charge of the selenite ion, glutathione is the thiol compound most likely to be oxidized by Se0,2- (17), and perhaps PCs can also serve as an appropriate reducing agent. ACKNOWLEDGMENTS The authors thank Lisa Kreppel for generous velopment of the gel assay and John Thomas helpful discussions.
assistance and Daniel
in the deOrtiz for
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ASSAY
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