Environ. Sci. Technol. 1992, 26, 1202-1205
Characterization of Cd Binding Sites on Datura innoxr'a Using li3Cd NMR Spectrometry Huel-Yang D. Ke and Gary D. Rayson" Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
The interactions between Cd(I1) and Datura innoxia cells have been examined using l13Cd NMR spectroscopy. A series of model solutions containing carboxylate, amine, hydroxyl, sulfhydryl, oxalate, sulfate, and sulfonate functionalities have been utilized to identify the moieties on the cell wall responsible for the observed chemical shifts. Carboxylate groups have been determined to be the dominant functional groups responsible for binding of Cd(I1) on the cell wall of D. innoxia at pH 1 5 . At pH 6, diamine groups have been observed to be involved in the binding of Cd(I1) to the biomass. Chemical shift data indicate carboxylate groups could interact with Cd(I1) in a 2:l ratio. The chemical shift of l13Cd NMR also suggests a rapid exchange equilibrium among Cd(I1)-containing species near the cell walls at pH 6. Introduction It has been known for some time that nonliving biomass materials could be used to remove toxic heavy and economically important metal ions from wastewaters and mining effluents by means of biosorption (adsorption) of metal ions onto the surface of microorganisms (1-5). A variety of strains of algae (1-3) have been successfully demonstrated to bind to Ag(I), Cu(II), Cd(II), Zn(II), Fe(11), Al(III), and Au(II1). Biosorption occurs mainly through interaction of the metal ions with functional groups contained in or on the cell wall biopolymers of either living or dead organisms (1). It has been proposed that complexation and electrostatic binding are the two main biological processes by which microorganisms remove metal ions from solutions (1, 6 ) . However, the chemical mechanisms responsible for the biosorption of metal ions by bioorganisms is still not clear. It has been suggested by Darnall et al. (1)and Crist et al. (7) that amino, thioether, sulflydryl, carboxyl, carbonyl, imidazole, phosphate, sulfate, phenolic, hydroxyl, and amide moieties are the possible functionalities responsible for the binding of metal ions on the algal cell walls. However, little characterizationof the binding of metal ions to cell walls has been reported, leaving the designation of these functionalities for binding to the appropriate metal ions relatively unclear. Compared with algal cell walls, little work has been published on the adsorption of metal ions by nonliving higher plant cells (8,9).Thus, it is still unknown whether higher plant tissues adsorb metal ions through the same processes as those observed in algae. However, since the higher plants possess similar biological molecules in their cell walls, it has been suggested that the same kind of adsorption on the plant cell walls is likely to occur. This paper discusses the investigation of the cadmium binding behavior of one type of higher plant cell, antheral cells from Datura innoxia, using I13Cd NMR. D. Innoxia, more commonly known as Datura meteloides or Sacred Datura, is a plant which is indigenous to Mexico and the southwestern United States. D. innoxia was selected because it belongs to the well-studied family Solanaceae (e.g., potatoes, tomatoes, and tobacco). The antheral cells from the plant were selected because of the 1202
Envlron. Scl. Technol., Vol. 26, No. 6, 1992
increased probability of isolating a pure cell line. Eaecauge of the inherent complexity of higher ordered (i.e. multicelled) plants, it was desirable to investigate the tiinding of metals to cell wall functionalization without t!,e corn. plication of multiple types of cells. The goal of thi, study was to investigate Cd binding sites on D. innoxia c: U W& using l13Cd NMR spectroscopy. It has been reported that I13Cd NMR spectrometry could be an excellent technique to probe the metal environment present in biological systems (2, 10, 11). The suitability of this probe is demonstrated by two characteristics: the broad range of chemical shifts and the relatively high isotope. The obnatural abundance of the spin I = served chemical shift dispersion covers a range of approximately 850 ppm, within which one can expect,to find resonance from l13Cd coordinated to the ligands fwnd in biological systems. This sensitivity of '13Cd chemic al shifts to variations in the local chemical environment makes it possible for l13Cd NMR to reflect subtle differences in the functional groups involved in cadmium ion binding. The natural abundance of l13Cd nuclei (nuclear spin of l/,J is 12.26%, thus making this isotope an ideal choitbe for a probe to the investigation of metal-ligand binding (2,10, 11). Recently, Majidi et al. (2) indicated that car: axylate functional groups on the cell walls of the alga Stic,'. ' COCCUS bacillaris are responsible for the binding of Cd(II1 Cu(II), Fe(II), Co(II), and Na(1). Although, nitrogen- ilnd sulfur-containing ligands have been also reported ta be capable of interacting with Cd(I1) (10, l l ) ,this type of interaction was not discussed by Majidi et al. (2) Interestingly, those researchers reported no significant impact of rapid ion-exchange equilibria between the bound and saturated cadmium ion, This type of complicating phenomenon has been reported elsewhere (10-12) for NMR studies of Cd binding. In this paper, several uifferent model ligands containing oxygen, nitrogen, or sulfclr have been examined by reacting them with Cd(I1). Thrwgh the generation of a table of observed chemical shi:is with various model ligands under the same binding corditions (Le., buffer, ionic strength, and pH), the chemical functionalities responsible for binding of Cd(I1) on the cell and the chemical-exchange phenomena have beer determined. I
Experimental Section Materials and Methods. A 0.1 M (2-N-morp!:olino)ethanesulfonic acid (MES; Sigma Chemical Co.) prepared with 50 v01 % D 2 0 was used as a noncomplexink buffer (12, 13). Distilled deionized water was used thrc--lghout the work. Hydrochloric acid (Baker, Inc.) and sodium hydroxide (Mallinckrodt) were used to adjust the st dutiom to the desired pH. A series of solutions contain(% o.05 M cadmium sulfate (Mallinckrodt) in 0.1 M ME2 buffer and a variety of acetate concentrationswere preparrd using glacial acetic acid (Baker). Sodium dodecyl sulfate (SDS) (International Biotech. nologies Inc.), p-toluenesulfonic acid (Eastman *7a), aD-(+)-glucose(Sigma), glycine (Bio-Rad), 1,5,8,12tetras.
0013-936X/92/0926-1202$03.00/0
0 1992 Amerlcan Chernicti' Sociev
loo
1
Cd
i
Table I. II3Cd Chemical Shifts Obtained from Reacting 0.05 M CdSO, with 50 mg mL-' Datura Cell Walls in 0.1 M MES Buffer Prepared with 50 vol 70 D,O at Different pH Conditions"
100 ppm
/ /
20i J 0
' 0
;
~
1
~
~
2
:
~
3
pH 1
2 3 :
4
;
~
5
6
chemical shift, PPm second first 5.0 4.1 1.3
chemical shift, PPm second first
pH
108.9 107.9 105.1
3.8 1.4 -50.5
4 5 6
107.6 105.3
"All chemical shifts are in parts per million with respect to external 0.1 M Cd(C10&
PH
mure 1. Effect of pH on the binding of Cd(I1) ions to Datura cells. Dwa cell wall materials (5 mg mL-') were suspended in 0.1 M MES buffer to react with 100 ppm Cd(I1) at different pH Conditions.
adodecane (Strem Chemical Co.), oxalic acid (Mallinckrdt), potato starch (E. H. Sargent), microcrystalline cellulose (Macherey Nagel), ethylenediaminetetraacetic acid (EDTA; Sigma), imidazole (Aldrich),ethylenediamine (udrich), ethanethiol (Aldrich), 1,Zethanedithiol (Aldrich), ammonium chloride (Baker), and L-(+)-glutamicacid (Baker) solutions were prepared as model ligand systems. These solutions were then used for l13Cd NMR analysis by mixing them with 0.05 M cadmium sulfate in a 1:l molar ratio in 0.1 M MES buffer at pH 6. For Cd(I1) binding site studies, a series of Cd(I1)-Datura samples were prepared for l13Cd NMR analysis by suspending 50 mg mL-l Datura cell material in 0.05 M CdS0, prepared in 0.1 M MES adjusted to different pH conditions. The pH dependence on the fraction of Cd(I1) bound to the cell material was determined. A sample of Datura cell material (50 mg) was suspended in 10 mL of a 0.1 M MES buffer solution (i,e,, no D20)with 0.89 mM (i.e., 100 ppm) CdS04at different pH conditions. After a contact time of 20 min to establish equilibrium, each solution was centrifuged. The final pH of the supernatant liquid was then recorded. Cd(I1) concentrations in the supernatant solutions were determined using an atomic absorption spectrophotometer (Therm0 Jarell Ash, Model 457) at 228.8 nm. The concentration difference between the initial and supernatant solutions represented the amount of Cd(I1) ions bound to the Datura cell material. The procedures used to grow and wash the D. innoxia cell material have been described previously (14). Briefly, Datura cells were grown in modified Gamborg's 1B5 medium supplemented with vitamins (15). Cells were washed twice with 95% ethanol and then dehydrated by heating at 42 "C. Dehydration was considered to be complete when there no additional weight lost with further heating. NMR Parameters. Each NMR sample was prepared $ a 5-mm borosilicate tube. A 0.1 M Cd(C104)2solution 0.1 M MES buffer was used as an external standard throughout the work. l13Cd NMR experiments were carried out on a Varian Unity 400 spectrometer with a 9.4-T mrow-bore superconducting magnet. A 5-mm broadband probe was tuned to observe the l13Cdsignal at 88.734 Samples were not spun. All solutions were prepared 50% D,O. Magnet shimming was accomplished by ?ing the proton decoupler channel to monitor the free uldWon decay (FID). The pulse sequence consisted of a 39" (Ernst angle) pulse, data acquisition over a *2?000-Hz bandwidth with 32K double-precision data and a 5.6-ps pulse width. The acquisition time for FID was fixed at 0.749 s. Acquisition times of 10-48 yielded acceptable signal-to-noise ratios.
a.
Cd-Datura
6
l1
. n w w w w W " ~ h ~ * ~W, ~ w ~ ~ J \ % * w , h ? A ~ w t d * I
I
I
I
120
80
40
0
I
I
-40
-80
I
-120
Chemical Shifl (ppm)
Figure 2. Typical '13Cd NMR spectra taken from table I. Datura cell wall materials (50 mg mL-') were suspended in 0.1 M MES buffer to react with 0.05 M CdSO, at different pH conditions.
Results and Discussion pH-Dependent Study on Cd-Datura Binding. The Cd-Datura binding behaviors as a function of various pH conditions (pH 1-6) are shown in Figure 1. The binding of Cd(I1) increased dramatically when the pH of the solutions was increased from 4 to 6. Near pH 6,90% of the Cd(I1) in solution was removed. The pH of the solution had a significant impact on the binding of Cd(I1) to the Datura cell material. A net negative charge on the cell material at solution pH values greater than the isoelectric point would be expected to lower any electrostatic energy barrier for the positive Cd(I1) to bind to cell material. This might explain the increased binding capacity shown in Figure 1 at pH >5. However, the identities of the functional groups responsible for binding of Cd(I1) on the cell walls at different pH conditions are not apparent. This question will be addressed in the following sections using l13Cd NMR data. '13Cd-Datura N M R Spectra. The chemical shifts of l13Cd-Datura NMR spectra obtained at different pH conditions with respect to the external standard of 0.1 M Cd(C10,)2 are summarized in Table I. Three typical l13Cd-Datura NMR spectra obtained at pH 1,5, and 6 are shown in Figure 2. The l13Cd NMR spectrum obtained from the solution containing only CdSO,,, ) reagent is shown in Figure 3. The central peak locate$ at 0 ppm is from the external standard. Thus, the peak located at 103.9 ppm shown in Figure 3 was assigned to be from free CdSO,,,, in 0.1 M MES buffer at pH 6.0. As a result, the peaks located from 105.3 to 108.9 ppm shown in Figure 2 and Table I are ascribed to the presence of free CdSO,,,,, Environ. Sci. Technol., Vol. 26, No. 6, 1992
1203
Table 11. lI3Cd Chemical Shifts Obtained from Reactin'gCd(I1) with Different Kinds of Ligands in 0.1 M MES Buffer Prepared with 50 vol % D,O at pH 6.0°
functional group
SO
120
0
40
-40
-80
-120
Chemical Shift (pprn)
Flgure 3. lT3CdNMR spectrum of 0.05 M CdSO, in 0.1 M MES buffer at pH 6 .
2:2
Cd:Acetate PH 6
iI!
2:4
11
v%q\&;,~w+qwp#p$+%~~ I*\'rb/A4,J+WlL#dMWWi~b#~W .
'
"
A
;,
Cd-Datura PH 5
"
;
"
I
*
. . ..
1;
unknown carboxylate carboxylate carboxylate carboxylate oxalate diamine diamine tetraamine amine amine and carboxylate diamine and tetracarboxylate amine and dicarboxylate hydroxyl hydroxyl hydroxyl sulfhydryl sulfhydryl sulfate sulfonate
complex Cd-Datura Cd-acetate (2:1) Cd-acetate ( 2 9 Cd-acetate (2:3) Cd-acetate (2:4) Cd-oxalate Cd-e thylenediamine Cd-imidazole Cd-1,5,8,12-tetraazadodecane Cd-ammonia Cd-glycine Cd-ethylenediaminetetraacetate Cd-glutamate
Cd-glucose Cd-potato starch Cd-cellulose Cd-ethanethiol Cd-ethanedithiol Cd-sodium dodecyl sulfate Cd-p-toluenesulfonic acid
I
I
I
I
I
I
80
40
0
-40
-80
95.2 2.3
38.9 103.8 -47.7 35.2 103.9 -59.8 -21.7 -103.8
82.2 -96.4
-91.3
-83.5
-32.4
71.5
-7.4
17.1
-7.8 -7.5
-41.1 -38.6
-5.2
62.7 65.2 19.3 17.2
'I
Chemical Shift (ppm)
Envlron. Scl. Technol.. Vol. 26, No. 6,1992
103.5
,
Figure 4. 'I3CdNMR spectra taken from Cd-acetate model solutions at pH 6 and Cd-Datura sample at pH 5. The concentration ratios of Cd to acetate vary from 2:2 to 2:4.
1204
-34.5 -8.7
I
I
-120
reagent. Because the l13Cd chemical shifts associated with free CdSO,(,,) are located from 105.3 to 108.9 ppm, it suggests that a 3.6 ppm difference of chemical shift is not significant in our l13Cd NMR measurements. Thus, it is reasonable that the peaks (i.e,, 1.3-5.0 ppm) located near the central external standard peak in Figure 2 are derived from the same Cd-Datura species present in similar environments at pH 1 5 conditions. It has been reported that l13Cd chemical shifts are extremely sensitive to the nature, number, and geometric arrangement of the ligands within the coordination metal sphere (IO). Consequently, the large peak shift occurring between pH 5 (Le,, 1.4 ppm) and 6 (i.e., -50.5 ppm) in Table I suggests that the functionalities responsible for the majority of binding of Cd(I1) at pH 5 and 6 are different. Also, the peak associated with free CdSO,,, ) near 105.3-108.9 ppm at pH 5 5 was not observed at PI%6. In order to investigate these two related observations, a series of Cd(I1)-containingmodel solutions were examined using I13Cd NMR spectra. '13Cd NMR Spectra Obtained from Model Solutions. In order to verify the functionalities responsible for binding of Cd(I1) on Datura cell walls, based on the reported data found from algal cells ( I , 2),a series of model solutions containing different concentration ratios of Cd to ligand were tested to see if their functional groups could be contributing to the observed l13Cd NMR spectra obtained from Cd-Datura samples. These include acetate as a model for a carboxylate, SDS for a sulfate, p toluenesulfonic acid for a sulfonate, ethylenediamine for a linear diamine, imidazole for a ring diamine, 1,5,8,12-
-50.5
"All chemical shifts are in parts per million with respe "L to ex. ternal 0.1 M Cd(C104)2.
+ -J w --
120
chemicai shift, PPI, --4 first iecond
11
Cd-Ethylenediamine
Cd-Datura
i
~
11
+ + y M w N + y w w ~ ~'i - w + + y + e q 120
EO
40
0
-40
-80
-120
Chemical Shift (ppm)
Figure 5. '13Cd NMR spectra taken from Cd-ethylenediamine and Cd-Datwa. A 0.05 M CdSO, solution was prepared to react with 0.05 M ethylenediamine and 50 mg mL-'Datura cells in 0.1 M M r S buffer at pH 6 .
tetraazadodecane for a tetraamine, glycine for singh amine and terminal carboxylate, cr-D-(+)-ghcose for hydroxY1i potato starch as a nonlinear polysaccharide, mil. rocrYstalline cellulose as a linear polysaccharide, oxalic Acid for carboxylates, ethanethiol and ethanedithiol for sdfhYdrY1 functionalities, ammonium chloride for single amlner EDTA, and L-(+)-glutamic acid as a model of binding to a P-carboxylate on an amino acid residue. All the Il3Cd NMR chemical shift data obtain6 d from these Cd(I1)-containing model solutions and the :d-Da' tura sample at pH 6 are summarized in Table I1 Some of the typical Il3Cd NMR spectra are shown in F.$res and 5. A comparison of the chemical shifts of lI3C; Nm spectra obtained for these model ligands and the d-Da.
tura sample suggests that carboxylate groups, as typified by acetate at a Cd(I1) to acetate concentration ratio of 2:4 (i,e,,1:2),provided the best fit to the Wd-Datura NMR dab at lower ( 5 5 ) pH conditions. AS shown in Table I1 and Figure 4,when the Cd(I1) to acetate concentration ratios were varied from 2:2 to 2:4 (1:l 1 9 , the lTd-acetate NMR peak positions shifted to lower field (-34.5 to 2.3 ppm, respectively). The chemical shifts observed for 113Cd-Daturaat pH 1 5 shown in Table 1 are comparable with those measured with a Cd(1I) to acetate model system at a concentration ratio of 1:2. This suggests that carboxylate groups are the dominant functiondities responsible for binding of Cd(I1) on Datura cells at pH 15. This result is consistent with our previous work, which suggests that carboxylate functionalities could bind to Eu(II1) in a 1:l or 2:l ratio (14,16). The interpretation of the additional peak at 95.2 ppm obtained from the 2:3 Cd(I1)-acetate model system, shown in Figure 4, is not clear. Because the chemical shift (95.2ppm) is near that assigned to the unbound CdSO,(,,), this peak may result from the presence of free CdSO,,,,) in a slightly different ionic environment. For the pH 6 condition, the chemical shift of l13Cdethylenediamine is located at -47.7 ppm. This is comparable with the -50.5 ppm shift measured for Cd-Datura (Table I1 and Figure 5). This suggests that diamine functionalities on Datura cell walls are significantly involved in the binding of Cd(I1) at pH 6. The fraction of Cd(I1) bound did not differ significantly between solutions of pH 5 and 6 (Figure 1). It is important to examine whether or not carboxylate and diamine functionalities are adjacent so that both functional groups could cooperatively bind to the same Cd(I1) ion. For this purpose, three model ligands containing amine and carboxylate functionalities, such as glycine, EDTA, and glutamate, were carefully examined. As shown in Table 11, two l13Cd NMR peaks were observed for each of the Cdglycine, Cd-EDTA, and Cd-glutamate complexes. However, none of these observed chemical shifts was comparable with the -50.5 ppm shift obtained from Cd-Datura at pH 6. Thus, it is unlikely that the carboxylate and diamine (or amine) functionalities are adjacent. In addition to carboxylate and diamine functionalities, polysaccharides, hydroxyl-containing components on the P h t cell walls, were also examined. As can be seen from Table 11, the chemical shifts obtained from Cd-glucose, Cd-potato starch, and Cd-cellulose are located from -7.8 to 17.1 ppm, which are not comparable with the observed Shift for the Cd bound to the cell material. This suggests that polysaccharides probably do not compete with carboxylate or diamine functionalities for binding of Cd(I1) On Datura cell wall material. Two sulfur-containing (i.e., e h e t h i o l and ethanedithiol), one sulfate-containing (i.e., SDS), and one sulfonate-containing (i.e., p-toluenesulfonic acid) ligands were also examined. On the basis of the chemical shift data for these model systems (Table 11), h e functionalities do not compete significantly with either carboxylate or diamine groups for binding of Cd(I1) under these conditions. However, one question remains; that is, why the peaks associated with Cd-carboxylate 8peciesnear the central standard peak present at pH 15 (Figure 2) are not observed at pH 6. Also, the peaks resulting from free CdSO,,,,, near 103 ppm observed at pH s5 are not present at pH 6. It would seem to be highly
improbable that Cd-carboxylate species are totally absent at pH 6. It is possible that the peak located at -50.5 ppm present at pH 6 might be the result of a fine averaged l13Cd NMR signal resulting from the presence of a rapid exchange equilibrium among the free CdSO,,,,), Cdcarboxylate, and Cd-diamine species. In order to bring this labile inorganic reaction into the slow-exchangeregime so that a detailed characterization of Cd binding sites could be obtained, a low-temperature environment provided by the use of supercooled aqueous solutions (10, 17,18) is required. Currently, a series of experiments using lowtemperature technique and competition reaction are being undertaken in this laboratory. Acknowledgments We thank Professors D. W. Darnall, E. R. Birnbaum, and W. Lwowski for informative discussions, Dr. P. J. Jackson for providing all the required Datura innoxia cell wall materials, and M. Mauldin for acquiring all W d NMR spectra. Registry No. Cd, 7440-43-9.
Literature Cited Gardea-Torresdey, J. L.; Becker-Hapak, M. K.; Hosea, J. M.; Darnall, D. W. Environ. Sci. Technol. 1990, 24, 1372-1378. Majidi, V.; Laude, D. A., Jr.; Holcombe, J. A. Environ. Sci. Technol. 1990, 24, 1309-1312. Harris, P. 0.;Ramelow, G. J. Enuiron. Sci. Technol. 1990, 24, 220-228. Gardea-Torresdey, J.; Darnall, D.; Wang, J. Anal. Chem. 1988,60, 72-76. Kubiak, W. W.; Wang, J.; Darnall, D. Anal. Chem. 1989, 61, 468-471. Watkins, J. W., 11;Elder, R. C.; Greene, B.; Darnall, D. W. Inorg. Chem. 1987,26, 1147-1151. Crist, R. H.; Oberholser, K.; Shank, N.; Nguyen, N. Environ. Sci. Technol. 1981, 15, 1212-1217. Delhaize, E.; Jackson, P. J.; Lujan, L. D.; Robinson, N. J. Plant Physiol. 1989, 89, 700-706. Delhaize, E.; Robinson, M. J.; Jackson, P. J. Plant Mol. Biol. 1989, 12,487-497. Armitage, I. M.; Boulanger, Y. In NMR of Newly Accessible Nuclei; Laszlo, P., Ed.; Academic Press: New York, 1983; Vol. 2, Chapter 13, pp 337-365. Armitage, I. M.; Otvos, J. D. In Biological Magnetic Resonance; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1981; Vol. 4, Chapter 2, pp 79-144. Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R. M. M. Biochemistry 1966,5,467-477. Good, N. E.; Izawa, S. Methods Enzymol. 1968,24,53-68, (part B). Ke, H. Y.; Birnbaum, E. R.; Darnall, D. W.; Rayson, G. D.; Jackson, P. J. Appl. Spectrosc. 1992, 46, 479-488. Chu, Y. E.; Lark, K. G. Planta 1976, 132, 259-268. Ke, H. Y.; Birnbaum, E. R.; Darnall, D. W.; Rayson, G. D.; Jackson, P. J. Enuiron. Sci. Technol. 1992, 26, 782-788. Jakobsen, H. J.; Ellis, P. D. J. Phys. Chem. 1981, 85, 3367-3369. Ackerman, M. J. B.; Ackerman, J. J. H. J. Phys. Chem. 1980, 84, 3151-3153.
Received for review October 28, 1991. Revised manuscript received February 12,1992. Accepted February 25,1992. Financial support by the U.S. Department of Energy through the New Mexico Waste-Management Education and Research Consortium is gratefully acknowledged.
Environ. Sci. Technol., Vol. 26, No. 6, 1992 1205
