Environ. Sci. Technol. 1994, 28, 586-591
Luminescence Studies of Metal Ion-Binding Sites on Datura innoxia Biomaterial Huel-Yang Davld Ke, Wendy L. Anderson, Robyn M. Moncrief, and Gary D. Rayson'
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003 Paul J. Jackson
Genomics and Structural Biology Group, Life Sciences Division, LS-2, MS M880, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 The metal ion-binding interactions between a series of metal ions and Datura innoxia cell wall fragments have been investigated using Eu3+ luminescence. An affinity order of Cu2+ Ag+ > Ni2+> Cd2+> Eu3+ > Sr2+> Ba2+ for total metal ion binding to D. innoxia cell material has been obtained when pH L 5. Both electrostatic and complexationinteractions have been verified to be involved in metal ion-binding interactions. Carboxylate groups have been demonstrated to be the dominant functional group responsible for the binding of most of the metal ions studied. At least two binding sites have been demonstrated to be involved in the binding of Ag+ at different Ag+ concentrations. One site is pH-independent and displays a greater affinity with a lower availability than the other, which is pH-dependent. The studies of competative uptake by carboxylate groups between Eu3+ and the metal ions studied demonstrate an affinity order of C U ~>+ Ag+ > Ni2+ > Sr2+> Ba2+> Cd2+.
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Introduction The binding of metal ions to nonliving biomass materials and the application of this phenomenon to water treatment have been amply investigated in recent years (1-9). The majority of these investigations have been focused on algal biomasses. However, previously reported results from our laboratory have demonstrated the ability of fragments of cultured cells from a higher order plant, specificallyDatura innoxia, to adsorb Eu3+ (IO),Gd3+,Cu2+ ( 1 0 , Cd2+(12), and U0z2+ (13-15) ions from aqueous solutions under a variety of conditions. This suggests that the same kind of biosorption (adsorption) processes as those observed in algae are likely to occur on the plant cell walls. It has been proposed that complexation and electrostatic binding are the primary biological processes by which microorganisms remove metal ions from solutions (5,161. Also, it has been suggested that amino, thioether, sulfhydryl, carboxylate, carbonyl, imidazole, phosphate, sulfate, phenolic, hydroxyl, sulfonate, and amide moieties are the possible functionalities responsible for the binding of metal ions on the algal cell walls (5, 17). However, relatively few reports of the characterization of the binding of metal ions to cell walls have appeared in the literature. The chemical nature of the metal binding interactions is left relatively unclear. In order to effectively use biomass materials in water purification and metal reclamation treatment processes, it is important to determine which chemical functionalities on the cell walls are responsible for the binding of different metal ions. One approach to gaining this information is the use of metal ion luminescence spectroscopy to directly probe the metal ion-binding interactions. 586
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Eu3+ possesses both nondegenerate ground (7F0) and excited emissive (5D0)states. This suggests that the 7F0 -,5D0electronic transition is a nondegenerate transition in any crystal field of any symmetry because neither the ground nor emissive level can be split by a ligand field (18, 19). Thus, the observation of more than one peak in the spectral region of this transition is an indication of the presence of more than one species. Consequently, useful spectral interpretations are possible without a detailed and difficult analysis of the crystal field splitting. This characteristic makes Eu3+luminescencean excellent probe for studying biological systems, which usually contain multiple binding sites. Several studies using Eu3+luminescence to probe metal-binding interactions on proteins have been described (20-23). Previously, carboxylate and sulfate groups have been determined to be the dominant functional groups on the cell walls of Datura innoxia that bind Eu3+ (10, 11). In other studies, carboxylate and diamine groups were found to be responsible for the binding of Cd2+ (12) and phosphoryl and dicarboxylate functionalities for the binding of U022+ (13-15). In this paper, through the investigation of competitive uptake by carboxylate groups between Eu3+ and each of a targeted group of metal ions (Le,, Ag+, CUB+,Ni2+,Ba2+,Sr2+,and Cd2+),the binding sites involving in the binding of these metal ions on Datura innoxia cell walls will be directly characterized using Eu3+ luminescence.
Experimental Section Materials and Methods. Stock europium(II1)chloride solutions, prepared from Eu2O3 (Molycorp), were standardized with ethylenediaminetetraacetic acid (EDTA) solution (Sigma)at pH 6.0by methods described elsewhere (10, 11, 20-23). Distilled deionized water was used throughout the work. A 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma) was used as a noncomplexing buffer (24, 25). Sodium hydroxide (Mallinckrodt) and nitric acid (Baker) were used to adjust the solutions to the desired pH. The procedures used to grow and wash Datura innoxia biomass materials have been described previously (10). Briefly,D. innoxia cells were grown in modified Gamborg's 1B5medium supplemented withvitamins (IO). Cells were washed twice with 95% ethanol and then dehydrated by heating at 42 "C. Dehydration was considered to be complete when there was no additional weight lost with further heating. In all metal-binding studies, blank and sample solutions were prepared in parallel. For blank solutions, 250 mg of the D. innoxia cell material was suspended in 50 mL of a metal-free buffer solution at the same pH as that used 0013-936X/94/092S-0586$04.50/0
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in the sample solutions. After a contact time of 1 h to establish equilibrium, the solution was centrifuged, and the supernatant liquid was stored. The blank and sample solutions (1mM) were then prepared by adding the same volume of the stock AgN03 (Mallinckrodt), Cu(NO3)z (Baker), Ni(NO& (Baker), Ba(NOd2 (Aldrich), Sr(N03h (Baker), Cd(N03)2 (Fisher Scientific), or EuCl3 solution to the supernatant liquid obtained above or the buffer solution containing the cell material (5 mg mL-l). After agitating for about 1 h, the blank and sample solutions were centrifuged, and the supernatant liquids were analyzed using either flame atomic absorption (AA) (Ag+, Cu2+,Ni2+,and Cd2+)or inductively coupled plasma atomic emission spectrometry (ICP-AES) (Ba2+,Eu3+,and Sr2+). The difference between the sample and blank solutions represents the amount of metal ions bound to D. innoxia cell materials. In the competitive binding experiments, the suspensions containing 1 mM of Eu3+ with 5 mg of D. innoxia biomaterial mL-l in 0.1 M MES buffer and various concentrations of each targeted metal ion were prepared at pH 5.0 by directly mixing the europium(II1) chloride solution with each metal nitrate salt solution. All solutions were agitated for about 1 h before centrifugation. The solid Eu(II1)-containing samples of D. innoxia material were air dried and stored for subsequent Eu3+luminescence measurements. Europium(II1) Luminescence. Excitation spectra associated with the electronic 7F0 5D0transition of the Eu(1II) ions were acquired with the laser spectrofluorimeter described previously (10, 11,20-23). Solid Eu(II1) Datura samples were placed in a sample cell (a 3-mm diameter Pyrex glass tube with one end sealed). All the luminescence experiments were performed at liquid nitrogen temperature. The dye laser was operated during these experiments using a 0.004 M Rhodamine 590 (Exciton Corp.) solution in absolute ethanol. Emission was monitored at 615 nm with an effective bandwidth of 1.67 nm (10). Luminescence spectra deconvolution was performed using a nonlinear least-squares regression routine. The spectra were fitted to a sum of peaks having a Lorentzian lineshape (10,11). The peakareasoftheexcitationspectra were calculated using a trapezoidal approximation to an integral. It should be emphasized that all luminescence measurements were made with the solid material at 77 K. Flame Atomic Absorption and Inductively Coupled Plasma-Atomic Emission Spectrometry. Metal analyses were performed with either AA (Ag+,Cu2+,Ni2+,Cd2+) or ICP-AES (Ba2+,Eu3+,Sr2+). The AA spectrometer was a Model 3030 B (Perkin-Elmer) with an air-acetylene flame. The ICP-AES system used in this study has been described elsewhere (10,261.A conventional tangentialflow torch was used with an outer argon gas-flow rate of 17 L min-', an intermediate flow of 1.5 L min-l, and a nebulizer flow rate of 1.1 L min-1. The plasma was operated at 1.0 kW of applied rf power with Ni2+ > Cd2+> Eu3+ > Sr2+> Ba2+when pH 15. The maximum binding occurred either at pH 5 or 6, and the minimum binding occurred at pH 1. The cell walls of D. innonia contain both proteins and polysaccharides, suggesting that there are several hard kinds (including carboxylate, hydroxyl, phosphoryl, sulfate, and amine groups) and soft kinds (including sulfhydryl groups, olefins, or aromatic groups) of binding sites. In considering the different kinds of metal ions which bind to this D.innoxia biomaterial, the principle of hard and soft acids and bases predicts that hard metals would bind most strongly to oxygen-containing or amine groups, whereas soft metals would bind most strongly to sulfhydryl Environ. Sci. Technoi., Vol. 28, No. 4, 1004 587
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Flgure 3. Excitation spectra derived from competitive binding experiments consisting of 1 mM Eu3+ and various concentrations of Cd2+, BaZf, (A) a = Eu, b = Eu 4- 1 mM Cd, c = Eu 2 mM Cd, and d = Eu 4- 4 mM Cd; (6) a = Eu, b = Eu 0.5 mM Ba, c = Eu 1 mM Ba, d = Eu -k 2 mM Ba, and e = Eu 4 mM Ba; (C) a = Eu, b = Eu 0.5 mM Sr, c = Eu 1 mM Sr, d = Eu 2 mM Sr, 1 mM Ni, d = Eu 2 mM Ni, and e = Eu 4 mM Ni; (E) a = Eu, b = and e = Eu 4- 4 mM Sr; (D)a = Eu, b = Eu 4- 0.5 mM Ni, c = Eu Eu 0.1 mM Ag, c = Eu 0.5 mM Ag, d = Eu 1 mM Ag, e = Eu 2 mM Ag, and f = Eu 4 mM Ag; and (F) a = Eu, b = Eu 0.5 mM Cu, c = Eu f 1 mM Cu, and d = Eu 4- 2 mM CIJ emission = 615 nm).
SrZf,Ni2+, Ag+ or Cu?
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groups. Metal ions of borderline classification probably bind, although less strongly, to either kind of binding site. This might explain why Cu2+ and Ni2+ bind to D. innoxia cell wall fragments better than the otherions. Theamount of Cd2+bound to this D. innoxia cell wall material is greater than that obtained from Eu3+, which prefers to bind to oxygen-containing groups (IO). This is consistent with our previous report in which Ca2+has been observed to bind to this biomaterial through either carboxylate or diamine groups (12). Divalent Sr2+and Ba2+ ions bind less to the D. innoxia cell wall fragments than trivalent Eu3+. Because these three ions are classified as hard acids, electrostatic effects might play an important role in these metal ion-binding interactions. However the identities of the functional groups responsible for the binding of these metals on the cell walls at different pH conditions are not apparent. This question will be addressed in the following sections using Eu3+ luminescence. The most striking metal ion-binding behavior is the observation of a concentration dependence of Ag+ binding on solution pH in Figure 2. This phenomenon has not been observed in the binding of Ag+ to algal biomasses, in which a pH independence on Ag+ binding has been reported by Darnall et al. (28). Because lower initial concentrations of Ag+ (0.1mM) were used in those studies, 588
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a similar study was undertaken using the cell wall material of D. innoxia (Figure 2). The data shown in Figure 2 suggest that at least two different sites for the binding of Ag+ on the D. innoxia cell wall material are present. One site is pH-independent and displays a greater affinity with a lower availability than the other, which is pH-dependent. This type of site would be consistent with the sulfhydryl functionality proposed for the binding of Ag+ to Chlorella vulgaris (28). The identity of functional groups responsible for the pH-dependent site is not clear. Further discussion regarding this type of binding site will be given below. In addition, the pH-independent site appears to be saturated when the cell material is exposed to a solution of 0.1 mM Ag+ (see Figure 2). This can be deduced from the near-identical binding of Ag+ at pH 1 observed for both the 0.1 and the 1 mM solutions (Figure 2). Surprisingly, this behavior was not observed for the binding of any of the other metal ions investigated. Characterization of the Binding Sites from the Analysis of Eu(III) Excitation Spectra Obtained from the Competative Binding Experiments. In order to characterize the binding sites responsible for the binding of each targeted metal ion on the D. innoxia cell wall fragments, various concentrations of these ions were separately used to compete with 1mM Eu(II1) to bind at
pH 5.0. The excitation spectra obtained by the excitation of the 7F0 5D0 electronic transition of the Eu(II1) in these metal-doped samples (Le., 4 mg) are shown in Figure 3. These excitation peak intensities of the 7F, 5D0 electronic transition decrease when the concentrations of Cd2+ (Figure 3A),Ba2+ (Figure 3B), Sr2+(Figure 3 0 , Ni2+ (Figure 3D), Ag+ (Figure 3E), or Cu2+ (Figure 3F) are increasing, suggesting that these ions are capable of competing with Eu3+to bind to carboxylate groups on the D. innoxia cell wall biomaterial. Specifically, mono- and dicarboxylate groups have been determined to be the two dominant functional groups responsible for the binding of Eu3+ at pH 5.0 on the biomaterial (IO). Thus, the data shown in Figure 3A-F indicate that these metal ions could bind to this biomaterial through carboxylate groups. This can be reinforced by the data shown in Figures 1 and 2 (see the solid circle line in Figure 2), which show the pHdependent binding profiles among these ions are similar. It should be noted that the bound Eu3+ions could also be removed from the biomass because of the electrostatic repulsion resulting from the presence of other cations near the material. This is likely to occur when a weak metal ion-binding interaction with the cell wall material is present. Figure 3B,C shows that the presence of both Ba2+and Sr2+, respectively, lowers the measured peak intensities more dramatically than Cd2+ (Figure 3A). However, the amount of Cd2+bound to the D. innoxia cell wall material at pH 5.0 is greater than those obtained from Ba2+ and Sr2+ (Figure 1). Because Cd2+can bind through either carboxylate or amine groups (12),the differences in lowering the excitation peak intensities of the Eu3+indicate that hard acid Ba2+ and Sr2+ions prefer to bind to the D. innoxia biomaterial through carboxylate groups rather than the amine groups involved in Cd2+binding. As shown in Figure 3D (Le., Ni2+) and F (i.e., Cu2+), Cu2+has much greater ability than Ni2+to replace E$+. Because both Ni2+and Cu2+ have the same charges and similar radii, the differences in binding to the D. innoxia cell wall material could be due to a complexation rather than an electrostatic effect. It has been reported that Cu2+ binds to the alga Stichococcus bacillaris mainly through carboxylate groups (6). This is consistent with the data shown in Figure 3F, which shows C U ~ can + significantly compete with Eu3+for binding. The differences in the amount of Ni2+ and Cu2+ bound to this biomaterial at pH 5.0 (Figure 1)are much less than the differences in lowering the excitation peak intensities of the Eu3+shown in Figure 3D,F. This indicates that Ni2+ might prefer to bind to the D. innoxia cell walls through functional groups other than carboxylate groups. For Ag+ ions, the pH-independent binding profile observed from 0.1 mM Ag+ conditions (see the solid triangle line in Figure 2) is different from those obtained from other metal ions in Figure 1. As mentioned earlier, the functional groups responsible for this pH-independent binding site have been suggested to be sulfhydryl groups (28). This type of soft acid to soft base interaction makes the Ag+-Datura bond so covalent that it cannot be affected by different pH conditions. However, as shown in Figure 3E, the excitation peak intensity of the Eu3+ decreased (see curve b) when 0.1 mM Ag+ was added to the solution to compete with Eu3+ for binding to the D.innoxia cell wall material. This indicates that electrostatic repulsion could be a significant effect for Ag+ to remove Eu3+from
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Dopant Concentration (mM) Figure 4. Relationship between dopant concentrations and excitation peak area ratios of pure Eu3+-containingsamples to Cd2+,Ba2+,Sr2+, Ni2+,Ag+, or Cu2+ doped samples in Figure 3 (R = Cd2+ doped, = Ba2+ doped, A = Sr2+ doped, 0 = Ni2+ doped, 0 = Ag+ doped, and A = Cu2+ doped).
the cell walls. At higher Ag+ (i.e,, 21 mM) concentration cases (curves c-0, Ag+ ions lower the excitation peak intensities of the Eu3+in a similar manner as those observed from Cu2+ in Figure 3F. Also the pH-dependent binding profile shown in Figure 2 (see the solid circle line) is similar to those shown in Figure 1. This suggests that the pHdependent binding site responsible for the binding of Ag+ on this biomaterialcould be formed by carboxylate groups. In order to compare the affinity for each of the targeted metal ions to bind to this biomaterial, all of the excitation peak areas shown in Figure 3A-F are integrated from 578.0 to 580.2 nm. The relationship between the dopant concentrations (i.e,, metal ions) and relative peak area ratios of pure Eu3+-containingsamples to the sample doped with each targeted metal ion are shown in Figure 4. As can be seen, the fluorescence intensity of the Eu3+ is affected by these ions in an order of Cu2+> Ag+ > Ni2+ > Sr2+ > Ba2+ > Cd2+. Characterization of the Carboxylate-Containing Binding Sites from the Analysis of the Excitation Spectra of Eu3+. The excitation spectra taken from a series of solid Eu3+-Datura samples (Le., 4 mg) containing 1mM of Eu3+and 1mM of Cd2+(Figure 5A), Ba2+(Figure 5B), Sr2+(Figure 5 0 , Ni2+(Figure 5D), Ag+ (Figure 5E), or Cu2+(Figure 5F) at pH 5.0 are shown in Figure 5A-F. The excitation spectra taken from solid Eu3+-Datura containing 1 mM Eu3+ and 0.1 mM Ag+, and from the pure Eu3+-Datura sample (Le., 1mM) are shown in Figure 5G and 5H, respectively. Because only two lifetimes were resolved from each of these Eu3+-containingsamples, the excitation spectra shown in Figure 5 were fitted with two Lorentzian functions (IO). The fitted excitation peak near 579.1 nm in Figure 5 has been previously assigned to the presence of both Eu3+-sulfate and Eu3+-carboxylate (Le., 1:l ratio species in which carboxylate groups bind to Eu3+ in a 1:l ratio) on a D. innoxia cell wall material (10). The other peak near 579.4 nm has been proposed to be indicative of the formation of another Eu3+-carboxylate species (Le., 1:2 ratio species) in which Eu3+ is bound by two carboxylate groups. Figure 5 shows that the two resolved peak positions in Figure 5A-G remain the same as those obtained from pure Eu3+-Datura samples (seethe dotted lines). This indicates Environ. Sci. Technol., Vol. 28. No. 4, 1994 589
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Flgure 5. Excitation spectra at pH 5.0 of solid Eu3+-Darura samples prepared by reacting 1 mM Eu3+ with (A) 1 mM Cd2+, (B)1 mM Ba2+, (C) 1 mM Sr2+, (D) 1 mM Ni2+, (E) 1 mM Ag+, (F) 1 mM Cu2+, or (G) 0.1 mM Ag+. The excitation spectrum of pure Eu3+-Dafura Is shown In (H) (++, data points; -, individual Lorentzian fitted curves or the sum of the resolved curves),,A,(, = 615 nm).
that the dominant functional groups responsible for the binding of Eu3+ on this biomaterial are not changed at these experimental conditions. Also, the peak intensity ratios between two resolved peaks (i.e., 579.1 and 579.4 nm) in these Eu3+-containingsamples are nearly the same as those observed from pure Eu3+-Datura samples (Figure 5H). It should be noted that this phenomenon has also been observed from other experimental conditions in which the targeted metal ion concentrations varying from 0.5 to 4 mM were used to compete with 1 mM Eu3+ for binding the D. innoxia biomass. This indicates that there is no competition for the binding of these metal ions between carboxylate-containing binding sites within this concentration range. If all the carboxylate groups on the cell walls were located in a similar chemical environment, some of which bind to Eu3+ in a 1:l ratio while others bind to Eu3+in a 2:l ratio, there would be a competition between the carboxylate groups for the binding of the targeted 590
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metal ions. This competitive binding between two carboxylate-containing binding sites would cause the 1:2 ratio species to convert to the 1:l ratio species when an excess amount of metal ions is used to react with the D. innoxia cell wall material. Thus, no competitive binding between carboxylate groups was observed, indicating that the carboxylate groups associated with the two binding sites on the D. innoxia cell walls are located in two different chemical environments. Surprisingly, the peak intensity ratio between two resolved peaks shown in Figure 5G (i.e., 0.1 m M Ag+) is similar to those observed in Figure 5A-F and 5H. It would be expected that an isotropic electrostatic interaction should be different from a more chemically selective complexation interaction. Thus, no preferential binding between two carboxylate-containing binding sites shown in Figure 5A-H suggests that the metal concentrations used in these competitive binding studies could be too
high to reflect the preferential differences in the binding of different metals. This is consistent with our previous report in which a preferential binding between two carboxylate-containing binding sites was observed when lower metal concentrations were used (11). It should be noted that although the concentration of Na+ from the MES buffer system is orders of magnitude greater than any of the targeted metal ions, significant binding of each metal ion was observed. If simple electrostatic attraction was the dominate mechanism of metal ion binding to this complex biomaterial, no variation in metal ion species or concentration would be expected. It can then be concluded that such a simplistic explanation cannot be accurately applied to explain the observed interactions.
Acknowledgments The authors thank Dr. E. R. Birnbaum for the use of the dye laser. Financial support by the U.S.Department of Energy through the New Mexico Waste-Management Education and Research Consortium is gratefully acknowledged.
Literature Cited (1) Crist, R. H.; Oberholser, K.; McGarrlty, J.; Crist, D. R.; Johnson, J. K.; Brittsna, J. M. Enuiron. Sci. Technol. 1992, 26, 496-502. (2) Crist, R. H.; Oberholser, K.; Wong, B.; Crist, D. R. Enuiron. Sci. Technol. 1992,26, 1523-1526. (3) Garnham G. W.; Codd, G. A.; Gadd, G. M. Enuiron. Sci. Technol. 1992,26, 1764-1770. (4) Wright, P. J.; Weber, J. H. Enuiron. Sci. Technol. 1991,25, 281-294. (5) Gardea-Torresdey, J. L.; Becker-Hapak, M. K.; Hosea, J. M.; Darnall, D. W. Enuiron. Sci. Technol. 1990,24, 13721378. (6) Majidi, V.; Laude, D. A., Jr.; Holcombe, J. A. Enuiron. Sci. Technol. 1990,24, 1309-1312. (7) Harris, P. 0. Ramelow, G. J. Enuiron. Sci. Technol. 1990, 24, 220-228. (8) Gardea-Torresdey, J.; Darnall, D.; Wang, J. Anal. Chem. 1988, 60, 72-76.
(9) Kubiak, W. W.; Wang, J.; Darnall, D. Anal. Chem. 1989,61, 468-471. (10) Ke, H. Y.; Birnbaum, E. R.; Darnall, D. W.; Rayson, G. D.; Jackson, P. J. Appl. Spectrosc. 1992,46, 479-488. (11) Ke, H. Y.; Birnbaum, E. R.; Darnall, D. W.; Rayson, G. D.; Jackson, P. J. Enuiron. Sci. Technol. 1992,26, 782-788. (12) Ke, H. Y.; Rayson, G. D. Enuiron. Sci. Technol. 1992,26, 1202-1205. (13) Ke, H. Y.; Rayson, G. D. Appl. Spectrosc. 1992,46, 11681175. (14) Ke, H. Y.; Rayson, G. D. Appl. Spectrosc. 1992,46,13761381. (15) Ke, H. Y.; Rayson, G. D. Appl. Spectrosc. 1992,47,44-51. (16) Watkins, J. W., 11; Elder, R. C.; Green, B.; Darnall, D. W. Inorg. Chem. 1987,26,1147-1151. (17) Crist, R. H.; Oberhoher, K.; Shank, N.; Nguyen, M. Enuiron. Sci. Technol. 1981,15, 1212-1217. (18) Horrocks, W. D., Jr.; Sudnick, D. R. Acc. Chem. Res. 1981, 14, 384-392. (19) Albin, M.; Horrocks, W. D., Jr. Znorg. Chem. 1985,24,895900. (20) Henzl, M. R.; Birnbaum, E. R. J. Biol. Chem. 1988,263, 10674-10680. (21) Hapak, R. C.; Lammers, P. J.; Palmisano, W. A.; Birnbaum, E. R.; Henzl, M. T. J . Biol. Chem. 1989,264,18751-18760. (22) Treviiio, C. L.; Palmisano, W. A.; Birnbaum, E. R.; Henzl, M. T. J. Biol. Chem. 1990,265, 9694-9700. (23) Palmisano, W. A.; Treviiio, C.L.;Henzl, M. R. J. Biol. Chem. 1990,265, 14450-14456. (24) Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R. M. M. Biochemistry, 1966,5,467-477. (25) Good, N. E.; Izawa, S. Methods Enzymol. 1968,24B, 53-68. (26) Rayson, G. D.; Shen, D. Y. Appl. Spectrosc. 1992,46,12451250. (27) Pearson, R. G. In Hard and Soft Acids and Bases; Pearson, R. G., Ed.; Dowden, Hutchinson, and Ross: Stroudsburg, PA, 1973; Part 11, pp 53-84. (28) Green, B.; McPherson, R.; Darnall, D. W. In Metals Speciation, Separation and Recovery; Patterson, J. W., Passino, R., Eds.; Lewis Publishers: Chicago, 1987;pp 315338.
Receiued for review May 4, 1993. Accepted January 3, 1994.' Abstractpublishedin Advance ACSAbstracts,February 1,1994.
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