Nuclear Magnetic Resonance Investigation of Cadmium 113 Substituted Pea and Lentil Lectins*

The lentil (LcH) and pea (PSA) lectins, which are members of the class of D-glUCOSe/D-mannOSe binding lectins, are Ca2+.Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. We have prepared a variety of Cd2+ deriva- tives of PSA and LcH, with Cd2+ in either the transition metal (Sl) or calcium (52) sites, or in both. Thus, Cd2+. Zn2+, Cd2+. Mn2+, and Ca2+. Cd2+ derivatives were prepared, in addition to the Cd2+.Cd2+ derivatives which we have recently reported. This is the first report of stable mixed metal Cd2+ complexes of lectins. The physical and saccharide binding properties of the Cd2+ de- rivatives of both lectins were characterized by a variety of physiochemical techniques and found to be the same as those of the corresponding native proteins. ‘I3Cd NMR spectra of mono- and disubstituted ‘13Cd2+ complexes of LcH and PSA were recorded and compared with ‘13Cd NMR data for concanavalin A (ConA) Bio- chemistry 5063-5070).

The lentil (LcH) and pea (PSA) lectins, which are members of the class of D-glUCOSe/D-mannOSe binding lectins, are Ca2+.Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. We have prepared a variety of Cd2+ derivatives of PSA and LcH, with Cd2+ in either the transition metal (Sl) or calcium (52) sites, or in both. Thus, Cd2+. Zn2+, Cd2+. Mn2+, and Ca2+. Cd2+ derivatives were prepared, in addition to the Cd2+.Cd2+ derivatives which we have recently reported. This is the first report of stable mixed metal Cd2+ complexes of lectins. The physical and saccharide binding properties of the Cd2+ derivatives of both lectins were characterized by a variety of physiochemical techniques and found to be the same as those of the corresponding native proteins.
'I3Cd NMR spectra of mono-and disubstituted '13Cd2+ complexes of LcH and PSA were recorded and compared with '13Cd NMR data for concanavalin A (ConA) (Palmer, A.R., Bailey, D.B., Behnke, W.D., Cardin, A.D., Yang, P.P., and Ellis, P.D. (1980) Biochemistry 19, 5063-5070). The data for the PSA and LcH derivatives were found to be very similar, indicating close homology of their metal ion binding sites. '13Cd resonances at 44.6 ppm and -129.4 ppm for Cd2+.LcH, and at 46.6 and -130.4 for the corresponding PSA derivative, are chemical shifts very similar to those observed for '13Cd2+. '13Cd2+. ConA. Assignment of the resonances to the transition metal (Sl) and calcium (52) sites were unambiguous since the Ca2+. '13Cd2+ and '13Cd2+.Zn2+ derivatives of both lectins showed single resonances characteristic of the S1 and 52 sites, respectively. The results indicate that, unlike ConA, l13Cd2+ binds tightly to PSA and LcH. Binding of monosaccharide to both lectins induce small (2 ppm) upfield shifts in their S2 l13Cd resonances, in contrast to the larger shift (8 ppm) observed in ConA. The '13Cd2+. Mn2+ complexes of PSA and LcH fail to show a '13Cd resonance characteristic of these derivatives, which provides evidence for the close proximity of the metal ions in the two proteins. The present findings indicate that the coordinating ligand atoms to the metal ions at the S1 and S2 sites in LcH, PSA, and ConA are the same.
113Cd2+. 113 * This work was supported in part by National Institutes of Health Grant GM 26295 (P. D. E.), National Science Foundation Grant CHE 82-07445 via its support of the RIF NMR facilities at the University of South Carolina, and National Institutes of Health Grants CA-16054 and Core Grant P30 CA-13339 (C. F. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Lentil (LcH)' and pea (PSA) lectins have been extensively employed as tools in many areas of biological research due to their unique carbohydrate specificities (1,2). Along with ConA, they have been classified as D-glucose/D-mannose specific lectins (3). However, unlike ConA, LcH and PSA display enhanced binding affinities toward certain fucosylated glycopeptides (4,5).
Metal ions are required for activities of a large number of lectins, including LcH, PSA, and ConA (3,6,7). However, only in the case of ConA has the role of metal ions been studied in detail (6). ConA possesses two metal-binding sites per monomer: S1, the so-called "transition metal" site, and S2, the "calcium" site (3). There are two conformational states of ConA which possess different binding properties: a "locked" conformation which strongly binds metal ions and saccharides; and an "unlocked" conformation which binds them very weakly (6,8). The apoprotein which exists predominantly in the unlocked conformation sequencially binds Mn'+ and Ca2+ and undergoes a first-order conformational transition to the locked or fully active conformation (6). Binary, ternary, and quaternary complexes of both conformations of ConA involving metal ions and saccharide have been characterized (6,8). Thus, the relationship between metal ion and saccharide binding properties of ConA are well established.
Although LcH and PSA also bind Mn'+ and Ca2+, little is known about the role of the metals in regulating the activities of the lectins. It is known that both proteins are dimers of molecular mass 47 kDa (9, lo), and that each monomer of both lectins consists of one a and one p chain, with molecular masses of 6.5 and 17 kDa, respectively ( 9 , l l ) . Each monomer of LcH and PSA also contains one Mn2+ and approximately two Ca'+ ions (7,12). Amino acid sequence data show conservation of the amino acids in LcH and PSA which constitute the S1 and S2 sites in ConA, except that Tyr-12 at S2 of ConA is replaced by a Phe residue in LcH (Phe-125) and PSA (Phe-123) (9,10). EPR (13)(14)(15) and magnetic circular dichroism data (16)' show the metals in the S1 sites to be in octahedral or slightly distorted octahedral environments in all three lectins, in agreement with the x-ray crystallographic data for ConA (18,19) and recent data for PSA (20) which show that the metal ions are located at positions similar to that in ConA. However, LcH and PSA differ from ConA in their metal ion exchange properties (7,12) and kinetics of The abbreviations used are: LcH, Lens culinuris hemagglutinin, lentil lectin; PSA, Pisum sativum agglutinin, pea lectin; ConA, concanavalin A, the jack-bean lectin; CD, circular dichroism; NMRD, nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, l/Tl, of solvent protons; 3-MDG, 3-0methyl-D-glucopyranose. E. Stafford, A. D. Cardin, W. D. Behnke, L. Bhattacharyya, and C. F. Brewer, manuscript in preparation. exchange of solvent water molecules at the Mn2+ and Caz+ sites (17,21). Furthermore, we have recently substituted Co2+ for Mn2+ in LcH and PSA (7), and observed differences in their visible CD spectra compared to Co'+-substituted ConA (22).2 Thus, it is important to characterize the metal ion binding properties of LcH and PSA in order to understand the role of the metals in the structure and activity of the lectins.
I13Cd NMR is an important tool for characterizing the metal ion-binding sites of metalloproteins because of its sensitivity to the nature of the coordinating ligands (cf. 23). This technique has been used to characterize the S1 and S2 sites of ConA (24, 25). However, in these studies, Cd" formed weak complexes with ConA and various mixed metal complexes of Cdz+ and the lectin were formed in solution under equilibrium conditions which did not yield well-defined complexes in certain instances (e.g. Cd2+ Mn2+ and Cd2+ .Zn2+).
We have recently reported the preparation of stable Cd2+.
CdZ+ derivatives of LcH and PSA (7). In the present study, we report the preparation and properties of stable Cd2+. Mn", Cd*+. Znz+, and Ca2+. Cd2+ derivatives of the two lectins, and I13Cd NMR spectra of these derivatives, as well as those of MATERIALS AND METHODS Seeds of lentil ( k m culinuris Med. sub. Macrosperma) and pea (Pisum satiuum L. var. Columbian) were purchased from a local food store. The respective lectins were purified by affinity chromatography on Sephadex G-100 (11,261. Salts of different metals were the highest purity products available from either Mallinckrodt or Fisher. Ix3Cd (95.8% enriched) was obtained from U. S. Services, Inc. Known weights of the metal were dissolved in 1:l diluted HCl or to get the corresponding 'Wd salt. Monosaccharides were obtained from Sigma and Pfanstiehl Laboratories. Polysaccharides PGM and GM of P. pinus were gifts from Dr. M. Slodki, Northern Regional Research Center, United States Department of Agriculture.
Preparation of Cd2+ Derivatives-A modification of the procedure described earlier for the preparation of other metal derivatives of LcH and PSA was used (7,12). LcH or PSA were dissolved at about 10 mg/ml in 50 mM sodium acetate buffer, pH 4.0, containing appropriate amounts of salts of different metals. The following combinations of salts were used 0.2 M CdSO, for the Cd2+.Cd2+ derivatives; 0.1 M each of CdCI2 and CaC12 for the Caz+. Cdz+ derivatives; 0.2 M CdSO, with 0.5 M MnSO, or ZnSo, for the Cd2+.Mn2+ or Cd2+.Zn2+ derivatives, respectively. The solutions were incubated at 37 "C for 16 days for LcH and 30 days for PSA. Any precipitate formed was removed by centrifugation, and the metal derivatives were then dialyzed against water at 4 "C and stored as salt-free lyophilizates. W d derivatives of the two lectins were obtained by using the appropriate 'I3Cd salts.
Protein Concentrations-The concentrations of LcH and its derivatives were determined using A:' & = 12.6 at 280 nm (7,27). The extinction coefficients at 280 nm of PSA and its derivatives were taken as 15.0 (7). Unless otherwise stated all protein concentrations are reported in terms of monomer concentration.
Metal Zon Analysis-The protein solutions of known concentrations were acidified to pH 1.2 with concentrated HCl and allowed to stand overnight at room temperature; the precipitates were removed by centrifugation. the supernatants were used for metal ion analysis by atomic absorption measurements using a Perkin-Elmer model 603 spectrophotometer (28). Mn2+ in these solutions was also determined by proton NMRD techniques (28).
Hemagglutination Assays-These were done at room temperature in phosphate-buffered saline at 1 mg/ml protein concentrations using 3% suspensions of rabbit erythrocytes (29).
Turbidity Reaction and Inhibition by Monosaccharides-These were done at room temperature in phosphate-buffered saline following Bhattacharyya et al. (7,12).
'I3Cd NMR Mea~urements--"~Cd NMR spectra were acquired on a Bruker WH-400 NMR spectrometer using a 10-mm broadband probe tuned to 88.756 MHz. Lyophilized protein samples (100-200 mg) were dissolved in 2.0 ml of pH 6.4 buffer, 0.1 M potassium acetate, and 0.1 M potassium chloride. To this solution was added 0.5 ml(20% v/v) of DzO. Spectra were acquired under deuterium field/frequency lock without proton decoupling at 22 'C. A 22" pulse and a 0.4-9 delay between scans was employed for a total of 130,000 scans/spectrum. Ligands to be added to the protein samples were dissolved in deionized water before being added to the samples. Dilution of protein samples by such additions was less than 10% in all cases. Typically, 50-Hz line broadening was applied before transforming each free induction decay. I13Cd chemical shifts were reported relative to 0.1 M cadmium perchlorate (0.0 ppm).
NMRD Measurements-Longitudinal (spin-lattice) relaxation rates of solvent water protons over a wide range of magnetic fields, corresponding to proton Larmor frequencies of 10 kHz to 60 MHz, were measured using a field-cycling apparatus as described (8,30). Solvent proton NMRD profiles of LcH and PSA and their derivatives were recorded in solutions of pH 6.4 buffers (0.1 M potassium acetate, 0.1 M potassium chloride) at 5 and 25 "C.

RESULTS
Preparation of Cd2' Derivatives-We have previously used 10 mM sodium acetate buffer, p H 4.0, to prepare Ca2+.Zn2+, Ca2+. Co2+, Ca2+. Ni2+, and Cd". Cd2+ derivatives of LcH and PSA (7, 12). However, preliminary experiments with LcH showed that the yields of Cd2+ derivatives listed in Table I were below 50% under these conditions. The yields of these derivatives were found to depend on the concentration of acetate in solution, with optimum yields of 70-80% obtained at 50 mM acetate. Therefore 50 mM sodium acetate buffer, p H 4.0, was used to prepare the derivatives reported here. This also allowed an increase in the protein concentrations used in the exchange reactions from the previously reported 5 mg/ml (7,12) to 10 mg/ml during the preparation of these derivatives. Table I shows the results of metal ion analysis of Cd". MnZ+, Cd2+.Zn2+, and Ca2+ Cd2+ derivatives of LcH and PSA.
The results show approximately 1:l stoichiometry between the metal ions and the 23.5-kDa monomer of the proteins.
Cd2+ .Mn2+ derivatives were found to have a slight excess of Cd2+ and less than the stoichiometric amounts of Mn2+, presumably due to the formation of small amounts of the corresponding Cd". Cd2+ derivatives during the exchange reactions (see below). The preparation and metal ion analysis of the Cd2+. Cd2+ derivatives of LcH and PSA have been reported earlier (7). Thus, conditions have been found in which Cd2+ occupies both the S1 and S2 sites of both lectins A mixture of all of the Cd2+ derivatives of LcH and the native protein coelutes in a single symmetrical peak (not shown) from a Bio-Gel P-100 column, indicating the same shape and size for the native lectin and Cd2+ derivatives. The same results are also obtained with native PSA and its Cd2+ derivatives. The near ultraviolet (240-320 nm) absorption and CD spectra of the Cd2+ derivatives are superimposable with those of the respective native proteins (7,22)' at the same protein concentrations (not shown). The results show that the conformations and overall structures of the Cd2+ derivatives of LcH or PSA are identical to those of the corresponding native lectins. The NMRD profiles of the Cd2+.Mn2+ derivatives of LcH and PSA were found to be identical to those of the corresponding native lectins containing Mn2+ and Ca2+ at the same temperature ( Fig. 2 and 3 of Ref. 21). The results indicate that the Cd2+-Mn2+ derivative of each lectin has the same coordination sphere of the Mn2+ ion as that of the corresponding native protein. Thus, Mn2+ is at the S1 sites and, as will be shown (below), Cd2+ at the S2 sites in these derivatives.
Hemagglutination, Precipitation, and Precipitation-lnhibition Assays-Native LcH and its Cd2+ derivatives are equally active in hemagglutinating rabbit red blood cells, each having a titer of 1024 at 1 mg/ml. The same results were obtained with the native PSA and its derivatives (Table I), each having a titer of 2048 at 1 mg/ml. The development of turbidity as a function of time was followed for Cd2+. Mn2+, Cd2+. Zn2+, and Ca2+. CdZ+ derivatives of LcH and PSA with the two polysaccharides from P. pinus PGM and GM, and compared with the corresponding curves obtained with the respective native lectins (Fig. 4 of Ref. 7). The native and the metal derivatives of each lectin were found to give overlapping curves.
For the determination of concentrations of monosaccharides required for 50% inhibition of precipitation, readings were taken 45 min after mixing (7). The concentrations of methyl a-D-glucopyranoside, methyl ,f3-D-glucopyranoside, and 3-MDG required for 50% inhibition of precipitation of PGM by native lectins and their Cd2+ derivatives are shown in Table 11. The results indicate that all of the Cd2+ derivatives  (Fig. lb, Table 111). Similar results were obtained for '13Cd2+ ll3CdZ+. LcH in the presence of 3-MDG (Table 111).
The 'l3Cd NMR spectrum (not shown) of 113Cd2+.Mn2+. LcH gave two small resonances of equal intensities which were nearly within the noise level and with the same chemical shifts as that of the corresponding double ' 13Cd derivatives. The same result was obtained with the PSA derivative. In each case, the intensity did not account for the Cd2+ incorporated in the proteins (Table I).

DISCUSSION
Native LcH and PSA consist of mixtures of two isolectins, A and B, which have the same molecular weight and amino acid compositions (11,26,27,31). The molecular and spectroscopic properties and biological activities of the isolectins of each protein are identical (15). Therefore, native mixtures of these lectins were used in this study.
Preparation of C8+ Deriuatiues of LcH and PSA-Bhattacharyya et al. (7,12) have previously shown that the Mn2+ and Ca2+ in PSA and LcH can be selectively substituted by other metal ions at pH 4.0 and 37 "C in the presence of high concentrations of the desired salts. Thus, Mn2+ in PSA and LcH was substituted with Co2+, Zn", Ni", and Cd2+ to give the corresponding Ca2+.M2+ complexes. Cd2+ was the only metal to replace both the Mn2+ and Ca2+ (at S1 and S2 sites, respectively) to give the double Cd2+ derivatives. All of the metal derivatives were found to be as active as the respective native proteins.
The present report shows that Cd2+ can be selectively substituted into the S1 and S2 sites of PSA and LcH to give either diamagnetic complexes, such as the Ca2+.Cd2+ and Cd2+ -Zn2+ complexes in which Cd2+ occupies S1 and S2, respectively, or paramagnetic complexes, such as the Cd2+. Mn2+ complexes, with Cd2+ at S2. These complexes are stable, fully active, and can be isolated and characterized. This is in contrast to Cd" complexes of ConA in which the metal is weakly bound to the protein and various mixed metal complexes of Cd" are formed in solution under equilibrium conditions (25). In fact, ConA does not yield well defined Cd2+. Zn2+ or Cd2+. Mn2+ complexes, and the interpretation of the ' 13Cd NMR results for such complexes have been tentative (25). Thus, the ability to prepare '13Cd2+ derivatives of PSA and LcH with selective substitutions at the S1 and S2 sites has permitted us to define fully the 'l3Cd2+ NMR spectral properties of the S1 and S2 sites in both lectins and compare these results with the 'l3Cd NMR data for ConA (25).
Properties of C&+ Deriuatiues-By a variety of criteria, the Cd2+ derivatives of LcH and PSA (Table I) are identical in their intrinsic molecular and spectroscopic properties to the respective Mn2+ and Ca2+ containing native lectin (12,21,22)? The near ultraviolet absorption and CD spectra indicate the same conformations of the derivatives as the respective native lectins. Gel-filtration chromatography show that the derivatives have the same hydrodynamic properties as the respective native lectins. NMRD studies indicate that solvent relaxation by Mn2+ in the Cd2+.Mn2+ complexes of LcH and PSA occurs via the same mechanisms as in the respective native proteins (21), which suggest that the Mn2+ in the former complexes have the same coordination environments as in the respective native proteins.
The results of hemagglutination, precipitation, and precipitation-inhibition studies (Table 11) indicate that saccharide binding activities of these derivatives are also equal to those of the respective native lectins. Similar results have previously been reported for other metal derivatives (7,12). These results for LcH and PSA are also similar to those observed for metalsubstituted ConA in which the derivatives are equally active as the native protein (32), indicating that the saccharide binding properties of these three lectins are not sensitive to the nature of the metals present at the S1 and S2 sites.
' 13Cd NMR of Double 'l3Cd Cornple~es--"~Cd NMR studies of ConA (24,25) showed that the ternary locked 'l3Cd2+. "3Cdz+-complex of ConA gave rise to resonances at 46 and -125 ppm, which were assigned to the S1 and S2 sites, respectively. The 46-ppm resonance is characteristic of nitrogen and oxygen-coordinated Cd2+ (33,34), and the -125-ppm '13Cd Nuclear Magnetic Resonance Studies of the Pea and Lentin Lectins resonance is characteristic of oxygen-coordinated Cd2+ (35-37), both in an octahedral environment. The data coroborate with the presence of Glu-8, Asp-10, Asp-19, and His-24 at the S1 site, and Asp-10, Tyr-12, Asp-14, and Asp-19 at the S2 site of ConA (18). The lI3Cd NMR spectrum of the double '13Cd2+ complex of PSA shows resonances at 46.6 and -130.4 ppm (Fig. 1, Table  111). 'l3Cd resonances at 44.6 and -129.4 ppm were observed for the double 113Cd2+ complex of LcH (Table 111). By analogy with ConA (25), the resonances at 46.6 and 44.6 ppm for PSA and LcH, respectively, can be tentatively assigned to '13Cd at the SI sites of the proteins, whereas the resonances at -130.4 and -129.4 ppm tentatively assigned to 'l3Cd at the S2 sites. Spin-echo EPR studies of native LcH and PSA3 show the presence of a His residue as the ligand for Mn2+ at the S1 site in both lectins, in agreement with the findings of conserved His residues at this site in the primary sequences of LcH, PSA,Fnd ConA (9,lO) and recent x-ray crystallographic data at 3 A resolution for PSA (20). Thus, the presence of '13Cd resonances at 44.6 and 46.6 ppm in LcH and PSA, respectively, is consistent with binding of '13Cd2+ to a His residue in the S1 sites of these proteins.
The resonances at -129. 4 and -130.4 ppm for the double W d complexes of LcH and PSA, respectively (Table III), are characteristic of purely oxygen-liganded '13Cd2+ (35-37) and indicate that these resonances are associated with binding of 'l3Cd to the S2 sites of LcH and PSA. Thus, substitution of a Phe residue in LcH and PSA for a Tyr residue in ConA at the S2 sites has little effect on the coordination environment of ll3Cd'+ at S2 in these proteins (9,10). Earlier 'l3Cd NMR studies of ConA (24, 25) observed that a fraction of the total '13Cd2+ added to a solution of apoCon A to form 113Cd2+.113Cd2+.Con A remained free. Further ' 13Cd NMR relaxation studies showed that free 'l3Cd2+ exchanged with Cd" bound at S1 and S2 of ConA due to the small binding constants of Cd" (3.1 X lo3 and 2.1 X 10' M-', respectively) at these sites (25). The absence of any detectable free II3Cd2+ in all spectra of LcH and PSA indicate much tighter binding of Cd2+ to both the S1 and S2 sites of these lectins.
'l3Cd NMR of C@+. Zn'+ Complexes-The '13Cd NMR spectrum of l13Cd2+. Zn'+. LcH (Fig. 2a) shows one resonance line at -122.2 ppm which is characteristic of purely oxygencoordinated Cd" (35-37). A single resonance at -123.7 ppm was also obtained with the corresponding complex of PSA (Table 111). Thus, the chemical shift of the single resonance in each spectrum confirms that the '13Cd is at the S2 site in each protein. Interestingly, the single resonances in the 'l3Cd2+. Zn2+ complexes of LcH and PSA are shifted downfield by approximately 7 ppm compared to the corresponding resonances in the respective double 'l3Cd complexes (Table 111).
These results indicate that the resonance due to 'l3Cd at the S2 site in both proteins is sensitive to whether Zn2+ or '13Cd2+ is present at the S1 sjte, which is consistent with the expected close proximity (5 A) of the two sites based on the x-ray crystallographic data of PSA (20) and ConA (cf. 19). Such an effect also appears to occur in the corresponding ll3Cd'+ complex of ConA, although the formation of Cd" . Zn2+. ConA complex is less well-defined (25). ' 13Cd NMR of Ca2+. '13Cd2+ Complexes-Single resonances were observed for the Ca2+.113Cd2+ complexes of LcH and PSA at positions nearly identical to the S1 resonances of the respective double 'l3Cd complexes of both proteins (Table  111). Ca2+. l13Cd2+. PSA has a resonance at 45.0 ppm (Fig. 3a) compared to 46.6 ppm for the double '13Cd derivative, while J. McCracken and C. F. Brewer, manuscript in preparation. Ca2+. 113 Cd'+.LcH has a resonance at 42.9 ppm compared to 44.6 ppm for the double '13Cd derivative (Fig. lb). Thus, there is a small chemical shift dependence for the 'l3Cd resonance at S1 on whether Cd2+ or Ca2+ is present at S2 for both proteins. These results also confirm the chemical shift assignment of the downfield resonances of the double 'I3Cd2+ complexes of LcH and PSA to the S1 sites.
'l3Cd NMR of '13Cd2+, Mn2+ Complexes-The 'l3Cd NMR spectra of the 1'3Cd2+. Mn2+ complexes of both LcH and PSA failed to show any resonance other than those associated with smaller amounts of double 'l3Cd complexes which were formed during the preparation of the former complexes. The presence of Mn" at the S1 sites in the 'l3Cd2+. Mn2+ complexes of both lectins was verified by NMRD analysis and the amount of Mn'+ and Cd2+ by NMRD and atomic absorption spectroscopy (Table I). Since divalent manganese is paramagnetic, nearby nuclei experience enhanced Tl and T2 relaxation, and thus undergo line-broadening of their resonances. The extent of line-broadening depends upon the distance between the two nuclei (39). The results obtained with the other Cd2+ derivatives already presented indicate close proximity between S1 and S2 sites. The distance between these two sites in LcH and PSA has been calculated (21) to be approximately 5.5 A, in agreement with recent x-ray crystallographic data for PSA (20). Thus, the broadening of '13Cd resonances in the '13Cd2+. Mn2+ derivatives are so large that they are unobservable. These results provide direct evidence for binding of '13Cd at the S2 sites in LcH and PSA. Effects of Saccharide Binding-Binding of the monosaccharide 3-MDG to the various ' 13Cd complexes of LcH and PSA produced essentially the same small effects. 'l3Cd resonances associated with the S2 sites in both proteins were shifted upfield by approximately 1-2 ppm, while resonances associated with the S1 sites were essentially unchanged (Table  111). This contrasts with the larger upfield shift of 8 ppm for the S2 resonance of the double 'l3Cd2+ complex of ConA (25). Like PSA and LcH, the '13Cd resonance for the S1 site of the ConA complex also was insensitive to saccharide binding.
These results suggest that the S2 sites of LcH and PSA undergo a larger conformational change than the S1 sites upon monosaccharide binding but that the magnitude of this change is somewhat less than that in ConA. The lack of any purturbation in the EPR spectra of the Mn2+ (15,38) and magnetic circular dichroic spectra of Co'+ (16)' at S1 sites in the three lectins supports the conclusion that there is little conformational change at S1 upon saccharide binding. The near ultraviolet CD spectra of LcH and PSA, however, does show that a conformational change occurs in the proteins upon saccharide binding (22).' Other Comments-Recent NMRD studies of Ca2+. Mn2+. LcH and .PSA indicate that the primary site of solvent relaxation occurred at the Ca2+ site (S2) and not at the Mn2+ site (Sl) (21). These results are in contrast to NMRD studies of Ca'+. Mn'+. ConA which showed solvent relaxation contributions at both the S1 and S2 sites (17). Since the x-ray crystallographic structure of ConA shows two water ligands of the Mn2+ (19), the question arises as to whether there are similar water ligands at the S1 sites in LcH and PSA. The present '13Cd NMR data show that complexes of LcH and PSA with Cd" at the S1 sites give resonances that are very close to the chemical shift position(s) of Cd2+ at the S1 sites of ConA (24, 25). Since l13Cd NMR chemical shifts are sensitive to the nature and number of ligand atoms, and geometry of the complex (23), these results provide evidence that the water ligands of the metal ion at S1 are present in LcH and PSA, as well as in ConA, and that their exchange kinetics must be slower in the former two lectins such that they do not contribute to the observed solvent proton relaxation rates in the respective Ca2+.Mn2+ complexes.
Similar considerations indicate that both ligand waters of Ca2+ in ConA (19) are also present in LcH and PSA.

SUMMARY
We have prepared and characterized a series of stable monoand disubstituted Cd2+ derivatives of LcH and PSA (the first time for any lectin) and found them to have the same physical and saccharide binding properties as the respective native Ca2+. Mn2+ lectins. '13Cd NMR spectra were recorded for derivatives of LcH and PSA, which allowed characterization of the transition metal (Sl) and calcium (S2) binding sites of both proteins. The '13Cd N M R data for LcH and PSA are similar, and also similar to that of ConA, which indicates conservation of the coordinating ligand atoms to metal ions at the S1 a n d S2 sites in all three proteins. These results can be compared to recent CD studies of Ca2+.Co2+ derivatives of the three lectins, which show some differences in the environments of the metal ions in LcH and PSA, as compared to ConA (22).' The results also agree with recent limited x-ray crystallographic data for PSA (20).