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Unfolding Studies on Soybean Agglutinin and Concanavalin A Tetramers: A Comparative Account

Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India

Correspondence: Address reprint requests to Avadhesha Surolia, Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India. Tel.: 91-80-22932714; Fax: 91-80-23600535; E-mail: surolia{at}mbu.iisc.ernet.in.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The unfolding pathway of two very similar tetrameric legume lectins soybean agglutinin (SBA) and Concanavalin A (ConA) were determined using GdnCl-induced denaturation. Both proteins displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provided values for conformational stability of the two proteins. It was found that the G of unfolding of SBA was much higher than ConA at all the temperatures at which the experiments were done. ConA had a T_g 18°C less than SBA. The higher conformational stability of SBA in comparison to ConA is largely due to substantial differences in their degrees of subunit interactions. Ionic interactions at the interface of the two proteins especially at the noncanonical interface seem to play a significant role in the observed stability differences between these two proteins. Furthermore, SBA is a glycoprotein with a GlcNac₂Man₉ chain attached to Asn-75 of each subunit. The sugar chain in SBA lies at the noncanonical interface of the protein, and it is found to interact with the amino acid residues in the adjacent noncanonical interface. These interactions further stabilize SBA with respect to ConA, which is not glycosylated.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Small monomeric proteins have been studied extensively for characterization of their folding reactions. This is mostly related to the high degree of reversibility and two-state unfolding reaction displayed by them. Additionally, these proteins also fold/unfold fairly fast, making them highly suitable systems for kinetic analyses of the reactions involved therein. Also, the folding process of small, monomeric globular proteins is amenable to study by NMR spectroscopy. Although the vast majority of proteins in nature exist as oligomers, quantitative study of their unfolding reactions have been few. Delineation of the unfolding of oligomeric proteins is relatively challenging as compared to the monomeric proteins, where only intrasubunit interactions are involved whereas the oligomeric proteins are stabilized by intersubunit interactions as well (Bowie and Sauer, 1989; Neet and Timm, 1994). Studying the unfolding of multimeric proteins provides an insight into the role and the contribution of intersubunit interactions to their stability. For example, under standard conditions viz. at pH 7.6, 298 K, the stability of dimeric tryptophan aporepressor, was found to be 23.3 kcal/mol as compared to the dissociated monomer at pH range 3.5–6.0, whose stability was only 5.4 kcal/mol monomer (Matthews, 1993; Gittelman and Matthews, 1990). Thus, we see that dimerization increases the stability of the protein by almost two times. It is, however, worth mentioning here that many oligomeric proteins show only a two-state transition, where the folded oligomer and the unfolded constituent polypeptide exist as the equilibrium species at any given time during the unfolding reaction. This suggests that a significant amount of stability is imparted to the system via intersubunit interactions.

This article reports the unfolding reaction of two tetrameric legume lectins, soybean agglutinin (SBA) and Concanavalin A (ConA). Legume lectins are a group of oligomeric proteins that bind carbohydrates reversibly and specifically. Although they differ in their specificities, the orientation of their binding site and mechanism of ligand recognition remains almost the same. This is perhaps due to the fact that all legume lectins share a common tertiary structure (Manoj and Suguna, 2001) although their quaternary structures differ considerably. The tertiary structural fold in legume lectins is described as the "jelly roll" motif. This is a kind of a ß-sandwich fold consisting of three antiparallel ß-sheets (Prabu et al., 1999; Chandra et al., 2001). The mutual disposition of the three ß-sheets is shown in Fig. 1 a. There is a six-stranded "back" ß-sheet, a curved seven-stranded "front" ß-sheet, and a five-stranded "top" ß-sheet, which forms a roof above the other two. There are several loop regions in the motif, which hold the sheets together. There are two strong hydrophobic cores stabilizing the structure; one lies at the center of the three sheets and the other in the curvature of the front ß-sheet. Our laboratory has been working on legume lectins as paradigms for the unfolding reactions of oligomeric protein (Mitra et al., 2002, 2003; Ahmad et al., 1998). Such reactions have been characterized to a considerable extent for this family of proteins that exists as dimers.

View larger version (41K): [in this window] [in a new window]   FIGURE 1  (a) A typical legume lectin monomer. (b) Tetramer of soybean agglutinin showing the N-linked glycan in ball and stick models. (c) Tetramer of Concanavalin A. In both tetramers the interface type is marked by arrows.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Materials
Ultrapure guanidinium hydrochloride (GdnCl) was purchased from Sigma Chemical (St. Louis, MO). All other reagents used for the study were of highest purity available. Stock GdnCl solutions were prepared fresh, in 25 mM HEPES (N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid) buffer pH 7 containing 15 mM CaCl₂ and MnCl₂. The concentration of GdnCl was determined by refractive index as described by Pace (1990).

Protein purification
Soybean agglutinin
In a typical preparation, 250 g of soybean seeds were homogenized and defatted. The defatted dry meal was extracted with 20 mM phosphate buffer pH 7.4 containing 150 mM sodium chloride (PBS) for 12 h at 4°C under constant stirring. The extract was subjected to ammonium sulfate fractionation of 30%. The precipitate was removed by centrifugation at 8000 rpm for 30 min. The supernatant was again subjected to 65% ammonium sulfate fractionation. The precipitate was collected this time by centrifugation at 8000 rpm for 45 min. The precipitate was dissolved in a minimum amount of the buffer and extensively dialyzed against the same buffer. The dialyzed solution was centrifuged at 8000 rpm for 15 min and the clear supernatant was loaded to a lactosylamine Biogel P-150 column preequilibrated with buffer (Baues and Gray, 1977). The column was then washed extensively with PBS until the washings have A₂₈₀ < 0.005. Elution was carried out in 0.2 M lactose in PBS. The concentration of the protein solution was determined from specific extinction coefficient of 12.8 for SBA (Lotan et al., 1974).

Concanavalin A
Jack bean seeds (250 g) were soaked overnight in water. After the removal of the seed coat the seeds were cut into small pieces and left in a homogenization buffer (20 mM Tris, 150 mM sodium chloride, 1 mM Ca²⁺, and Mn²⁺, pH 7.4). The seeds were then homogenized in six volumes of homogenized buffer and centrifuged at 5000 rpm for 10 min. The pellet was discarded and the supernatant was subjected to ammonium sulfate fractionation of 65%. The precipitate was collected by centrifugation at 8000 rpm for 30 min. The pellet was resuspended in a minimum amount of buffer and extensively dialyzed against the same buffer. The dialyzed solution was centrifuged at 8000 rpm for 15 min and the clear supernatant was loaded onto a Sephadex G-75 column preequilibrated with buffer (Agrawal and Goldstein, 1967). The concentration of the protein solution was determined from specific extinction coefficient of 12.4 for ConA (Mitra et al., 2002).

Isothermal GdnCl-induced denaturation
Equilibrium unfolding studies as a function of guanidinium hydrochloride concentration was monitored using fluorescence spectroscopy and far-ultraviolet (UV) circular dichroism (CD). The fluorescence measurements were done on a Jobin Yvon Horiba fluorometer (Jobin Yvon (Spex division), Cedex, France) in a 1-cm water-jacketed cell using a protein concentration of 2 µM. The protein concentration used for all the experiments was 2 µM in terms of the monomer, unless otherwise mentioned. The excitation and emission wavelengths were fixed at 280 and 370 nm, respectively for SBA and at 280 and 323 nm for ConA. These wavelengths were determined by obtaining the maxima of the difference spectra of the native and unfolded forms of the respective proteins. This is very well seen in Fig. 2, a and c. The maximum difference between the native and the denatured spectra occurs at 370 nm for SBA and 323 nm for ConA. This has been indicated by a dashed line drawn at those wavelengths in the figure. In all experiments, the slit width was fixed at 3 and 5 nm for excitation and emission, respectively. All the CD experiments were done in a JASCO-J715 polarimeter (JASCO, Tokyo, Japan) in a 0.1-cm pathlength cell, with a slit width of 1 nm, response time of 4 s and scan speed of 50 nm/s. Each data point was an average of three accumulations. The data for SBA were collected at 222 nm and for ConA at 226 nm. The reversibility of unfolding was checked by refolding the unfolded protein by both dialyzing the unfolded protein in HEPES buffer containing 15 mM metal ions and diluting the unfolded protein from 7.8 M GdnCl to 0.5 M GdnCl or less. Eight and 12 GdnCl-induced isothermal denaturation curves were collected between the temperature range 273–323 K for SBA and ConA, respectively. A Julabo water bath was used to maintain the sample temperature within 0.1 K of the set temperature.

View larger version (26K): [in this window] [in a new window]   FIGURE 2  Refolding of SBA monitored by (a) fluorescence and (b) CD. Refolding of ConA monitored by (c) fluorescence and (d) CD. The CD spectra are normalized with respect to protein concentration (2 µM) and pathlength of the cuvette used (0.1 cm). Fluorescent spectra are normalized to the highest intensity observed.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Both SBA and ConA showed >90% reversibility upon refolding of the unfolded protein obtained by GdnCl denaturation. To obtain the condition of total reversibility we have used different concentrations of metal ions and found that at 15 mM metal ion concentration the unfolding reaction is almost reversible for both proteins. Figs. 2 and 3 show the refolding of the two proteins monitored by spectroscopy (fluorescence and CD) and gel filtration, respectively.

View larger version (30K): [in this window] [in a new window]   FIGURE 3  Refolding of SBA and ConA as observed by gel filtration. The protein concentration used for the refolding study was 40 µM. The protein sample was denatured in 6 M GdnCl and then renatured by dialyzing in 25 mM HEPES buffer for 12 h. The samples were then loaded to a P-150 Biogel column (40 ml bed volume; void volume was 11 ml as determined by blue dextran). The flow rate was maintained at 5 ml/h throughout the experiment. The gel filtration was conducted at 293 K.

where K₁ and K₂ are the equilibrium constants for the formation of the partially unfolded and fully unfolded protein, respectively. Here, the intermediate could be a partially folded/unfolded tetramer or dimer or even a monomer.

Transitions that occur with the formation of intermediate(s) are associated with biphasic denaturation curves or nonsuperimposable transitions when different spectroscopic probes, monitoring different properties of the protein, are used. In this case the respective denaturation curves for both ConA and SBA monitored by fluorescence and far-UV CD were cooperative and superimposable (Fig. 4). This ruled out the possibility for a non-two-state process (Barrick and Baldwin, 1993). In addition to this, the overlay of the fluorescence spectra of the two proteins in different GdnCl concentrations (0–7.3 M range) shows the existence of an isoemissive point in both cases (Fig. 5). This suggests the existence of only two species in the transition (Gualfetti et al., 1999). Furthermore, analyses of the gel filtration profiles suggested that in the pretransition region both proteins elute in their native form and as an unfolded form in the posttransition region (Fig. 6). The unfolded form is unfolded monomers whose size is often bigger than that of the folded tetramer; this property is in general observed in proteins. In the transition region, different concentrations of the native and unfolded form are seen, which are almost similar to what is observed in the spectroscopic studies. Because it was not possible to do the gel filtration experiment at low protein concentrations of 2 µM, denaturation transition of the two proteins were studied by both fluorescence and CD in the presence of 40 µM protein concentration. It was observed that under this condition the transitions monitored by the two spectroscopic probes overlay (Fig. 7). This also strongly supports the two-state assumption. Although the proteins show concentration dependence in their profile they do not deviate from their two-state behavior (Fig. 8, b and c). At this point, only one scenario seems possible and that is N₄ 4U transition, i.e., the native tetramer dissociates to unfolded monomers. A general consequence of a two-state process is that the fraction of molecules populating the native state increases with protein concentration. This is very well evident from Fig. 8, b and c. The equilibrium constant for the above reaction may be given as:

(1)

where is the fraction of folded protein molecules, n is the number of subunits in the oligomeric protein, and p is the protein concentration in n-mer equivalents.

View larger version (12K): [in this window] [in a new window]   FIGURE 4  Overlap of GdnCl-induced unfolding profiles obtained using fluorescence () and far UV-CD (•) at 298 K. The completely superimposable profiles confirm the two-state unfolding reaction for both proteins.

View larger version (37K): [in this window] [in a new window]   FIGURE 5  Fluorescence emission spectra of SBA and ConA in different GdnCl concentrations (0–7.5 M range). The gradient is indicated by an arrow near the wavelength, which was used to monitor the isothermal melts. All the curves are equally spaced with respect to Gdncl concentration. The spectra are normalized to the highest intensity.

View larger version (29K): [in this window] [in a new window]   FIGURE 6  Gel filtration profiles of SBA and ConA at different GdnCl concentrations. The denaturant concentration is indicated at the side of each profile. The protein concentrations used were 40 µM in each case. The optical densities were taken at 280 nm. The column used was a P-150 Biogel column (40 ml bed volume; void volume was 11 ml as determined by blue dextran). The flow rate was maintained at 5 ml/h throughout the experiment. The gel filtration was conducted at 293 K. During the experiment the column volume changed at the most by 0.75 ml at 3.5 M GdnCl, 2 ml at 4.9 M GdnCl, and 2.7 ml at 6.2 M GdnCl. Each time the elution volume was normalized with respect to the starting column volume.

View larger version (15K): [in this window] [in a new window]   FIGURE 7  Overlap of GdnCl-induced unfolding profiles obtained using fluorescence () and far UV-CD (•), with a protein concentration of 40 µM at 298 K. The superimposable curves indicate the two-state folding behavior of the transition.

View larger version (13K): [in this window] [in a new window]   FIGURE 8  (a) GdnCl-induced unfolding of SBA () and ConA () at 298 K. The solid line passing through the points is the fit of Eq. 7 to the experimental data. (b) Protein concentration-dependent unfolding of ConA at 298 K, () 2 µM, and () 10 µM. (c) Protein concentration-dependent unfolding of SBA at 298 K, () 2 µM, and () 10 µM.

(2)

G_u was calculated at various denaturant concentrations using the equation:

(3)

The free energy of unfolding of the two proteins at different temperatures is calculated according to the LEM model (Schellman, 1990). According to this model the changes in free energy, enthalpy, entropy, and heat capacity, which accompany the unfolding reaction, bear a linear relationship to the molar concentration of the denaturant, i.e.,

(4)

(G_u represents the free energy of unfolding obtained in presence of a known concentration of denaturant and G_o is the free energy of unfolding in the absence of any denaturant).

Thus, from Eqs. 3 and 4,

(5)

By substituting Eq. 5 in Eq. 2 we get four roots for , two of which are real and two imaginary. Out of the two real roots one is a positive fraction, which corresponds to the fraction unfolded in this investigation. The software MATHEMATICA was used to determine the desired root. The root is shown below:

(6a)

where

(6b)

and

(6c)

Finally, this root was used in the following equation and fitted to the normalized spectroscopic data obtained experimentally (fluorescence or CD).

(7)

From the fit of Eq. 7, the parameters of interest G_o, m, m_u, m_n, y_n, and y_u were obtained. The free energies of unfolding of ConA and SBA at each temperature thus obtained from the fit are listed in Table 1. The fit of the plot of fraction native against denaturant concentration is shown in Fig. 8 a.

(8a)

x

(8b)

where, T_g is the temperature at which G = 0 and H_g is the unfolding enthalpy at T_g; T_m is the melting temperature for the protein, and H_m is the enthalpy at T_m. Values of the parameters obtained from the fit of Eqs. 8a and 8b are summarized in Table 2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

Unfolding results in demetallization, which will introduce perturbation in the system, and prevent the protein from folding back to its native conformation, when attempts to refold in sparse metal ion conditions are made. However, if the system contains enough metal ions, the unfolded protein can get remetallized leading to recovery of the native state as indeed is observed in these studies.

One of the most important features of protein unfolding is the exposure of its hydrophobic core. This exposure is reflected in the positive change in heat capacity of the protein during the unfolding reaction. Thus, the change in heat capacity is directly proportional to the extent of unfolding and conversely the extent to which the apolar residues are buried in the native protein. Both SBA and ConA show a positive change in heat capacity upon unfolding, thus confirming the exposure of nonpolar surfaces to the polar aqueous environment. It was shown by Spolar et al. (1992) that the transfer of hydrocarbons from water to pure liquid state showed similar proportionality in the reduction of water-accessible surface area as is shown by the folding of globular proteins (Livingstone et al., 1991; Spolar et al., 1992; Ganesh et al., 1997). The theoretical calculation of C_p is done according to the equation given by Spolar:

(9)

We calculated the change in accessible surface area (ASA) using NACCESS (Hubbard, 1996; Manoj et al., 2000) for both proteins and it was found that they were very similar at 32,583.8 A² and 33,739.7 A² for SBA and ConA, respectively. The contribution of the polar and apolar regions (ASA_p and ASA_np) in the above figures is 40 and 60%, respectively, for SBA. For ConA they are 48 and 52%, respectively. The proportion of ASA buried at the interface is 15% in SBA and 25% in ConA. C_p values obtained from Eq. 9 are 19.6 kcal/mol/K for SBA and 17.7 kcal/mol/K for ConA. The greater value for C_p for SBA suggests that the bulk of the buried hydrophobic core is greater in SBA when compared to ConA. This strong hydrophobic core may be a contributing factor for the higher stability of SBA (Table 1; Fig. 9). The percentage of hydrophobic residues in SBA is 50% as compared to ConA where it is 40%, the actual numbers being 512 and 448 for SBA and ConA, respectively. This suggests that the hydrophobic core is stronger in SBA than in ConA. The experimentally obtained values of C_p for SBA and ConA are 5.0 and 4.2 kcal/mol/K, respectively. Although the experimentally determined values of C_p for both lectins are lower than the calculated values they follow, the trend of the theoretically determined ones viz. values for SBA are greater than that for ConA. Why does a difference between the theoretically and experimentally determined values exist? This is perhaps a reflection of the fact that some proteins do not undergo complete unfolding, i.e., they do not attain the complete random coil structure (on which the theoretical calculations are based) upon denaturant-induced unfolding reactions. This has been previously observed in many oligomeric proteins (Backmann et al., 1998).

View larger version (11K): [in this window] [in a new window]   FIGURE 9  Stability curve for SBA and ConA obtained from GdnCl-induced isothermal unfolding experiments done at different temperatures indicated in Table 1. The continuous line shows the fit of the points to Eq. 8a. The data points () for SBA, and () for ConA have been obtained using individual melts done at different temperatures.

Although the main force driving protein stability is hydrophobic interactions, many mutagenesis studies have revealed that hydrogen bonds also play an important role in stabilizing proteins (Shirley et al., 1992; Pace, 1995, 2001; Pace et al., 1996; Myers and Pace, 1996). In this study we have used HBPLUS to determine the number of hydrogen bonds in both proteins (McDonald and Thornton, 1994). When we analyzed the relative strength of the bonds it was observed that the number of strong hydrogen bonds (donor acceptor distance D–A < 2.5 Å) were 18 times greater in SBA than in ConA, enhancing the stability of the SBA tetramer. The distributions of the hydrogen bond lengths in both proteins are shown in Fig. 10. The protein data bank (PDB) coordinates of 1cvn and 2sba have been used for the above and all other calculations.

View larger version (17K): [in this window] [in a new window]   FIGURE 10  Plot of the number of hydrogen bonds as a function of the donor-acceptor distance in SBA and ConA.

The amino acid composition of the two proteins show that the population of charged residues is 6% more in SBA than in ConA; the actual numbers being 240 and 224, respectively. The increase in charged residues in SBA probably results in an enhanced number of electrostatic interactions in SBA as compared to ConA. Contrary to expectations, the total number of ionic interactions are greater in ConA than in SBA. But, a further analysis of the interactions in the interface revealed that the noncanonical interface in SBA, was significantly stronger than in ConA. There were 10 intersubunit interactions across this interface in SBA (giving rise to a total of 20 in the tetramer), whereas in ConA only six such interactions (a total of 12 in the tetramer) were observed across the corresponding interface. The details of the interactions are summarized in Table 3. All the intersubunit interactions are shown in Figs. 11 and 12.

View larger version (44K): [in this window] [in a new window]   FIGURE 11  (a) The noncanonical interface in ConA showing the intersubunit salt bridges. Six such salt bridges are formed per noncanonical interface so the tetramer has a total of 12 salt bridges. (b) Enlarged view of the area where the interactions take place. The salt bridges are shown by dashed lines. Only three pairs are shown in the figure. The other three are just the mirror images of the three shown. Figure was generated using ViewerLite42. All the interactions are listed in Table 3.

View larger version (66K): [in this window] [in a new window]   FIGURE 12  (a) The noncanonical interface in SBA showing the intersubunit salt bridges. Ten such salt bridges are formed per noncanonical interface so the tetramer has a total of 20 salt bridges. (b) Enlarged view of the area where the interactions take place, which are marked by a square frame in panel a. The salt bridges in both the frames are identical with the only difference that the chain identity of the amino acids involved is reversed; i.e., the Arg-182 shown in the enlarged version is changed to Arg'-182 in the other window. The salt bridges are shown by dashed lines. Figure was generated using ViewerLite42. All the interactions are listed in Table 3.

Between SBA and ConA, the total area buried at the interface is greater in ConA. Despite this, SBA shows greater stability. Thus, we see that SBA makes up for the relatively smaller buried area at the interface by other interactions that strengthen the interface. This is in contrast to what was observed in dimeric ConA and WBA II. Here, ConA with a greater buried area at the interface was also found to be more stable (Mitra et al., 2002).

Another factor that probably accounts for the greater stability of SBA over ConA is the presence of an N-linked glycan in SBA. SBA has three potential glycosylation sites out of which only one, Asn-75, is glycosylated due to the perfect geometry of this site. It has been reported that glycosylation affects both the structural elements and the global stability of proteins. The N-linked glycans are large hydrophilic structures. The molecular weight of the N-linked glycan in SBA (GlcNac₂Man₉) is 1884. The volume of such a big carbohydrate moiety is 1800 ų, excluding the solvation sphere. This size is therefore significant in comparison to the monomeric protein size, which is 30,000 ų. If the solvent sphere is also taken into consideration, one can imagine the enormous stability imparted to the system due to its enlarged solvent cage as compared to ConA (Wyss and Wagner, 1996; O'Connor and Imperiali, 1996; Rudd and Dwek, 1997; Imperiali and O'Connor, 1999). N-linked glycan induced stability has also been shown in another legume lectin, Erythrina corallodendron lectin (Mitra et al., 2003).

By a careful analysis of the SBA structure (1g9f in the PDB database) we found that the glycan is found at the noncanonical interface of the protein. This gives rise to interactions between the glycan of one of the subunits with the adjacent noncanonical subunit. This glycan-mediated interaction, gives added stability to the noncanonical interface of the protein (Fig. 13). No such stabilizing interactions are expected in ConA because it is not glycosylated.

View larger version (63K): [in this window] [in a new window]   FIGURE 13  (a) The interactions between the glycan of one subunit and the residues of the other subunit at the noncanonical interface in SBA. (b) The enlarged view of the interactions. The solid ones are hydrogen bond interactions and the dashed ones are the hydrophobic interactions. Figure was generated using ViewerLite42. Contacts of structural units (CSU) were derived with CSU software (Sobolev et al., 1999). The PDB structure 1g9f was used for the analyses of glycan protein interactions.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
S.S. and G.K. thank the Council of Scientific and Industrial Research, of India, for the award of Senior Research Fellowships. This work is supported by a grant from the Department of Biotechnology, government of India, to A.S.

Submitted on August 6, 2004; accepted for publication November 3, 2004.

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