**4. Structural heterogeneity of the constant region domains of light chains (CLs)**

In the previous section, we focused on a full-length light chain, which is consisted of the variable and the constant domain. It is noteworthy to study which domain, the former or latter, causes the structural diversity problem. Although there are many studies on the role of the constant domains (especially for a Fc region) of the heavy chain of the antibody, the reports on the role of the constant region domain of the light chain are scarcely seen. From this point of view, we investigated the role of the constant domain as described in the following.

#### **4.1. Sequence of the constant region domain of a human antibody light chain**

**Figure 8** shows the amino acid sequence of the recombinant constant region domain (kappa type) of a human antibody light chain employed in this study. Methionine (M) and alanine (A) at position nos. 1 and 2 of the aa sequence have been inserted by cloning using the restriction enzyme *Nco I*. Underlined is the sequence of the constant region domain. Arginine (R) at position no. 3 is the first amino acid residue of the constant region. Leucine (L) and glutamic acid (E) before His × 6 were also inserted by using the restriction enzyme (*Xho I*).

#### *4.1.1. Chromatography and SDS-PAGE analysis*

The expression and purification of the kappa type constant domain were similarly conducted as made in the full-length light chain. Ni-NTA chromatography was also performed to purify the recovered constant region domain. The result was also similar with that obtained in the case of full-length light chain except for the molecular size. The SDS-PAGE analysis for the collected fraction in the Ni-NTA chromatography is shown in **Figure 9**. Under non-reduced condition, a strong band was detected in the monomeric form at 15 kDa as well as a weak band in the dimeric form at 30 kDa. Under reduced condition, only the monomeric form was observed and the purity was over 95%. This sample was applied to cation exchange chromatography.

The results are shown in **Figure 10** along with the SDS-PAGE analysis under non-reduced condition. Several peaks were observed at retention times from 5 to 25 min while it was a single material of the constant domain. In **Figure 10**, peak 1 appearing at the retention time of 7.5 min is a monomer, and peaks 2, 3, and 4 appearing at 14–17 min contain mainly monomers. The dimers or/and trimer were detected for peaks 2 and 4. Peaks 5 and 6 appearing at 21–23 min are the dimer. These results mean that differently charged molecules of the constant region domain as well as differently sized molecules coexisted in solution at the same time. It is obvious that a constant region molecule shows molecular heterogeneity (structural diversity) from the viewpoint of both electrical charge and molecular size, which are very

**Figure 9.** SDS-PAGE of the constant region domain after Ni-NTA chromatography. Under non-reduced condition, a strong band was detected in the monomeric form at 15 kDa as well as a weak band in the dimeric form at 30 kDa. Under

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reduced condition, only the monomeric form was observed and the purity was over 95%.

As stated previously, copper ion (Cu2+) hugely influenced the structural diversity of the fulllength light chain. The same experiment was performed with the constant region domain molecule. The results are summarized in **Figure 11a**–**f**. In the case of 0.1 eq. addition of Cu2+ for the constant region domain molecule, we observed a small peak 7 and one main peak

similar with those observed in the full-length light chain.

**4.2. Effect of copper ions**

**Figure 8.** Amino acid sequence of the constant region domain of a human antibody light chain (kappa type). Underlined is the aa sequence of the constant region domain of the kappa light chain. Methionine (M) and alanine (A) of the aa sequence at the first and second position were inserted by cloning using the restriction enzyme *Nco I*. Arginine (R) at the third position is the first amino acid residue of the constant region. Leucine (L) and glutamic acid (E) before His × 6 were also inserted by cloning using the restriction enzyme *Xho I.*

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**Figure 9.** SDS-PAGE of the constant region domain after Ni-NTA chromatography. Under non-reduced condition, a strong band was detected in the monomeric form at 15 kDa as well as a weak band in the dimeric form at 30 kDa. Under reduced condition, only the monomeric form was observed and the purity was over 95%.

The results are shown in **Figure 10** along with the SDS-PAGE analysis under non-reduced condition. Several peaks were observed at retention times from 5 to 25 min while it was a single material of the constant domain. In **Figure 10**, peak 1 appearing at the retention time of 7.5 min is a monomer, and peaks 2, 3, and 4 appearing at 14–17 min contain mainly monomers. The dimers or/and trimer were detected for peaks 2 and 4. Peaks 5 and 6 appearing at 21–23 min are the dimer. These results mean that differently charged molecules of the constant region domain as well as differently sized molecules coexisted in solution at the same time. It is obvious that a constant region molecule shows molecular heterogeneity (structural diversity) from the viewpoint of both electrical charge and molecular size, which are very similar with those observed in the full-length light chain.

#### **4.2. Effect of copper ions**

approximate length of 20 nm, the width of 10 nm, and the height of 4 nm. The lateral and height length are comparable with the AFM image of IgG by Querghi et al. [33]. We could not identify the position of the copper ion residing in the light chain from this AFM analysis.

In the previous section, we focused on a full-length light chain, which is consisted of the variable and the constant domain. It is noteworthy to study which domain, the former or latter, causes the structural diversity problem. Although there are many studies on the role of the constant domains (especially for a Fc region) of the heavy chain of the antibody, the reports on the role of the constant region domain of the light chain are scarcely seen. From this point of view, we investigated the role of the constant domain as described in the following.

**Figure 8** shows the amino acid sequence of the recombinant constant region domain (kappa type) of a human antibody light chain employed in this study. Methionine (M) and alanine (A) at position nos. 1 and 2 of the aa sequence have been inserted by cloning using the restriction enzyme *Nco I*. Underlined is the sequence of the constant region domain. Arginine (R) at position no. 3 is the first amino acid residue of the constant region. Leucine (L) and glutamic acid

The expression and purification of the kappa type constant domain were similarly conducted as made in the full-length light chain. Ni-NTA chromatography was also performed to purify the recovered constant region domain. The result was also similar with that obtained in the case of full-length light chain except for the molecular size. The SDS-PAGE analysis for the collected fraction in the Ni-NTA chromatography is shown in **Figure 9**. Under non-reduced condition, a strong band was detected in the monomeric form at 15 kDa as well as a weak band in the dimeric form at 30 kDa. Under reduced condition, only the monomeric form was observed and the purity was over 95%. This sample was applied to cation exchange chromatography.

**Figure 8.** Amino acid sequence of the constant region domain of a human antibody light chain (kappa type). Underlined is the aa sequence of the constant region domain of the kappa light chain. Methionine (M) and alanine (A) of the aa sequence at the first and second position were inserted by cloning using the restriction enzyme *Nco I*. Arginine (R) at the third position is the first amino acid residue of the constant region. Leucine (L) and glutamic acid (E) before His × 6 were

**4. Structural heterogeneity of the constant region domains of light** 

**4.1. Sequence of the constant region domain of a human antibody light chain**

(E) before His × 6 were also inserted by using the restriction enzyme (*Xho I*).

*4.1.1. Chromatography and SDS-PAGE analysis*

also inserted by cloning using the restriction enzyme *Xho I.*

**chains (CLs)**

240 Antibody Engineering

As stated previously, copper ion (Cu2+) hugely influenced the structural diversity of the fulllength light chain. The same experiment was performed with the constant region domain molecule. The results are summarized in **Figure 11a**–**f**. In the case of 0.1 eq. addition of Cu2+ for the constant region domain molecule, we observed a small peak 7 and one main peak

**Figure 10.** Cation exchange chromatography for the constant region domain molecule of the light chain (CL). Peak 1 appearing at the retention time of 7.5 min is a monomer and peaks 2, 3, and 4 appeared at 14–17 min contain mainly monomers. The dimers or/and trimer were detected for peaks 2 and 4. Peaks 5 and 6 appearing at 21–23 min are the dimer. These results mean that differently charged molecules of the constant region domain as well as differently sized molecules coexisted in solution at the same time. This result was very similar with that observed with the full-length light chain.

8, which were eluted at the retention time around 16 and 22 min, respectively (**Figure 11a**). Peak 8 was the dimer by the SDS-PAGE analysis under non-reduced condition (peak 7 was not analyzed because of the small peak). In the case of 0.2 eq. addition of copper, mainly two peaks (9 and 10) were obtained (**Figure 11b**). The elution times of peaks 9 and 10 were identical with those of peaks 7 and 8, respectively. Peak 9 included mainly the dimer with a very slight amount of the monomer. Peak 10 was the dimer. For these two peaks, UV/VIS spectroscopy was performed. The results are presented in **Figure 12**. Peak 9 showed an absorbance of around 580 nm, which was based on the interaction of Cu2+ and amino acids of the constant region domain molecule. On the other hand, no absorbance was detected for peak 10. Namely, protein of peak 9 bound to Cu2+ but peak 10 did not. Though the peaks are dimeric forms of the constant region domain, they were separated by the cation exchange column chromatography whether or not the peak contains Cu2+. For the case of 0.3 eq. addition, the main peak was peak 11 observed at the retention time of 16 min, which included the dimer along with a slight monomer and trimer forms (**Figure 11c**). In the case of 0.4 eq. addition, a clear single peak of the dimer form was detected at the retention time of 16 min (**Figure 11d**). In **Figure 11e** and **f**, peaks 13 and 14 were observed as single peak at the retention time of 16 min. And they were the dimer. It seems that enough content of added copper ion was 0.4 eq. to induce mono-form formation from the multi-forms of the constant region domain molecule.

The amount of Cu2+ bound to the constant region domain molecule was also quantified using a commercially available copper detection kit (Copper, Low Concentration, Assay Kit, AKJ, CU21M, Metallogenics Co., Ltd., Chiba, Japan). For the peak appearing at the retention time of 16 min and containing Cu2+, the ratio of Cu2+: constant region domain was around 0.55. This result agreed with those of the UV/VIS spectroscopy, suggesting that two constant region domain molecules bind one copper ion.

**4.3. Binding affinity of copper ions**

multi-molecular forms of the constant region domain molecule.

The UV/VIS spectrum changed as different concentrations of Cu2+ was added to the Ni-NTA elution after the samples were dialyzed against PBS. The results are presented in **Figure 13**. The absorbance of 580 nm became larger along with an increase in the amount of added Cu2+, as showing a slight red shift. In **Figure 14**, the values for the concentration of added Cu2+ were plotted vs. the maximum absorbance, which is the absorption isotherm curve for Cu2+ binding to constant region domain molecules. The Langmuir plot is shown in the inset of **Figure 14**, indicating a good linear relationship. The binding constant was estimated to be 48.0 μM−1.

**Figure 11.** Effect of copper ions. (a) 0.1 eq. addition of Cu2+: a small peak 7 and one main peak 8 were observed at the retention time around 16 and 22 min, respectively. Peak 8 was the dimer. (b) 0.2 eq. addition of Cu2+: the eluted times of peaks 9 and 10 were identical with those of peaks 7 and 8, respectively. Peak 9 included mainly the dimer with a very slight contamination of the monomer. Peak 10 was the dimer. (c) 0.3 eq. addition of Cu2+: peak 11 was observed as the main peak at the retention time of 16 min. (d) 0.4 eq. addition of Cu2+: A clear single peak of the dimer form was detected at the retention time of 16 min. (e) 1.0 eq. addition of Cu2+: only peak 13 was observed at the retention time of 16 min. It was the dimer. (f) 10.0 eq. addition of Cu2+: only peak 14 was observed at the retention time of 16 min. It was also the dimer. It seems that the amount of 0.4 eq. added copper ions is sufficient to induce the mono-molecular form from the

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**Figure 11.** Effect of copper ions. (a) 0.1 eq. addition of Cu2+: a small peak 7 and one main peak 8 were observed at the retention time around 16 and 22 min, respectively. Peak 8 was the dimer. (b) 0.2 eq. addition of Cu2+: the eluted times of peaks 9 and 10 were identical with those of peaks 7 and 8, respectively. Peak 9 included mainly the dimer with a very slight contamination of the monomer. Peak 10 was the dimer. (c) 0.3 eq. addition of Cu2+: peak 11 was observed as the main peak at the retention time of 16 min. (d) 0.4 eq. addition of Cu2+: A clear single peak of the dimer form was detected at the retention time of 16 min. (e) 1.0 eq. addition of Cu2+: only peak 13 was observed at the retention time of 16 min. It was the dimer. (f) 10.0 eq. addition of Cu2+: only peak 14 was observed at the retention time of 16 min. It was also the dimer. It seems that the amount of 0.4 eq. added copper ions is sufficient to induce the mono-molecular form from the multi-molecular forms of the constant region domain molecule.

#### **4.3. Binding affinity of copper ions**

8, which were eluted at the retention time around 16 and 22 min, respectively (**Figure 11a**). Peak 8 was the dimer by the SDS-PAGE analysis under non-reduced condition (peak 7 was not analyzed because of the small peak). In the case of 0.2 eq. addition of copper, mainly two peaks (9 and 10) were obtained (**Figure 11b**). The elution times of peaks 9 and 10 were identical with those of peaks 7 and 8, respectively. Peak 9 included mainly the dimer with a very slight amount of the monomer. Peak 10 was the dimer. For these two peaks, UV/VIS spectroscopy was performed. The results are presented in **Figure 12**. Peak 9 showed an absorbance of around 580 nm, which was based on the interaction of Cu2+ and amino acids of the constant region domain molecule. On the other hand, no absorbance was detected for peak 10. Namely, protein of peak 9 bound to Cu2+ but peak 10 did not. Though the peaks are dimeric forms of the constant region domain, they were separated by the cation exchange column chromatography whether or not the peak contains Cu2+. For the case of 0.3 eq. addition, the main peak was peak 11 observed at the retention time of 16 min, which included the dimer along with a slight monomer and trimer forms (**Figure 11c**). In the case of 0.4 eq. addition, a clear single peak of the dimer form was detected at the retention time of 16 min (**Figure 11d**). In **Figure 11e** and **f**, peaks 13 and 14 were observed as single peak at the retention time of 16 min. And they were the dimer. It seems that enough content of added copper ion was 0.4 eq. to induce mono-form

**Figure 10.** Cation exchange chromatography for the constant region domain molecule of the light chain (CL). Peak 1 appearing at the retention time of 7.5 min is a monomer and peaks 2, 3, and 4 appeared at 14–17 min contain mainly monomers. The dimers or/and trimer were detected for peaks 2 and 4. Peaks 5 and 6 appearing at 21–23 min are the dimer. These results mean that differently charged molecules of the constant region domain as well as differently sized molecules coexisted in solution at the same time. This result was very similar with that observed with the full-length light chain.

The amount of Cu2+ bound to the constant region domain molecule was also quantified using a commercially available copper detection kit (Copper, Low Concentration, Assay Kit, AKJ, CU21M, Metallogenics Co., Ltd., Chiba, Japan). For the peak appearing at the retention time of 16 min and containing Cu2+, the ratio of Cu2+: constant region domain was around 0.55. This result agreed with those of the UV/VIS spectroscopy, suggesting that two constant region

formation from the multi-forms of the constant region domain molecule.

domain molecules bind one copper ion.

242 Antibody Engineering

The UV/VIS spectrum changed as different concentrations of Cu2+ was added to the Ni-NTA elution after the samples were dialyzed against PBS. The results are presented in **Figure 13**. The absorbance of 580 nm became larger along with an increase in the amount of added Cu2+, as showing a slight red shift. In **Figure 14**, the values for the concentration of added Cu2+ were plotted vs. the maximum absorbance, which is the absorption isotherm curve for Cu2+ binding to constant region domain molecules. The Langmuir plot is shown in the inset of **Figure 14**, indicating a good linear relationship. The binding constant was estimated to be 48.0 μM−1.

**Figure 12.** UV/VIS spectra. Peak 9 showed the absorbance of around 580 nm, which was based on the interaction of Cu2+ and amino acids of the constant region domain molecule. On the other hand, no absorbance was detected for peak 10.

under non-reduced conditions, respectively. In the former case, many spots were observed: two spots at pI = 6.9 for the dimer and three spots at pI = 6.2, 6.5, and 6.9 for the monomer. In contrast, one strong spot was observed at pI = 6.9 for the dimer in the case with the addition of Cu2+, while very faint spots were detected in the positions of the monomer. Note that Cu2+ can facilitate changes from the multimeric form to the monomeric form as well as from different

**Figure 14.** Kinetic analysis. The values for the concentration of added Cu2+ were plotted vs. the absorbance at 580 nm, which is the isothermal curve for copper binding to the CL protein. The Langmuir plot is presented in the inset of the

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graph, indicating a good linear relationship. The binding constant was estimated to be 48.0 μM−1.

**Proteins (metal ion) K (M−1) Affinity** Hemocyanin (Cu2+) 1017–1019 Very strong Metallothionein (Cu2+) 1017–1019 Very strong Carbonic anhydrase (Zn2+) ~1012 Strong Aminopeptidase III (Co2+) 2 × 10<sup>4</sup> Weak CL (Cu2+) 4.8 × 10<sup>7</sup> Medium

About 1.0 eq. of a metal ion such as Ca2+, Mg2+, Ni2+, and Zn2+was added in each Ni-NTA elution and incubated overnight. **Figure 16** shows the results of the cation exchange chromatography and SDS-PAGE (non-reduced) for peaks P3 and P5. For the cases of Ca2+, Mg2+, and Ni2+ (**Figure 16a**–**c**, respectively), a large peak P3 was observed at 17 min along with a small peak P5 at 23 min. Interestingly, a large peak P3 was observed and peak P5 became very small in the case of Zn2+ (**Figure 16d**). The chromatogram resembled the case of Cu2+ (**Figure 16e**). From the results of the SDS-PAGE, the peak P3 was mostly in a monomeric form for all the cases of Ca2+, Mg2+, Ni2+, and Zn2+. On the other hand, P3 of Cu2+ was the dimer. The molecular form (size) of P3 in the case of Ca2+, Mg2+, Ni2+, and Zn2+ was quite different from that of

electrical charges to a single electrical charge.

**Table 1.** Comparison of the binding affinities of some proteins with metal ions.

**4.4. Other metal ions**

**Figure 13.** Spectrum changes with the concentration of added Cu2+. Along with an increase of the concentration of added Cu2+, the absorbance at around 580 nm became larger.

The binding affinity from several proteins incorporating divalent metal ions was investigated. The values (K) are presented in **Table 1**. Hemocyanin and metallothionein have very strong affinity to bind Cu2+. Carbonic anhydrase-binding Zn2+ shows a strong affinity. Aminopeptidase III binding Co2+ possesses a weak affinity. In the case of CL, the value (48.0 μM−1) seems to be intermediate among those metalloproteins.

In order to further investigate the molecular heterogeneity of the constant region domain molecule, two-dimensional (2D) electrophoresis was performed using samples with or without Cu2+. **Figure 15a** and **b** shows the results for the cases without and with the addition of Cu2+

**Figure 14.** Kinetic analysis. The values for the concentration of added Cu2+ were plotted vs. the absorbance at 580 nm, which is the isothermal curve for copper binding to the CL protein. The Langmuir plot is presented in the inset of the graph, indicating a good linear relationship. The binding constant was estimated to be 48.0 μM−1.


**Table 1.** Comparison of the binding affinities of some proteins with metal ions.

under non-reduced conditions, respectively. In the former case, many spots were observed: two spots at pI = 6.9 for the dimer and three spots at pI = 6.2, 6.5, and 6.9 for the monomer. In contrast, one strong spot was observed at pI = 6.9 for the dimer in the case with the addition of Cu2+, while very faint spots were detected in the positions of the monomer. Note that Cu2+ can facilitate changes from the multimeric form to the monomeric form as well as from different electrical charges to a single electrical charge.

#### **4.4. Other metal ions**

The binding affinity from several proteins incorporating divalent metal ions was investigated. The values (K) are presented in **Table 1**. Hemocyanin and metallothionein have very strong affinity to bind Cu2+. Carbonic anhydrase-binding Zn2+ shows a strong affinity. Aminopeptidase III binding Co2+ possesses a weak affinity. In the case of CL, the value

**Figure 13.** Spectrum changes with the concentration of added Cu2+. Along with an increase of the concentration of added

**Figure 12.** UV/VIS spectra. Peak 9 showed the absorbance of around 580 nm, which was based on the interaction of Cu2+ and amino acids of the constant region domain molecule. On the other hand, no absorbance was detected for peak 10.

In order to further investigate the molecular heterogeneity of the constant region domain molecule, two-dimensional (2D) electrophoresis was performed using samples with or without Cu2+. **Figure 15a** and **b** shows the results for the cases without and with the addition of Cu2+

(48.0 μM−1) seems to be intermediate among those metalloproteins.

Cu2+, the absorbance at around 580 nm became larger.

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About 1.0 eq. of a metal ion such as Ca2+, Mg2+, Ni2+, and Zn2+was added in each Ni-NTA elution and incubated overnight. **Figure 16** shows the results of the cation exchange chromatography and SDS-PAGE (non-reduced) for peaks P3 and P5. For the cases of Ca2+, Mg2+, and Ni2+ (**Figure 16a**–**c**, respectively), a large peak P3 was observed at 17 min along with a small peak P5 at 23 min. Interestingly, a large peak P3 was observed and peak P5 became very small in the case of Zn2+ (**Figure 16d**). The chromatogram resembled the case of Cu2+ (**Figure 16e**). From the results of the SDS-PAGE, the peak P3 was mostly in a monomeric form for all the cases of Ca2+, Mg2+, Ni2+, and Zn2+. On the other hand, P3 of Cu2+ was the dimer. The molecular form (size) of P3 in the case of Ca2+, Mg2+, Ni2+, and Zn2+ was quite different from that of

**Figure 15.** 2D electrophoresis for the constant region domain (CL). (a) Without Cu2+: two spots at pI = 6.9 for the dimer and three spots at pI = 6.2, 6.5, and 6.9 for the monomer were found. (b) With Cu2+: one strong spot was observed at pI = 6.9 for the dimer. It is revealed that copper ion accelerates both dimerization and generation of a mono-molecular form of the light chain.

of Zn2+. Although there are several reports on the relationship between metal ions and the enzymatic activity of catalytic antibodies, details of the contributions of metal ions to the molecular structure of catalytic antibodies are unclear at present [20, 34]. Paul et al. reported an interesting function regarding Zn2+, which was essential for exhibiting the catalytic function of the antibody light chain to cleave beta-amyloid peptides, while the ion will not affect

was observed and peak P5 became very small. The chromatogram seemed to be like the case of Cu2+.

**Figure 16.** Other metal ions. In all cases, 1.0 eq. metal ion was added. Cation exchange chromatograms are presented with the results of SDS-PAGE (under non-reduced condition). m: monomer; di: dimer. (a) Addition of Ca2+. (b) Addition of Mg2+. (c) Addition of Ni2+. (d) Addition of Zn2+. (e) Addition of Cu2+. Peak P3 was mostly in a monomeric form for all the cases of Ca2+, Mg2+, Ni2+ and Zn2+. In the cases of addition of Ca2+, Mg2+, and Ni2+ (**Figure 16a**–**c**, respectively), a large peak, P3, was observed at 17 min along with a small peak, P5, at 23 min. In the case of Zn2+ (**Figure 16d**), a large peak P3

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For the reason why the addition of copper hugely effects the formation of a mono-form structure of the constant light chain domain, we postulated one of the situations from the viewpoint of potential energy and the wall height as illustrated in **Figure 17**. It is likely that the energy

**4.5. Consideration about unstable forms and a stable form of CL**

the catalytic site [14].

Cu2+. It must be considered that the Zn2+ could not accelerate the dimerization of the constant region domain molecule, while the ion decreased the peak P5 and showed a large peak P3. Zn2+ could have some ability to unify the structural diversity, but the effect is different from that of copper.

Zn2+ did not accelerate the dimerization of the constant region domain molecule but has some functions that may contribute to solve the heterogeneity problem. Out of the several metals analyzed, Zn2+ exhibited an interesting behavior, which must be a characteristic feature

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**Figure 16.** Other metal ions. In all cases, 1.0 eq. metal ion was added. Cation exchange chromatograms are presented with the results of SDS-PAGE (under non-reduced condition). m: monomer; di: dimer. (a) Addition of Ca2+. (b) Addition of Mg2+. (c) Addition of Ni2+. (d) Addition of Zn2+. (e) Addition of Cu2+. Peak P3 was mostly in a monomeric form for all the cases of Ca2+, Mg2+, Ni2+ and Zn2+. In the cases of addition of Ca2+, Mg2+, and Ni2+ (**Figure 16a**–**c**, respectively), a large peak, P3, was observed at 17 min along with a small peak, P5, at 23 min. In the case of Zn2+ (**Figure 16d**), a large peak P3 was observed and peak P5 became very small. The chromatogram seemed to be like the case of Cu2+.

of Zn2+. Although there are several reports on the relationship between metal ions and the enzymatic activity of catalytic antibodies, details of the contributions of metal ions to the molecular structure of catalytic antibodies are unclear at present [20, 34]. Paul et al. reported an interesting function regarding Zn2+, which was essential for exhibiting the catalytic function of the antibody light chain to cleave beta-amyloid peptides, while the ion will not affect the catalytic site [14].

#### **4.5. Consideration about unstable forms and a stable form of CL**

Cu2+. It must be considered that the Zn2+ could not accelerate the dimerization of the constant region domain molecule, while the ion decreased the peak P5 and showed a large peak P3. Zn2+ could have some ability to unify the structural diversity, but the effect is different from

**Figure 15.** 2D electrophoresis for the constant region domain (CL). (a) Without Cu2+: two spots at pI = 6.9 for the dimer and three spots at pI = 6.2, 6.5, and 6.9 for the monomer were found. (b) With Cu2+: one strong spot was observed at pI = 6.9 for the dimer. It is revealed that copper ion accelerates both dimerization and generation of a mono-molecular

Zn2+ did not accelerate the dimerization of the constant region domain molecule but has some functions that may contribute to solve the heterogeneity problem. Out of the several metals analyzed, Zn2+ exhibited an interesting behavior, which must be a characteristic feature

that of copper.

form of the light chain.

246 Antibody Engineering

For the reason why the addition of copper hugely effects the formation of a mono-form structure of the constant light chain domain, we postulated one of the situations from the viewpoint of potential energy and the wall height as illustrated in **Figure 17**. It is likely that the energy potential of each molecular form is at a comparable level after the preparation of the molecule (CL) without Cu2+, as shown in **Figure 17a**. In this case, transfer of the potential well A to B (or C) is easy because the walls of the potential energies of the wells are low (**Figure 17b**). However, the energy potential is drastically changed when copper ions are added. The multimolecular forms of the constant region domain, which are sitting in each potential well, drop in one deep potential energy level, as shown in **Figure 17c**, resulting in the formation of a mono-molecular form from the multi-molecular forms. Once the molecule dropped into the deep potential well, the form would be no longer able to transfer to other forms. As a consequence, the monomolecular form of the constant region domain molecule became stable. This situation can be achieved by the presence of copper ion in a ratio of more than 0.5 eq. of Cu2+ to the constant region domain molecule.

**5. Binding of copper ions in the constant region domain**

In order to clarify the copper-binding site, two mutants were prepared from the C51 light chain, because the light chain has no histidine residues in the variable region compared to the sequence of the constant region domain comprising 2 His residues (**Figure 8**). Both histidine and cysteine residues are considered as the most plausible candidates for the binding site. Therefore, the residues of His195, His204, and Cys220 present in the constant region domain of the C51 light chain were mutated to Ala. As the consequence, two mutants were made. One is Cys220Ala (C220A: mono-mutant) and another is His195Ala, His204Ala, and Cys220Ala (H195A/H204A/C220A: triple-mutant; **Figure 18a**). The locations of the mutated residues are

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**Figure 18.** C51 mutants and locations of His and Cys residues. There are no histidine residues in the variable region of the C51 light chain. (a) Location of Cys220, His195 and His204 in wild type. The mutated positions, C220A and H195A/ H204A/C220A, are also indicated with green colored character. (b) Three-dimensional structure of the C51 light chain.

Light blue is sheet structure and red is helix structure.

**5.1. Preparation of mutants and their uptake of copper ions**

shown in **Figure 18b**.

**Figure 17.** Consideration about conversion of unstable forms to a stable form of CL. (a) State A (corresponding to peak 1 in **Figure 10**), State B (corresponding to peaks 2, 3, and 4 in **Figure 10**), and State C (corresponding to peaks 5 and 6 in **Figure 10**) may stay in a chemical equilibrium. (b) Assumed situation in potential energy for the case without Cu2+: each potential energy level for the case without Cu2+ may be comparable in wells of A, B, and C. The walls among the potential energy wells are not high. (c) Assumed situation of potential energy for the case with Cu2+: when Cu2+ is incorporated, a deep potential level can be generated, and all molecules showing a different heterogeneity may drop into the well and exist as a stable form.
