GUIMIN LUO,b LAN DING,c ZHI LIU,d TONGSHU YANG,e
AND JIAZHAN NIc
bNational Laboratory of Enzyme Engineering
Changchun 130023, China
cInstitute of Applied Chemistry
Chinese Academy of Sciences
Changchun 130022, China
dInstitute of Military Veterinary Sciences
Changchun University of Agriculture and Animal Husbandry Sciences
Changchun 130021, China
eThe Endemic Institute
Bethune Medical University
Changchun 130021, China
ABSTRACT: Using two different glutathione derivatives as hapten, we have prepared
two abzymes, which display glutathione peroxidase (GPX) activity. Their GPX activities are 0.2 and 1.6 times that of natural GPX from rabbit liver, respectively. Selenium content analysis indicates that the activity difference between the two abzymes is possibly attributed to the conformation difference of the abzymes.
Glutathione peroxidase (GPX, EC 220.127.116.11) is one of the important members of the antioxidative enzyme family. It can scavenge active oxygen, can prevent lipid from peroxidating, and has the potential to cure tumorous, cardiovascular, and endemic diseases. However, the stability of native GPX is poor and its availability is limited. Therefore, its application has been greatly restricted. Early in the 1980s, the biological function of GPX was imitated by organic molecules with low molecular weight,1 but the GPX activities of these mimics are very low compared with native GPX. Preparation of abzymes is one of the important ways to imitate enzyme function. Although abzyme study has been rapidly developing since the late 1980s, the activities of abzymes prepared so far are usually 100每1000 times lower than that of respective native enzymes. Thus, it is critical to increase abzyme activity.
On the basis of glutathione (GSH) structure, we designed and synthesized a hapten Hp1 and prepared a selenium-containing abzyme (Se-abzyme) m4A4, the activity of which is 20% of the GPX activity from rabbit liver.2 Now, we have further modified GSH; obtained a more reasonable structure of hapten Hp2; and for the first time prepared an Se-abzyme, the activity of which surpassed the level of rabbit liver GPX.
MATERIALS AND METHODS
GSH, NADPH, PMSF, SeCys, and papain were purchased from Sigma. Glutathione reductase was obtained from Boehringer Mannheim GmbH. Rabbit IgG [anti-human IgM (Fc_)] antibody (rIgG) and HiTrap protein G affinity column were Pharmacia products. Bio-gel P200 was from Bio-Rad. Enzyme-labeled antibodies, BALB/c mice, and myeloma cells (NS-1) were from Bethune Medical University. 2,4-Dinitrochlorobenzene (DNCB) was from Beijing Chemical Plant. Bovine serum albumin (BSA) was from the Shanghai Institute of Biochemistry. Rabbit liver GPX (RL-GPX) was prepared according to reference 3. All other chemicals were of analytical
Synthesis of Haptens Hp1 (GSH-S-DNP) and Hp2 (GSH-S-DNP Methyl Ester)
Hp1 was synthesized according to reference 4 and esterified by methanol to obtain Hp2 with a yield of 87%.
Preparation of Antigen Ag2 and Determination of the Amount of Hp2 Bound to BSA
Hp2 was cross-linked with BSA using glutaraldehyde to obtain antigen Ag2.5 Hp2 has a maximum absorbance at 410 nm and the concentration of Hp2 is in proportion to the absorbance in the range of 0每0.7 mg/mL. Thus, the average Hp2 amount bound to BSA can be determined.
Determination of Dissociation Constants between Ligands and Antibodies
The dissociation constants were determined by using competitive ELISA6 and their values were calculated by using the following equation:
A0/(A0 每 A) = 1 + KD/a0.
A0 and A are the absorbances in the absence and presence of ligands, respectively. a0 is the total concentration of ligand.
Determination of GPX Activity
The activity was determined according to reference 7. One unit of activity is defined as the amount of compound that utilizes 1 _mol of NADPH per minute.
Determination of the SeCys Amount in Se-Abzyme
The total amount of -SH and -SeH groups in the mutated antibody and the amount of -SH groups in the unmutated antibody were determined by the DTNB method.8
The difference between the two amounts is the amount of -SeH groups in the mutated antibody.
Determination of Antibody Titer
The antibody titers before and after chemical mutation were determined by ELISA.9
Principle of Chemical Mutation
Antibody structure analysis indicates that, in the variable region of antibody Lchain,there are 3每4 serine residues. These serine residues can be chemically mutated
into SeCys,5 which is one of the catalytic groups of GPX.10 Therefore, we can incorporate catalytic groups into the antibody combining sites to obtain Se-abzyme.
Characterization of Hp2
Hp2 was characterized with elemental analysis, IR, and 1H-NMR, and its structure
is as shown in FIGURE 1.
Characterization of Antigen Ag2
It was shown from PAGE analysis that the molecular weight of Ag2 is 85 ㊣ 2 kDa and it was found that 30.2 haptens were bound to each BSA molecule on average by using the absorbance spectrum method.
Preparation of Se-Abzyme and Its GPX Activity
Hp2 was used to immunize BALB/c mice for production of McAb. Hybridoma was produced by fusion of immunized mouse spleen cells (NS-1) in the HAT medium.
Hp2-specific McAb 4G3 (4G3 IgG) was screened out by ELISA. The 4G3 IgG was cloned, propagated in ascites, and purified by salt fractionation, DEAE-52, and Bio-gel P200 column chromatography. The purity of 4G3 IgG was >90% by SDS-PAGE analysis. The serine residues in the variable region of 4G3 IgG were chemically mutated into SeCys by using PMSF and NaHSe to obtain the Se-abzyme (m4G3).The GPX activity, selenium content, and titer of m4G3 and 4G3 are listed in TABLE 1.
Results of KD Determination
The KD values of McAb and ligands are listed in TABLE 2. The KD of m4G3-GSH and m4A4-GSH cannot be determined by ELISA because of their GPX activity, so we only determined the KD values between 4G3, 4A4, and GSH haptens.
Se-Abzyme Titer and KDValue
TABLE 1 shows that the titer of 4G3 is almost the same as m4G3. The titer of 4A4 and m4A4 is also almost the same, and the KD values between McAb and their respective haptens before and after mutation are also similar, indicating that chemical mutation has no obvious effect toward the immune activity and spatial conformation of the antibodies. This property may lead to valuable applications.
The KD of rIgG-GSH is 4.8 ℅ 10每4 M, indicating that there is no GSH binding site in the combining site of McAb; thus, its GPX activity is only 1.2% of RL-GPX. The KD values of 4G3-GSH and 4A4-GSH approached 10每7 M, indicating that there is a GSH binding site in the combining site of 4G3 and 4A4; hence, their activities are much more than that of RL-GPX and approach 1.6 and 0.2 times that of RL-GPX.The KD of 4G3-GSH is higher than that of 4A4-GSH, but the GPX activity of m4G3 is 7.5 times that of m4A4 GPX activity, indicating that the affinity for GSH should have a limit.
Relationship between Se-Abzyme Activity and Its Structure
We can see from TABLES 1 and 2 that, first, although 4G3 and 4A4 have substrate binding sites, there is no catalytic group in them, so they have no GPX activity. Although mrIgG has SeCys, there is no GSH binding site, so its activity is low. Second, the SeCys number of m4G3, m4A4, and mrIgG is 1.9每2.0, but their activities are very different, indicating that the activity difference does not result from the SeCys number; instead, it all depends on the spatial conformation of the McAb active site. The antigen of rIgG is human IgM (Fc污); the antigen combining site (i.e., active site) of mrIgG does not match with GSH, so its activity is low. Although m4G3 and m4A4 have a GSH binding site (it is known from the KD and titer), the spatial conformation of the active site of m4G3 is more suitable to SeCys functioning. Thus, the activity of m4G3 is 7.5 times that of m4A4 and 1.6 times that of RL-GPX. This is the first Seabzyme, the GPX activity of which surpassed that of the native enzyme.
Experimental results have demonstrated that an Se-abzyme with higher than native enzyme activity can be generated by combination of McAb preparation technique with simple chemical mutation. It is critical to design a proper hapten with reasonable affinity for the substrate. This kind of Se-abzyme with high activity will find extensive applications.
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