化学修饰单克隆抗体模拟谷胱甘肽过氧化物酶

化学修饰具有底物谷胱甘肽（ＧＳＨ）结合部位的单克隆抗体（４Ａ４）使其结合部位上的丝氨酸（Ｓｅｒ）转变成谷胱甘肽过氧化物酶（ＧＰＸ）的催化基因硒代半胱氨酸（ＳｅＣｙｓ）因而产生高活力的含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）突变的４Ａ４（ｍ４Ａ４）的ＧＰＸ活力达到了天然酶活力的１９％并对ｍ４Ａ４的酶学性质和动力学性质进行了研究;硒代谷胱甘肽（ＧＳｅＨ）连到４Ａ４结合部位,其ＧＰＸ活力由３．８６Ｕ／μｍｏｌ提     

含硒抗体酶性质的研究

在成功地制备了具有谷胱甘肽过氧化物酶（ＧＰＸ）活性含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）的基础上,我们筛选了制备Ｓｅ－ａｂｚｙｍｅ的最佳条件,并对其理化性质及酶学性质和稳定性进行了深入的研究。结果表明,Ｓｅ－ａｂｚｙｍｅ的等电点为６．９５和７．０８,一为１５８ｋｄ;适ＰＨ和最适温度范围比天然酶宽广;抗体酶的贮藏稳定性比天然酶高。高Ｘ射线光电子能谱技术测得在Ｓｅ－ａｂｚｙｍｅ中含硒量为５ｍｏｌＳｅ／ｍｏｌ     

GPX－abzyme对受XO／HX系统损伤的心肌线粒体的保护作用

探讨了研制的具有谷胱甘肽过氧化物酶活力的含硒抗体酶（ＧＰＸ－ａｂｚｙｍｅ）对于受损心肌线粒体的保护作用,利用牛的心肌线粒体为实验材料,通过线粒体的膨胀度、脂质过氧化物含量、ＣＣＯ活力变化及电镜观察等几个方面证明ＧＰＸ－ａｂｚｙｍｅ能抵抗ＸＯ／ＨＸ系统产生的自由基的损伤作用,ＥＳＲ研究也表明ＧＰＸ－ａｂｚｙｍｅ能明显降低ＸＯ／ＨＸ损伤系统中的自由基含量。     

GPX—abzyme对受XO／HX系统损伤的心肌线粒体的保护作用

探讨了研制的具有谷胱甘肽过氧化物酶活力的含硒抗体酶（ＧＰＸ－ａｂｚｙｍｅ）对于受损心肌线粒体的保护作用。利用牛的心肌线粒体为实验材料,通过线粒体的膨胀度、脂质过氧化物含量、ＣＣＯ活力变化及电镜观察等几个方面证明ＧＰＸ－ａｂｚｙｍｅ能抵抗ＸＯ／ＨＸ系统产生的自由基的损伤作用,ＥＳＲ研究也表明ＧＰＸ－ａｂｚｙｍｅ能明显降低ＸＯ／ＨＸ损伤系统中的自由基含量。     

化学修饰抗体增加模拟酶的GPX活性

将丝氨酸共价偶联到兔抗人ＩｇＭ（Ｆｃμ）抗体上,以增加抗体结合部位丝氨酸残基含量．在中性条件下,丝氨酸被对苯甲基磺酰氟活化,再经ＮａＨＳｅ亲核取代,将丝氨酸诱变为硒代半胱氨酸（ＳｅＣｙｓ）．其谷胱甘肽过氧化物酶（ＧＰＸ）活性由未增加丝氨酸前的诱变抗体的７０Ｕ／μｍｏｌ提高到２１８Ｕ／μｍｏｌ,其活性为模拟物ＰＺ５１的２００多倍．     

化学突变具有底物结合部位的单克隆抗体制备含硒抗体酶

开发了一种制备抗体酶的新方法。用二硝基氯苯（ＤＮＣＢ）专一地与谷胱甘肽（ＧＳＨ）的巯基反应,合成出半抗原ＧＳＨ－Ｓ－ＤＮＰ。用戊二醛将半抗原偶联到牛血清白蛋白（ＢＳＡ）上,制成全抗原。再用标准的单抗制备法获得具有ＧＳＨ结合部位的单抗（４Ａ４ＩｇＧ）。用苯甲基磺酞氟（ＰＭＳＦ）和Ｈ２Ｓｅ相继处理该单抗,则将单拉结合部位上的丝氨酸（Ｓｅｒ）突变成硒代半胱氨酸（ＳｅＣｙｓ,因而在单抗结合部位上引入了谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团。突变后的单抗具有ＧＰＸ活性,其活力已达到天然ＧＰＸ的数量级水平。动力学行为也与天然ＧＰＸ类似。这种新的含硒抗体酶有优于ＧＰＸ的一些特点。     

硒对培养人胚肝细胞Ⅲ型前胶原,羟脯氨酸合成的影响

原代培养人胚肝细胞经１．１５６×１０￣(－７）ｍｏｌ／Ｌ硒预处理４ｈ,加入２０ｍｍｏｌ／Ｌ四氟化碳作用２０ｈ,观察硒对其Ⅲ型前胶原（ＰＣⅢ）和羟脯氨酸（Ｈｙｐ）生成的影响。结果培养液中ＰＣⅢ水平、细胞内Ｈｙｐ含量及细胞内外丙二醛（ＭＤＡ）水平均降低,与未加硒对照组比较差别有显著性（Ｐ＜０．０１）。而硒谷腕甘肽过氧化物酶（Ｓｅ－ＧＳＨ－ＰＸ）活性则较对照组显著增高（Ｐ＜０．００１）,且ＰＣⅢ水平与Ｓｅ－ＧＳＨ－Ｐ＿Ｘ／ＭＤＡ比值呈负相关（ｒ＝－０．９１５６,Ｐ＜０．０１）。提示硒可提高Ｓｅ－ＧＳＨ－Ｐ＿Ｘ／ＭＤＡ比值,抑制脂质过氧化激发的肝细胞胶原合成。     

慈菇（Sagittaria safittifolia）α－半乳糖苷酶的纯化与性质

以对硝基苯糖苷基为底物,测定了慈菇的１２种糖苷酶,其中α－甘露糖苷酶、α－和β－半乳糖苷酶活力较高;经硫酸铵分级沉淀,ＳｅｐｈａｄｅｘＧ－１５０分子筛层析,ＣｏｎＡＳｅｐｈａｒｏｓｅ４Ｂ亲和层析,ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析,从慈菇抽提液纯化了α－半乳糖苷酶。纯化酶的比活提高１０７２倍,活力回收１５．６％,在圆盘聚丙烯酰胺凝胶电泳和ＳＤＳ－ＰＡＧＥ上均显示１条蛋白质带,在α－半乳糖苷酶浓度为１５０ｍＵ／ｍｌ的溶液中测不到其他糖苷酶的活力。慈菇α－半乳糖苷酶的分子量用ＳｅｐｈａｄｅｘＧ－１００凝胶过滤柱测定或在ＳＤＳ－ＰＡＧＥ上测定均为６０ｋＤ,酶反应的最适ｐＨ在５．８附近,最适温度为６０℃。该酶分解对硝基苯基－α－半乳糖苷的Ｋ＿ｍ值为３．７×１０￣（－４）ｍｏｌ／Ｌ,Ｖ＿ｍ值为２．１×１０￣（－４）ｍｏｌ／Ｌ。银离子、汞离子显著抑制酶活力,Ｄ－半乳糖和密二糖均竞争性地抑制该酶水解对硝基苯基α－Ｄ－半乳糖苷的活力,根据Ｄｉｘｏｎ作图求得其Ｋ＿ｉ值分别为０．９２×１０￣（－３）ｍｏｌ／Ｌ和１．９８×１０￣（－３）ｍｏｌ／Ｌ。２－脱氧－Ｄ－半乳糖和Ｌ－岩藻糖为酶活力的非竞争性抑制剂。化学修饰     

模拟谷胱甘肽过氧化物酶活性三肽的制备及其性质研究

开发一种制备硒代谷胱甘肽（ＧｓｅＨ）的新方法。以合成的谷氨酰－γ－丝氨酰－甘氨酸（Ｇｌｕ－Ｓｅｒ－Ｇｌｙ）三肽为原料,经苯甲基磺酰氟（ＰＭＳＦ）活化,用Ｈ＿２Ｓｅ突变Ｓｅｒ成硒代半脱氨酸（ＳｅＣｙｓ）制成ＧＳｅＨ。用元素分析及氨基酸分析确定此三肽的组成并推导出此三肽的结构。研究了ＧＳｅＨ的性质．结果表明,此三肽具有谷胱甘肽过氧化物酶（ＧＰＸ）的活性,其活力比其它一些小分子有机模拟物高,在性质上也有优于它们的一些特点,其动力学行为与天然ＧＰＸ类似。     

胞外青霉素酰化酶的纯化及部分理化性质

巨大芽孢杆菌产胞外青霉素酰化酶发酵液经硫酸铵分级抽提及ＳｅｐｈａｄｅｘＧ－１００、羟基磷灰石、ＤＥＡＥ纤维素ＤＥ５２等层析步骤,提纯了青霉素酰化酶,得到电泳均一的酶制剂。纯酶比活力约为２５Ｕ／ｍｇ蛋白,纯化４９倍,活力回收５８％,经ＰＡＧＥ及ＳＤＳ－ＰＡＧＥ测知该酶不含亚基,其分子量约为１４０ｋＤ。该酶最适ｐＨ为９．０,最适温度４７℃,用底物ＮＩＰＡＢ测活,其Ｋｍ值为６．２×１０~（－４）ｍｏｌ／Ｌ,Ｖｍ值为１．２４×１０４ｍｏｌ／Ｌ。此外还探讨了部分金属离子对该酶的影响。     

Improving GPX activity of selenium‐containing human single‐chain Fv antibody by site‐directed mutation based on the structural analysis

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se‐scFv‐B3 (selenium‐containing single‐chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv‐B3 was carried out. A three‐dimensional (3D) structure of scFv‐B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer‐aided docking and energy minimization (EM) calculations of the antibody‐GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni²⁺‐immobilized metal affinity chromatography (IMAC), then transformed to selenium‐containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se‐scFv‐B3‐A180S was significantly increased compared to the original Se‐scFv‐B3. Copyright © 2009 John Wiley & Sons, Ltd.     

Conformational changes at the active site of adenylate kinase detected using a fluorescent probe and monoclonal antibody binding

A fluorescent probe, IAEDANS, was introduced into the active site of adenylate kinase (AK) by specifically modifying Cys-25. During modification, enzyme activity was greatly diminished. This probe allowed observation of conformational changes at the active site during denaturation that could not be detected directly in previous studies. The binding ability of modified AK with its monoclonal antibody (McAb3D3) was identical to that of native AK and the fluorescence of modified AK was quenched by interaction with McAb3D3. The relative fluorescence changes during the binding of modified AK with McAb3D3 in different concentrations of guanidine hydrochloride were monitored. The combination of this active site modification with the use of a conformation specific monoclonal antibody has potential for use in the study of the kinetics of folding of AK and in the detection of folding intermediates.     

Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t-butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni(2+)-immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0-3.0 mg of proteins from 1 L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288 U/micromol. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Conversion of trypsin into a selenium-containing enzyme by using chemical mutation

A chemical derivative of trypsin, selenotrypsin, was prepared to imitate glutathione peroxidase (GPX) by converting active serine residues in the active site of trypsin into selenocysteines. The strategy for preparation of selenotrypsin contained selective sulfonation by phenylmethanesulfonyl fluoride and nucleophilic substitution by NaHSe. Seleno-trypsin displayed enzyme activity of GPX, which is 150U/(mol. The kinetic properties of selenotrypsin was demonstrated to be similar to that of native GPX. © Rapid Science Ltd. 1998     

Selenium-containing 15-mer peptides with high glutathione peroxidase-like activity

Glutathione peroxidase (GPX) is one of the most crucial antioxidant enzymes in a variety of organisms. Here we described a new strategy for generating a novel GPX mimic by combination of a phage-displayed random 15-mer peptide library followed by computer-aided rational design and chemical mutation. The novel GPX mimic is a homodimer consisting of a 15-mer selenopeptide with an appropriate catalytic center, a specific binding site for substrates, and high catalytic efficiency. Its steady state kinetics was also studied, and the values of k(cat)/K(m)(GSH) and k(cat)/ K(mH(2)O(2)) were found to be similar to that of native GPX and the highest among the existing GPX mimics. Moreover, the novel GPX mimic was confirmed to have a strong antioxidant ability to inhibit lipid peroxidation by measuring the content of malondialdehyde, cell viability, and lactate dehydrogenase activity. Importantly, the novel GPX mimic can penetrate into the cell membrane because of its small molecular size. These characteristics endue the novel mimic with potential perspective for pharmaceutical applications.     

Generation of selenoprotein with glutathione peroxidase activity by chemical modification of the single-chain variable fragment expressed in a single-protein production system and its antioxidant ability

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. Single-chain variable fragment (scFv) can be converted into seleniumcontaining single-chain variable fragment (Se-scFv) by chemical modification of the hydroxyl groups in scFv, thus Se-scFv possesses GPX activity and becomes a prodrug. To improve the expression of scFv and simplify its purification steps, Single-protein production (SPP) system was used to express scFv and chemical modification was used to synthesize Se-scFv. Therefore, we must construct a new scFv-WCD1-lessACA gene, which can express its mRNA not containing any ACA sequences and express its amino acid sequence of target protein (scFv) being same to scFv-WCD1. In this way, the scFv-WCD1-lessACA can be only expressed in SPP system and no other background proteins in the cells could be expressed. The expression results showed that high level of scFv-WCD1-lessACA synthesis was at least sustained for 96 h in the virtual absence of background protein synthesis. Then, selenocysteine (Sec) was incorporated into the scFv-WCD1-lessACA by chemical modification and resulted in Se-scFv-WCD1-lessACA. The enzymatic characteristics of Se-scFv-WCD1-lessACA were determined. GPX activity was 2,563 U/μmol, its binding constant for GSH was 0.687 ×10⁵/mol. Moreover, Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability to protect mitochondria against oxidative damage induced by Vc/Fe²⁺ (mitochondrial damage model), suggesting that Se-scFv-WCD1-lessACA has potential application for protection of mitochondrial damage induced by reactive oxygen species (ROS).     

Characterization of selenium‐containing glutathione transferase zeta1–1 with high GPX activity prepared in eukaryotic cells

Accumulating evidence shows that glutathione peroxidase (GPX, EC.1.11.1.9), one of the most important antioxidant selenoenzymes, plays an essential role in protecting cells and tissues against oxidative damage by catalyzing the reduction of hydrogen peroxide by glutathione. Unfortunately, because of the limited availability and poor stability of GPX, it has not been used clinically to protect against oxidative stress. To overcome these problems, it is necessary to generate mimics of GPX. In this study, we have used directed mutagenesis and the inclusion of a selenocysteine (Sec) insertion sequence to engineer the expression in eukaryotic cells of human glutathione transferase zeta1–1 (hGSTZ1–1) with Sec in the active site (seleno‐hGSTZ1–1). This modification converted hGSTZ1–1 into an active GPX and is the first time this has been achieved in eukaryotic cells. The GPX activity of seleno‐hGSTZ1–1 is higher than that of GPX from bovine liver, indicating Sec at the active site plays an important role in the determination of catalytic specificity and performance. Kinetic studies revealed that the ping–pong catalytic mechanism of Se‐hGSTZ1–1 is similar to that of the natural GPX. Copyright © 2012 John Wiley & Sons, Ltd.