具有加卤过氧化物酶活性含卟啉辅基的抗体酶研究
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摘要
游离酪氨酸和蛋白质中酪氨酸的碘化是由甲状腺匀浆液和亚细胞部分中存在的酶催化的。Alexander已经证明在动物甲状腺组织中有一种加碘过氧化物酶，并相信该酶对加碘反应起作用，它是通过形成一个氧化态的碘中间体而起作用。该酶是加氯过氧化物酶(CPO)(EC1.11.10)。它是一个相对分子量大约42000Da的血红蛋白，每个分子含有一个铁(Ⅲ)原卟啉Ⅸ。对Cl~-、Br~-、I~-和合适卤素受体之间碳-卤键的过氧化形成，该酶是使用过氧化氢作为氧化剂。
甲状腺球蛋白或它的亚基中的酪氨酸残基的碘化是甲状腺素形成的基本步骤之一。在加氯过氧化物酶催化的碘化过程中，碘能很快地结合形成3-碘酪氨酸、3，5-二碘酪氨酸和甲状腺素。这样该酶在甲状腺素的代谢和生理功能上起重要作用。加氯过氧化物酶的缺乏会导致一些严重的甲状腺疾病。由于加氯过氧化物酶在甲状腺组织中含量低及在试剂中催化活性不稳定等原因，利用常规蛋白质化学技术分离和纯化加氯过氧化物酶，已经证明是非常困难的。由于这个原因，对该酶的生化性质和结构-功能的研究大大受阻。因此，制备具有加氯过氧化物酶活性的催化抗体用于机制研究和治疗应用是相当有意义的。另外，催化抗体的选择性卤化在有机合成等方面也有广阔的前景。
用邻硝基苯甲醛和吡咯反应，生成了meso-四(邻-硝基苯)卟啉(H_2TNO_2PP)，再还原生成了meso-四(邻-胺基苯)卟啉(H_2TamPP)。根据薄层色谱结果分离异构体，得到α，α，α，α-H_2NH_2PP。它与苯乙酰氯反应生成meso-四(α，α，α，α-o-苯乙酰胺苯)卟啉。对合成的抗原，经紫外可见光谱、核磁、红外、元素分析及质谱证明得到的meso-四(α，α，α，α-O-苯乙酰胺苯)卟啉就是所需要的抗原。
meso-四(α，α，α，α-o-苯乙酰胺苯)卟啉的相对分子量Mr只有1147.3，仍可作为全抗原免疫Balb／c小鼠，通过细胞融合等技术筛选得到了杂交瘤细胞株1 F 2和3 E 4。经E L I S A法得到鼠血清抗体效价为1：320，细胞株1F2的腹水效价为1：3200，细胞株3E4的腹水效价为1：6400。通过HiTrap Protein A纯化得到了McAb 1F2和McAb 3E4纯品。利用高效液相色谱法证明纯化得到的McAb 1F2很纯，亚型为IgG2a。经MALDI／TOFMS测定，McAb 1F2的相对分子量为156678.8Da。卟啉与McAb 1F2形成复合物后，卟啉Sorer带最大吸收峰从408nm红移到416nm，并有增色效应，反映了卟啉一抗体刚性且紧密的结合。从CD和UV吸收强度在对pH6～12范围内基本不变，说明抗原与抗体结合很紧
密，抗体酶稳定性很高，反映了抗原的苯乙酚苯基与抗体结合位点之间
的强烈疏水相互作用是稳定的主要因素。
通过对抗体酶的加卤过氧化物酶活性及过氧化物酶活性的测定，得
到加卤过氧化物酶的几、Kc。t、Kc。丫K。分别是 1．50 X 10“、0．5185‘、
3．45 X 10“M．S－’；过氧化物酶的 K。、Ke。t、Kc。；从分别是 20．29InM、
396．82min－’、l．9557X10’。这是因为 meso一四（。，a，a，a－o一苯乙酸
胺苯）叶琳有8个苯环，极大地增强了它的疏水性，因此也就增加了它
的免疫原性，使机体产生了较强的免疫应答。Thomas在总结成功的半抗
原设计方案后提出，半抗原结构中应含有芳香结构。实验表明，含有芳
香结构的半抗原其免疫原性就较强，用其免疫可使机体产生了较强的免
疫应答，从而增加了抗体酶筛选的概率。说明小分子物质只要设计合理，
有一定的化学组成和较为复杂的结构，即使不连接载体，也可具有免疫
凉性。因此，我们用小分于meso一四（a，a，a，a－0一苯乙酸苯）叶琳
（Mr＝11 47．3）作为全抗原，诱导产生了具有一定加卤过氧化物酶及过氧化
物酶活力的抗体酶。
随着叶咐／抗体比率的增加，紫外可见差八 A。。。n。越来越大，有较显著
的增色效应，反映了叶琳与抗体刚性且紧密的结合，抗体的抗原结合部
位有芳香簇氨基酸存在。荧光光谱法通过荧光淬灭测定叶琳与抗体的相
I［作用，得到叶琳与抗体的结合比为1：1，离解常数为
了 084土0．246X 10－“moVL，可见叶琳与 McAb IF2有很高的亲合力。亲
合力高，抗体酶催化活性也高，因此说明了小分子meso一四
（。，a；a，a－0一苯乙酸苯）叶琳为什么能诱导出具有较高催化活性的
抗体酶。用同步荧光光谱法结合荧光淬灭，选择西人二20urn时，得到结
合位点及附近有酪氨酸，但大部分处于结合位点较远的位置上。选择八
入。80n。时，得到结合位点及附近有较多色氨酸存在，但NBS仅能淬灭抗
体r 分子表面及次表面的色氨酸残基荧光。说明了IF2分子中大部分
色氨酸残基处于疏水性很强的内核环境中。
Preparation and Properties of A Chloroperoxidase-like Catalytic
Antibody Doctor Candidate: Qi Chao
Advisor: Prof. Zhang Yu-jing
lodination of free tyrosine and tyrosine in protein is mediated by an enzyme present in thyroid homogenates and subcellular fractions. Alexander has demonstrated an iodide poroxidase in animal thyroid tissue and believes this enzyme is responsible for the iodination reactions through formation of an oxidized iodinating intermediate. This enzyme is chloroperoxidase(CPO)(EC1. 11. 10). It is a heme protein of Mr -42000Da containing one ferriprotoporphyrin IX per molecule. The enzyme uses hydrogen peroxide as the oxidant for the peroxidative formation of a carbon-halogen bond between Cl, Br-, Y and a suitable halogen acceptor. But it is unable to utilize F for this reaction.
lodination of tyrosine residues in thyroglobulin, or in its subunits, is one of the essential steps in the formation of thyroxine in the thyroid gland. Iodide can be rapidly bound as 3-iodotyrisine, 3, 5-diiodotyrosine, and thyroxine during the chloroperoxidase-catalyzed iodination. Thus, theenzyme plays an important role in the metabolism and physiological function of thyroxine. Deficiency of chloropeeroxidase will result in some serious thyroid diseases.
Because of the low abundance of chloroperoxidase in thyroid tissue and the instability of the catalytic activity in detergent, etc, attempts to isolate and purify chloroperoxidase by conventional techniques of protein chemistry, have been difficult. For this reason, the studies of biochemical properties and structure-function of the enzyme have been impeded. Therefore, it will be of considerable interest to prepare a catalytic antibody with chloroperoxidase activity for mechanistic studies and therapeutic application. In addition, the antibody-catalyzed selective halogenation also will have broad prospects for organic synthesis, etc.
In short, condensation of o-nitrobenzaldehyde and pyrrole
followed by reduction of the meso-tetra(o-nitrophenyl)porphyrin led to a satisfactory yield of meso-tetra(o-aminophenyl) porphyrin, H2TamPP. Thin-layer chromatography on silica gel gave excellent separation of four components which, in ratio 1:2:4:1, were presumed to be the four atropisomers in statistical abundance. With Rf values based on their expected polarity and considering the relative amounts of the individual atropisomers these are provisionally assigned as , with the most polar tetra- a -atropisomer, moving most slowly. Isolation of a, a , a , a -H2TamPP by silica gel '.column chromatography afforted gram quantities of product. The mixture containing the remaining three atropiaomers was reequilibrated in boiling tobuene followed by chromatography to isolate more of the a , a , a , a -atropisomer. Repetition of these steps leads to ultimate conversion of nearly all of the H2TamPP into the desired a , a , a , a - H2TamPP. The four amino atropisomers are rather stable in solution at 25?C. The amino groups of H2TamPP are also more reactive. The a , a , a , a -atropisomer of H2TamPP could be further modified and the configurational energy barrier raised through reaction with phenylacetyl chloride forming amide. Phenylacetyl chloride gave 72% yield of meso-tetra( a , a , a , a -o- phenylacetylamide phenyl)porphyrin. McAb was raised against meso-Tetra( a , a , a , a -o-phenylacetylamide phenyl)porphyrin through hybridoma methology.
Small molecule meso-Tetra (a , a-, a, a -0-phenylacetylamide phenyl)porphyrin'could be used as complete antigen to immunize Balb/c mice and induce McAb 1F2. The purity of McAb 1F2 was indicated very high by MALDI/TOF MS and SDS-PAGE. The subtype of McAb 1F2 is IgG2a. A relative molecular weight of McAb 1F2 was determined to be 156678.8Da by MALDI/TOF MS. The intensities of UV and CD spectra over a pH range between 6 and 12 almost remain constant, which reveals that the tight binding of Fe porphyrin and McAb 1F2 and the high stability of abzyme. The chloroperoxidase activity of McAb lF2-Fe porphyrin complex appears thermostable until 60C
甲状腺球蛋白或它的亚基中的酪氨酸残基的碘化是甲状腺素形成的基本步骤之一。在加氯过氧化物酶催化的碘化过程中，碘能很快地结合形成3-碘酪氨酸、3，5-二碘酪氨酸和甲状腺素。这样该酶在甲状腺素的代谢和生理功能上起重要作用。加氯过氧化物酶的缺乏会导致一些严重的甲状腺疾病。由于加氯过氧化物酶在甲状腺组织中含量低及在试剂中催化活性不稳定等原因，利用常规蛋白质化学技术分离和纯化加氯过氧化物酶，已经证明是非常困难的。由于这个原因，对该酶的生化性质和结构-功能的研究大大受阻。因此，制备具有加氯过氧化物酶活性的催化抗体用于机制研究和治疗应用是相当有意义的。另外，催化抗体的选择性卤化在有机合成等方面也有广阔的前景。
用邻硝基苯甲醛和吡咯反应，生成了meso-四(邻-硝基苯)卟啉(H_2TNO_2PP)，再还原生成了meso-四(邻-胺基苯)卟啉(H_2TamPP)。根据薄层色谱结果分离异构体，得到α，α，α，α-H_2NH_2PP。它与苯乙酰氯反应生成meso-四(α，α，α，α-o-苯乙酰胺苯)卟啉。对合成的抗原，经紫外可见光谱、核磁、红外、元素分析及质谱证明得到的meso-四(α，α，α，α-O-苯乙酰胺苯)卟啉就是所需要的抗原。
meso-四(α，α，α，α-o-苯乙酰胺苯)卟啉的相对分子量Mr只有1147.3，仍可作为全抗原免疫Balb／c小鼠，通过细胞融合等技术筛选得到了杂交瘤细胞株1 F 2和3 E 4。经E L I S A法得到鼠血清抗体效价为1：320，细胞株1F2的腹水效价为1：3200，细胞株3E4的腹水效价为1：6400。通过HiTrap Protein A纯化得到了McAb 1F2和McAb 3E4纯品。利用高效液相色谱法证明纯化得到的McAb 1F2很纯，亚型为IgG2a。经MALDI／TOFMS测定，McAb 1F2的相对分子量为156678.8Da。卟啉与McAb 1F2形成复合物后，卟啉Sorer带最大吸收峰从408nm红移到416nm，并有增色效应，反映了卟啉一抗体刚性且紧密的结合。从CD和UV吸收强度在对pH6～12范围内基本不变，说明抗原与抗体结合很紧
密，抗体酶稳定性很高，反映了抗原的苯乙酚苯基与抗体结合位点之间
的强烈疏水相互作用是稳定的主要因素。
通过对抗体酶的加卤过氧化物酶活性及过氧化物酶活性的测定，得
到加卤过氧化物酶的几、Kc。t、Kc。丫K。分别是 1．50 X 10“、0．5185‘、
3．45 X 10“M．S－’；过氧化物酶的 K。、Ke。t、Kc。；从分别是 20．29InM、
396．82min－’、l．9557X10’。这是因为 meso一四（。，a，a，a－o一苯乙酸
胺苯）叶琳有8个苯环，极大地增强了它的疏水性，因此也就增加了它
的免疫原性，使机体产生了较强的免疫应答。Thomas在总结成功的半抗
原设计方案后提出，半抗原结构中应含有芳香结构。实验表明，含有芳
香结构的半抗原其免疫原性就较强，用其免疫可使机体产生了较强的免
疫应答，从而增加了抗体酶筛选的概率。说明小分子物质只要设计合理，
有一定的化学组成和较为复杂的结构，即使不连接载体，也可具有免疫
凉性。因此，我们用小分于meso一四（a，a，a，a－0一苯乙酸苯）叶琳
（Mr＝11 47．3）作为全抗原，诱导产生了具有一定加卤过氧化物酶及过氧化
物酶活力的抗体酶。
随着叶咐／抗体比率的增加，紫外可见差八 A。。。n。越来越大，有较显著
的增色效应，反映了叶琳与抗体刚性且紧密的结合，抗体的抗原结合部
位有芳香簇氨基酸存在。荧光光谱法通过荧光淬灭测定叶琳与抗体的相
I［作用，得到叶琳与抗体的结合比为1：1，离解常数为
了 084土0．246X 10－“moVL，可见叶琳与 McAb IF2有很高的亲合力。亲
合力高，抗体酶催化活性也高，因此说明了小分子meso一四
（。，a；a，a－0一苯乙酸苯）叶琳为什么能诱导出具有较高催化活性的
抗体酶。用同步荧光光谱法结合荧光淬灭，选择西人二20urn时，得到结
合位点及附近有酪氨酸，但大部分处于结合位点较远的位置上。选择八
入。80n。时，得到结合位点及附近有较多色氨酸存在，但NBS仅能淬灭抗
体r 分子表面及次表面的色氨酸残基荧光。说明了IF2分子中大部分
色氨酸残基处于疏水性很强的内核环境中。
Preparation and Properties of A Chloroperoxidase-like Catalytic
Antibody Doctor Candidate: Qi Chao
Advisor: Prof. Zhang Yu-jing
lodination of free tyrosine and tyrosine in protein is mediated by an enzyme present in thyroid homogenates and subcellular fractions. Alexander has demonstrated an iodide poroxidase in animal thyroid tissue and believes this enzyme is responsible for the iodination reactions through formation of an oxidized iodinating intermediate. This enzyme is chloroperoxidase(CPO)(EC1. 11. 10). It is a heme protein of Mr -42000Da containing one ferriprotoporphyrin IX per molecule. The enzyme uses hydrogen peroxide as the oxidant for the peroxidative formation of a carbon-halogen bond between Cl, Br-, Y and a suitable halogen acceptor. But it is unable to utilize F for this reaction.
lodination of tyrosine residues in thyroglobulin, or in its subunits, is one of the essential steps in the formation of thyroxine in the thyroid gland. Iodide can be rapidly bound as 3-iodotyrisine, 3, 5-diiodotyrosine, and thyroxine during the chloroperoxidase-catalyzed iodination. Thus, theenzyme plays an important role in the metabolism and physiological function of thyroxine. Deficiency of chloropeeroxidase will result in some serious thyroid diseases.
Because of the low abundance of chloroperoxidase in thyroid tissue and the instability of the catalytic activity in detergent, etc, attempts to isolate and purify chloroperoxidase by conventional techniques of protein chemistry, have been difficult. For this reason, the studies of biochemical properties and structure-function of the enzyme have been impeded. Therefore, it will be of considerable interest to prepare a catalytic antibody with chloroperoxidase activity for mechanistic studies and therapeutic application. In addition, the antibody-catalyzed selective halogenation also will have broad prospects for organic synthesis, etc.
In short, condensation of o-nitrobenzaldehyde and pyrrole
followed by reduction of the meso-tetra(o-nitrophenyl)porphyrin led to a satisfactory yield of meso-tetra(o-aminophenyl) porphyrin, H2TamPP. Thin-layer chromatography on silica gel gave excellent separation of four components which, in ratio 1:2:4:1, were presumed to be the four atropisomers in statistical abundance. With Rf values based on their expected polarity and considering the relative amounts of the individual atropisomers these are provisionally assigned as , with the most polar tetra- a -atropisomer, moving most slowly. Isolation of a, a , a , a -H2TamPP by silica gel '.column chromatography afforted gram quantities of product. The mixture containing the remaining three atropiaomers was reequilibrated in boiling tobuene followed by chromatography to isolate more of the a , a , a , a -atropisomer. Repetition of these steps leads to ultimate conversion of nearly all of the H2TamPP into the desired a , a , a , a - H2TamPP. The four amino atropisomers are rather stable in solution at 25?C. The amino groups of H2TamPP are also more reactive. The a , a , a , a -atropisomer of H2TamPP could be further modified and the configurational energy barrier raised through reaction with phenylacetyl chloride forming amide. Phenylacetyl chloride gave 72% yield of meso-tetra( a , a , a , a -o- phenylacetylamide phenyl)porphyrin. McAb was raised against meso-Tetra( a , a , a , a -o-phenylacetylamide phenyl)porphyrin through hybridoma methology.
Small molecule meso-Tetra (a , a-, a, a -0-phenylacetylamide phenyl)porphyrin'could be used as complete antigen to immunize Balb/c mice and induce McAb 1F2. The purity of McAb 1F2 was indicated very high by MALDI/TOF MS and SDS-PAGE. The subtype of McAb 1F2 is IgG2a. A relative molecular weight of McAb 1F2 was determined to be 156678.8Da by MALDI/TOF MS. The intensities of UV and CD spectra over a pH range between 6 and 12 almost remain constant, which reveals that the tight binding of Fe porphyrin and McAb 1F2 and the high stability of abzyme. The chloroperoxidase activity of McAb lF2-Fe porphyrin complex appears thermostable until 60C
引文
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