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蛋白质和多肽聚乙二醇修饰过程中活性区域的保护
Alternative TitleProtection of the active region in PEGylation of protein and peptide
王卿卿
Subtype博士
Thesis Advisor苏志国
2014-05
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
KeywordPegylation   hemoglobin   heme   glucagon-like Peptide-1   active Sites
Abstract聚乙二醇修饰可以有效地延长药物在体内的循环半衰期,但其修饰过程同时也会带来药物活性的损失或造成药物分子的氧化。本文以血红蛋白和胰高血糖素样肽-1(GLP-1)的聚乙二醇修饰为研究对象,分别探索了小分子一氧化碳(CO)在血红蛋白修饰过程中的抗氧化作用、大分子聚乙二醇(PEG)对血红素区域的保护作用以及金属螯合介质对GLP-1的N末端活性中心的保护作用。 向氧合血红蛋白中持续通入CO气体,将氧合血红蛋白转化为碳氧血红蛋白,然后再进行PEG修饰。并考察了修饰产物的结构和稳定性,以及修饰过程中高铁血红蛋白的生成情况。实验发现,在聚乙二醇修饰之前先使血红蛋白与CO结合,能有效的抑制修饰过程中高铁血红蛋白(metHb)的生成,将metHb含量控制在1%以内,明显低于未结合CO修饰过程(15%)。对碳氧血红蛋白(HbCO)的PEG修饰产物在近紫外区域(250-500 nm)的圆二色谱研究,发现CO分子的结合可以减少PEG修饰对血红素结构和血红素非极性微环境的影响。通过对HbCO修饰产物的稳定性研究,发现CO的结合使PEG修饰产物的热稳定性提高了35-40%,pH稳定性提高了5%,修饰产物二级结构变性一半的温度(Tm)明显增加,其中分子量5kDa的聚乙二醇双琥珀酰亚胺碳酸酯(PEG5K-SC)修饰HbCO的Tm值高达85oC,比未结合CO的修饰产物提高了18 oC。CO的结合还能明显的降低修饰产物的自氧化速率。对HbCO修饰产物的分子形态和分子结构的分析表明,CO与血红蛋白结合后能够使蛋白四级结构更紧密,增加了蛋白四聚体的凝聚力。对HbCO修饰产物重新氧合,检测P50和Hill系数,发现CO的结合对血红蛋白的载氧活性没有影响。 将分子量20kDa的聚乙二醇(PEG20K)通过可解离的二硫键和腙键连接在血红蛋白血红素附近的Cys-93(β)上,然后再用分子量为5kDa的聚乙二醇对血红蛋白进行修饰,修饰完成后再将PEG20K解离下来。随后对修饰产物的修饰位点、自氧化性质、结构和功能进行了研究,并与直接修饰产物进行了比较,通过对液质联用对酶解肽段进行分析,并与蛋白一级结构进行对比发现,被保护的修饰位点包括:Val-1(α)、Lys-16(α)和 Lys-95(β)。保护修饰产物的自氧化速率(kox)为0.017 h-1,明显低于直接修饰产物的0.030 h-1。利用圆二色谱对修饰产物结构的研究发现,PEG大分子保护血红素区域的方法,能明显降低修饰剂对血红素区域结构和微环境的影响。通过分析超离心对修饰产物的研究,发现保护修饰产物中四聚体的解离系数仅为直接修饰产物的1/3。保护修饰产物较直接修饰产物的氧亲和力低,更利于氧的释放。可逆连接大分子保护剂的方法,能够成功的保护几个重要的位点不被修饰,进而保护了蛋白活性中心血红素区域的结构,提高了蛋白稳定性。 利用胰高血糖素样肽-1 N末端的组氨酸,使其吸附在铜离子螯合介质上,然后再对其进行修饰。分别考察了分子量为5kDa、10kDa和20kDa的PEG分子对吸附在介质上的GLP-1的修饰情况,发现只有分子量为5kDa的PEG能够成功的对GLP-1进行修饰,而且得到的均为单修饰产物。对GLP-1修饰产物酶解图谱的分析,发现固相单修饰产物中不含有N末端修饰产物,而液相单修饰产物中含有N末端修饰产物,证明金属螯合介质成功的保护了GLP-1的N末端活性中心。对单修饰产物的药代动力学研究,发现GLP-1固相单修饰产物的半衰期为119.0 ± 4.2 min,较GLP-1提高了16倍。通过葡萄糖耐受实验对修饰产物的体内药效的研究,发现GLP-1固相单修饰产物的降糖效果优于液相单修饰产物,说明利用金属螯合介质保护N末端活性中心的方法,使GLP-1的修饰产物保留了较高的活性。
Other AbstractPEGylation, an effective method to prolong thein vivo half-life of protein and peptide, may induce the loss of activity and the oxidation of some products during their PEGylation processes. In this study, we investigated the protection methods for hemoglobin (Hb) and glucagon-like peptide-1(GLP-1), includingpre-binding of carbon monoxide (CO) and reversible conjugation of PEG20K at the Cys-93(β) during Hb PEGylation, as well as the protection at the N-terminal active site using metal-ion media for GLP-1. Hemoglobin may undergo oxidation of ferrous Hb into a non-functional methemoglobin(metHb) in conventional PEGylation process.It was found thatconventional PEGylation may result in 15%metHb formation. However, by pre-binding of CO,the metHb could be minimized to 1%even under long mixing condition. The circular dichroism spectra in the near UV and Soret band region (250-500 nm) showed that the hememicroenvironment was not affected by the PEG chains for the CO binding protein while the hemoglobin without CO binding would have its microenvironment perturbed. The physical and chemical parameters of the PEGylated products showed that CO binding increased thermo-stability for 35-40%,pH stabilityfor 5%, and the melting temperature (Tm) up to 85oC from 67 oC.CO binding can also inhibit the autoxidation of the PEGylatedHb. Dynamic light scatteringand sedimentation velocityanalysis indicated that the conformation of PEG-HbCOwas compacted slightly after CO binding. The PEGylatedHbCO could regain its oxygen delivery ability after PEGylation through removing CO by feeding O2under visible light irradiation. The protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylatedHb. In this study, 20 kDa polyethylene glycol (PEG20K) as protective reagent was reversibly conjugated at Cys-93(β) of Hb through hydrazone and disulfide bonds, followed by PEGylation with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry. Then, PEG20K was released from Hb by hydrazone hydrolysis and disulfide reduction. The autoxidation, structure and function of the PEGylatedHb were investigated. Reversible conjugation of PEG20K at Cys-93(β) can protect Lys-95(β), Val-1(?) and Lys-16(?) of Hb from PEGylation with PEG5K-SC. The autoxidation rate was 0.017 h-1,obviouly lower than that of PEGylated product witout protection 0.030 h-1. The circular dichroism spectrashowed that the PEGylation with protection could reduce the perturbation inhememicroenvironment. The results of the analytical ultracentrifugation showed that the tetramer stability of the PEGylatedHb was improved upon protection with PEG20K. The N-terminal active site of GLP-1 was chelated on the copper-ion chromatographic media, as there was a histidine on the N-terminus. Sequentially, succinimidyl carbonate mPEGs (5, 10 and 20 kDa) were introduced to the media and allowed to react with the bounded GLP-1. It was found that only the PEG5K-SC could react with the bounded GLP-1 and get the monoPEG5K-(GLP-1). The tryptic peptide mapping of the monoPEG5K-(GLP-1) suggested that there was no PEG5K-(N-terminus)-(GLP-1) which was appeared in the monoPEG5K-(GLP-1) without protection of the N-terminus. This revealed that the copper-ion media could protect the N-terminal active sites from PEGylation through its interaction with the peptide. The half-life was 119.0 ± 4.2 minand increased 16 times compared to the unmodified GLP-1. The glucose-stabilizingcapability was superior to that of the monoPEG5K-(GLP-1) without protection of the N-terminus. Thus, the method of protecting N-terminal active sitethrough chelating with copper-ion media could well retain the activity of GLP-1 during PEGylation process.
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/15516
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
王卿卿. 蛋白质和多肽聚乙二醇修饰过程中活性区域的保护[D]. 中国科学院研究生院,2014.
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