整体柱—电泳技术在蛋白核苷活性及含量分析的应用
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摘要
毛细管电泳是一种强大的分析技术,从分析小分子到生物大分子都具有很高的效率。它具有高分离效率,高分辨率,快速,易自动化等优点,毛细管电泳在化学,生物技术,环境等方面得到了极大的应用。
毛细管电泳不仅是一种有效的分离工具而且在酶的研究和药物开发方面也是一个有效的平台。在众多的毛细管电泳技术中,电泳媒介微分析技术(EMMA)在酶抑制剂筛选,酶动力学研究等方面是一个十分有效的工具。这种筛选通过使用二极管阵列检测器接合多通道毛细管电泳能实现高通量。毛细管中固定酶制作微反应器和微流控芯片在酶研究、蛋白组学、诊断学等方面也是一个的十分有希望的微型化的方法。
高血压是最常见的心血管疾病之一,严重威胁着人类的健康。血管紧张素转换酶(ACE)能将血管紧张素I转换为有高活性的血管紧张素II,引起血压升高。在抗高血压药物中,血管紧张素转换酶抑制剂(ACEI)是一类常见的有效的药物,如卡托普利,赖诺普利。但合成ACEI类降压药物的使用常伴随着多种不良反应。近年来,大量研究发现ACEI广泛存在于天然产物中,天然产物中的ACEI与合成ACEI相比有安全性高、降压持久、无副作用等优点。从中草药和食品中间筛选没有副作用的新抗高血压药引起了广大学者的兴趣。为此本学位论文主要做了以下工作：1、利用毛细管电泳技术,结合酶微反应器从中草药和食品中间筛选没有副作用的筛选ACEI。将ACE直接固定在整体基质上,制成了酶微反应器,结合毛细管电泳在线分离,筛选ACEI.制备了将酶直接共价固定于整体基质的ACE微反应器,考查卡托普利,赖诺普利,和各种中草药提取物的抑制作用,从中草药中筛选出没有副作用的新抗高血压药。通过筛选发现山楂,决明子,槐米等水提物有一定的抑制效果,分别得出了其抑制率。
2、毛细管电泳分析肽和蛋白质时,由于肽和蛋白质的表面的不均一性,肽和蛋白质特别是碱性蛋白／肽易于被毛细管壁吸附,使结果到了一个不能接受的水平。针对这种情况,本文建立了高效毛细管电泳快速分离IgG等蛋白质及肽的方法。用化学共价改性毛细管成功抑制了碱性蛋白及肽在空白毛细管上的吸附,实现了毛细管电泳对蛋白样品的高效的分离、得到了较高的蛋白回收率、良好的重现性。较非共价包覆毛细管有稳定性高,更宽的pH范围等优点。
3、建立简单、快速测定冬虫夏草和人工蛹虫草子实体中虫草素、腺嘌呤、鸟嘌呤、尿嘧啶、次黄嘌呤、腺苷、鸟苷、尿苷、次黄苷9种核苷及碱基成分的高效毛细管区带电泳法,比较天然冬虫夏草与人工蛹虫草子实体中核苷类化合物的含量。采用未涂层弹性石英毛细管,以硼砂-硼酸溶液作为缓冲体系,分离电压20 kv,温度20℃,检测波长258 nm,进样量为3445 Pa,进样5 s。在优化的实验条件下,冬虫夏草及人工蛹虫草子实体中9种核苷类在15 min内达到基线分离；得出人工蛹虫草子实体与天然虫草有相似的核苷类成分,但在含量上有较大差异的结论。新建的方法操作简便,实验消耗低,高效而快速,可以成为冬虫夏草及代用产品中核苷类物质检测的一种简便、价廉、有效的分析方法。
Capillary electrophoresis (CE) is a powerful analytical technique for the analysis of small molecules and large biopolymers. Due to its high separation efficiency, high resolution, rapid separation time and ease of automation, CE has got wide application in many areas such as chemistry, biotechnology, environmental sciences.
Capillary electrophoresis (CE) is not only a separation tool with high separation performance but also a versatile platform for enzyme research and drug discovery. Among the various CE techniques, electrophoretically mediated microanalysis (EMMA) technique is a useful miniature tool for the study of enzymes and inhibitor screening.. The high-throughput screening can be dramatically improved by using multiplex capillary electrophoresis with diode array detector.Alternatively, immobilized enzyme microreactors fabricated on capillaries and microfluidic chips represent another promising miniature approach for enzyme study, peptide mapping in proteomics, and diagnostics.
Hypertension, one of the most common chronic diseases, has become a significant public health problem worldwide. Angiotensin I converting enzyme (ACE) plays a very important role in the control of blood pressure in mammals. It catalyzes the conversion of the decapeptide angiotensin I to the biologically potent octapeptide angiotensin II (vasopressor, aldosterone regulator, and dipsogen), and inactivates the vasodepressor bradykinin. One class of antihypertensive drugs is known as angiotensin I converting enzyme inhibitors (ACEI), such as captopril and lisinopril. However, in present, many synthesized antihypertensive drugs can cause serious side effects such as cough and angioedema. For that, many studies discovered that abundance ACEI exist in natural products. Compared with synthesized antihypertensive drugs, ACEI which existing in natural products have high security, lasting effected and no side effects. To develop rapid and highly efficient methods for screening ACE inhibitors from more huge range attracts many researchers.
This paper is mainly composed of the following parts:
1. ACEI was fast screened from Chinese herbal extracts and foods using the enzyme microreactor, which direct on-column immobilized ACE on the monolithic matrix combined with reactor-separator system. The conditions, which including the preparation of monolithic column and the immobilized enzyme was optimized. A small library of compounds containing captopril, lisinopril arid natural extracts were tested. We found that some compounds had ACE inhibiting activity such as the water extractive of hawthorn, Chinese Taxillus twing, Japanese Pagodatree Flower-bud and got its inhibition.
2. The results is unacceptable when analysis peptides and proteins by Capillary electrophoresis for capillary wall adsorption. The reason is the heterogeneous surface of the peptides and proteins. In response, we established a capillary electrophoresis approach for rapid analysis IgG and other protein and peptide. The covalent modified capillary inner successfully inhibited the absorption of basic protein, which achieve high separation efficiency and higher protein recovery and good reproducibility for protein sample. Compared with non-covalent coated capillary, this method has high stability and a wider range of pH.
3. To determination of nucleosides in natural Cordyceps sinensis and cultured Cordyceps militaris and compared the contents of nucleosides from them, a simple and rapid high performance capillary zone electrophoresis (HPCZE) method was developed. The contents of nucleosides were determined using HPCE equipped with PDA detector and a fused-silica capillary was adopted. The running buffer solution was 0.05 mol-L-1 sodium borate buffer(pH=9.0). Running voltage was 20 kV and the column temperature was set at 20℃during separation and the wavelength of detector was 258 nm and the sampling condition was 3.445 kPa,5.0 s. Under the optimized conditions, the nine nucleosides reach the baseline separation in 15 minutes and the nucleosides contents demonstrated good linear relationship in the range of 1.0-205.5μg·mL-1(r≥0.9995). There are have similar nucleosides from natural Cordyceps sinensis and cultured C. militaris, but the contents of the nucleosides showed relatively large difference. Results show that this method can be utilized for the quantitative analysis of nucleosides in natural and cultured Cordyceps militaris and its substitutes, which is helpful to control their quality. It has the advantages of accuracy, fastness, convenience and low sample consumption.
毛细管电泳不仅是一种有效的分离工具而且在酶的研究和药物开发方面也是一个有效的平台。在众多的毛细管电泳技术中,电泳媒介微分析技术(EMMA)在酶抑制剂筛选,酶动力学研究等方面是一个十分有效的工具。这种筛选通过使用二极管阵列检测器接合多通道毛细管电泳能实现高通量。毛细管中固定酶制作微反应器和微流控芯片在酶研究、蛋白组学、诊断学等方面也是一个的十分有希望的微型化的方法。
高血压是最常见的心血管疾病之一,严重威胁着人类的健康。血管紧张素转换酶(ACE)能将血管紧张素I转换为有高活性的血管紧张素II,引起血压升高。在抗高血压药物中,血管紧张素转换酶抑制剂(ACEI)是一类常见的有效的药物,如卡托普利,赖诺普利。但合成ACEI类降压药物的使用常伴随着多种不良反应。近年来,大量研究发现ACEI广泛存在于天然产物中,天然产物中的ACEI与合成ACEI相比有安全性高、降压持久、无副作用等优点。从中草药和食品中间筛选没有副作用的新抗高血压药引起了广大学者的兴趣。为此本学位论文主要做了以下工作：1、利用毛细管电泳技术,结合酶微反应器从中草药和食品中间筛选没有副作用的筛选ACEI。将ACE直接固定在整体基质上,制成了酶微反应器,结合毛细管电泳在线分离,筛选ACEI.制备了将酶直接共价固定于整体基质的ACE微反应器,考查卡托普利,赖诺普利,和各种中草药提取物的抑制作用,从中草药中筛选出没有副作用的新抗高血压药。通过筛选发现山楂,决明子,槐米等水提物有一定的抑制效果,分别得出了其抑制率。
2、毛细管电泳分析肽和蛋白质时,由于肽和蛋白质的表面的不均一性,肽和蛋白质特别是碱性蛋白／肽易于被毛细管壁吸附,使结果到了一个不能接受的水平。针对这种情况,本文建立了高效毛细管电泳快速分离IgG等蛋白质及肽的方法。用化学共价改性毛细管成功抑制了碱性蛋白及肽在空白毛细管上的吸附,实现了毛细管电泳对蛋白样品的高效的分离、得到了较高的蛋白回收率、良好的重现性。较非共价包覆毛细管有稳定性高,更宽的pH范围等优点。
3、建立简单、快速测定冬虫夏草和人工蛹虫草子实体中虫草素、腺嘌呤、鸟嘌呤、尿嘧啶、次黄嘌呤、腺苷、鸟苷、尿苷、次黄苷9种核苷及碱基成分的高效毛细管区带电泳法,比较天然冬虫夏草与人工蛹虫草子实体中核苷类化合物的含量。采用未涂层弹性石英毛细管,以硼砂-硼酸溶液作为缓冲体系,分离电压20 kv,温度20℃,检测波长258 nm,进样量为3445 Pa,进样5 s。在优化的实验条件下,冬虫夏草及人工蛹虫草子实体中9种核苷类在15 min内达到基线分离；得出人工蛹虫草子实体与天然虫草有相似的核苷类成分,但在含量上有较大差异的结论。新建的方法操作简便,实验消耗低,高效而快速,可以成为冬虫夏草及代用产品中核苷类物质检测的一种简便、价廉、有效的分析方法。
Capillary electrophoresis (CE) is a powerful analytical technique for the analysis of small molecules and large biopolymers. Due to its high separation efficiency, high resolution, rapid separation time and ease of automation, CE has got wide application in many areas such as chemistry, biotechnology, environmental sciences.
Capillary electrophoresis (CE) is not only a separation tool with high separation performance but also a versatile platform for enzyme research and drug discovery. Among the various CE techniques, electrophoretically mediated microanalysis (EMMA) technique is a useful miniature tool for the study of enzymes and inhibitor screening.. The high-throughput screening can be dramatically improved by using multiplex capillary electrophoresis with diode array detector.Alternatively, immobilized enzyme microreactors fabricated on capillaries and microfluidic chips represent another promising miniature approach for enzyme study, peptide mapping in proteomics, and diagnostics.
Hypertension, one of the most common chronic diseases, has become a significant public health problem worldwide. Angiotensin I converting enzyme (ACE) plays a very important role in the control of blood pressure in mammals. It catalyzes the conversion of the decapeptide angiotensin I to the biologically potent octapeptide angiotensin II (vasopressor, aldosterone regulator, and dipsogen), and inactivates the vasodepressor bradykinin. One class of antihypertensive drugs is known as angiotensin I converting enzyme inhibitors (ACEI), such as captopril and lisinopril. However, in present, many synthesized antihypertensive drugs can cause serious side effects such as cough and angioedema. For that, many studies discovered that abundance ACEI exist in natural products. Compared with synthesized antihypertensive drugs, ACEI which existing in natural products have high security, lasting effected and no side effects. To develop rapid and highly efficient methods for screening ACE inhibitors from more huge range attracts many researchers.
This paper is mainly composed of the following parts:
1. ACEI was fast screened from Chinese herbal extracts and foods using the enzyme microreactor, which direct on-column immobilized ACE on the monolithic matrix combined with reactor-separator system. The conditions, which including the preparation of monolithic column and the immobilized enzyme was optimized. A small library of compounds containing captopril, lisinopril arid natural extracts were tested. We found that some compounds had ACE inhibiting activity such as the water extractive of hawthorn, Chinese Taxillus twing, Japanese Pagodatree Flower-bud and got its inhibition.
2. The results is unacceptable when analysis peptides and proteins by Capillary electrophoresis for capillary wall adsorption. The reason is the heterogeneous surface of the peptides and proteins. In response, we established a capillary electrophoresis approach for rapid analysis IgG and other protein and peptide. The covalent modified capillary inner successfully inhibited the absorption of basic protein, which achieve high separation efficiency and higher protein recovery and good reproducibility for protein sample. Compared with non-covalent coated capillary, this method has high stability and a wider range of pH.
3. To determination of nucleosides in natural Cordyceps sinensis and cultured Cordyceps militaris and compared the contents of nucleosides from them, a simple and rapid high performance capillary zone electrophoresis (HPCZE) method was developed. The contents of nucleosides were determined using HPCE equipped with PDA detector and a fused-silica capillary was adopted. The running buffer solution was 0.05 mol-L-1 sodium borate buffer(pH=9.0). Running voltage was 20 kV and the column temperature was set at 20℃during separation and the wavelength of detector was 258 nm and the sampling condition was 3.445 kPa,5.0 s. Under the optimized conditions, the nine nucleosides reach the baseline separation in 15 minutes and the nucleosides contents demonstrated good linear relationship in the range of 1.0-205.5μg·mL-1(r≥0.9995). There are have similar nucleosides from natural Cordyceps sinensis and cultured C. militaris, but the contents of the nucleosides showed relatively large difference. Results show that this method can be utilized for the quantitative analysis of nucleosides in natural and cultured Cordyceps militaris and its substitutes, which is helpful to control their quality. It has the advantages of accuracy, fastness, convenience and low sample consumption.
引文
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