Study On Proteins Activity And Nucleosides Content Analysis By Capillary Eletrophoresis Combined With Monolithic Column Technology

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.