毛细管区带电泳技术研究龙血竭有效单体与核酸适配体及凝血酶之间的相互作用
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摘要
利用毛细管区带电泳技术(CZE)研究5个龙血竭有效单体与牛凝血酶(B-Thr)、29碱基凝血酶核酸适配体(Apt29)之间的相互作用.电泳条件为有效长度是40 cm的非涂层石英毛细管,气压进样3.45 kPa,进样时间5 s,分离电压+15 kV,分离温度15℃,检测波长分别为214 nm(牛凝血酶体系)和330 nm(核酸适配体体系).结果表明,龙血素A和B-Thr之间的结合常数K_b为4.73×10~4 L/mol,白藜芦醇、龙血素B、7,4′-二羟基黄酮、龙血素C与B-Thr没有显著结合;龙血素B和7,4′-二羟基黄酮与核酸适配体的结合常数K_b分别为1.98×10~4,1.83×10~4 L/mol,白藜芦醇、龙血素A、龙血素C与核酸适配体之间没有表现出明显的结合现象.本文结果对进一步研究龙血竭在凝血酶上的潜在结合位点,以及核酸适配体能否向凝血酶靶向转运龙血竭,提供了科学数据支持.
Interactions between Apt29, bovine thrombin(B-Thr) and five active molecules in Dragon's blood were investigated based on capillary zone electrophoresis(CZE). The CZE conditions were set as following: effective capillary length of 40 cm, injection volume of 3.45 kPa, injection time of 5 s, voltage of +15 kV, temperature of 15 ℃, detection wavelength of 214 nm and 330 nm. The results show that K_b between Loureirin A and B-Thr is 4.73×10~4 L/mol, and Loureirin B, Loureirin C, Resveratrol 7,4-dihydroxyflavanone are no bound with B-Thr obviously. The K_b between Loureirin B, DHF and Apt29 are 1.98×10~4, 1.83×10~4 L/mol, respectively. Loureirin A, Loureirin C and Resveratrol are no bound with Apt29 obviously. The study indicates the interactions between B-Thr, Apt29 and active molecules in Dragon's blood. It can provide a basis for speculating the potential binding site of dragon's blood with thrombin, and delivering Dragon's blood to thrombin with aptamer as target delivery tool.
Interactions between Apt29, bovine thrombin(B-Thr) and five active molecules in Dragon's blood were investigated based on capillary zone electrophoresis(CZE). The CZE conditions were set as following: effective capillary length of 40 cm, injection volume of 3.45 kPa, injection time of 5 s, voltage of +15 kV, temperature of 15 ℃, detection wavelength of 214 nm and 330 nm. The results show that K_b between Loureirin A and B-Thr is 4.73×10~4 L/mol, and Loureirin B, Loureirin C, Resveratrol 7,4-dihydroxyflavanone are no bound with B-Thr obviously. The K_b between Loureirin B, DHF and Apt29 are 1.98×10~4, 1.83×10~4 L/mol, respectively. Loureirin A, Loureirin C and Resveratrol are no bound with Apt29 obviously. The study indicates the interactions between B-Thr, Apt29 and active molecules in Dragon's blood. It can provide a basis for speculating the potential binding site of dragon's blood with thrombin, and delivering Dragon's blood to thrombin with aptamer as target delivery tool.
引文
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[3] 陈惠琴,梅文莉,左文健,等.海南血竭的化学成分研究[J].中国药物化学杂志,2011,21(4):308-311.Chen Huiqin,Mei Wenli,Zuo Wenjian,et al.Study on chemical constituents of Dragon’s blood in Hainan [J].Chin J Med Chem,2011,21(4):308-311.(in Chinese)
[4] 黄树莲,陈学芬,陈晓军,等.广西血竭的活血化瘀研究[J].中药材,1994,17(9):37-39.Huang Shulian,Chen Xuefen,Chen Xiaojun,et al.Study on promoting blood circulation and removing blood stasis of Guangxi dragon’s blood [J].Chin Med Mat,1994,17(9):37-39.(in Chinese)
[5] Darmostuk M,Rimpelova S,Gbelcova H,et al.Current approaches in SELEX:An update to aptamer selection technology[J].Biotechnol Adv,2015,33(6):1141-1161.
[6] Kanwar J R,Shankaranarayanan J S,Gurudevan S,et al.Aptamer-based therapeutics of the past,present and future:from the perspective of eye-related diseases[J].Drug Discov Today,2014,19(9):1309-1321.
[7] Radom F,Jurek P M,Mazurek M P,et al.Aptamers:Molecules of great potential[J].Biotechnol Adv,2013,31(8):1260-1274.
[8] Bock L C,Grif C,Vermaas E H,et al.Selection of single-stranded-DNA molecules that bind and inhibit human thrombin[J].Nature,1992,355(6360):564-566.
[9] Macaya R F,Schultze P,Smith F W,et al.Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution[J].Proc Natl Acad Sci,1993,90(8):3745-3749
[10] Tasset D M,Kubik M F,Steiner W.Oligonucleotide inhibitors of human thrombin that bind distinct epitopes[J].J Mol Biol,1997,272(5):688-698.
[11] Liu R,Yu X,Gao W,et al.Study on the interaction between salvianic acid a sodium and bovine serum albumin by spectroscopic methods [J].Spectrochim Acta Part A,2011,78(5):1535-1539.
[12] Liu J,Wang X,Cai Z,et al.Effect of tanshinone ⅡA on the noncovalent interaction between warfarin and human serum albumin studied by electrospray ionization mass spectrometry [J].J Am Soc Mass Spectrom,2008,19(10):1568-1575.
[13] Wang Y L,Yuan J F,Shang W,et al.Dialysis sampling on-line coupled with high-performance liquid chromatography for simultaneous investigation of the interaction between multi-components in herbs and the albumin [J].Analyst,2011,136(4):823-828.
[14] SunS J,Long C J,Tao C Y,et al.Ultrasonic microdialysis coupled with capillary electrophoresis electrochemiluminescence study the interaction between trimetazidine dihydrochloride and human serum albumin [J].Anal Chim Acta,2014,851(2):37-42.
[15] Liang A Y,Liu X J,Du Y G,et al.Further characterization of the binding of heparin to granulocyte colony-stimulating factor:importance of sulfate groups [J].Electrophoresis,2008,29(6):1286-1290.
[16] Kuroda Y,Bo C,Akimasa S,et al.Effect of oxidation of low-density lipoprotein on drug binding affinity studied by high performance frontal analysis-capillary electrophoresis[J].Electrophoresis,2001,22(16):3401-3407.
[17] Shibukawa A ,Yoshikawa Y,Kimura T,et al.Binding study of desethyloxybutynin using high-performance frontal analysis method[J].J Chromatogr B,2002,768(1):189-197.