Effect of Factors on Pepsin and Trypsin Fibrinolytic Activity and Deactivation of Fibrinolytic Activity

WANG Zheng-jun1,2 LI Bo1,2,3,4 GUO Li-wei1,2 WU Mian-hua3,4 ZHU Hua-xu1,2

(1.Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing, China 210023)
(2.Key Laboratory of Separation Engineering for Chinese Medicine Compound, Nanjing University of Chinese Medicine, Nanjing, China 210029)
(3.The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, China 210046)
(4.Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing University of Chinese Medicine, Nanjing, China 210023)

【Abstract】 Objective To investigate the method for study on the effect of factors on pepsin and trypsin fibrinolytic activity and deactivation of fibrinolytic activity and to eliminate the interference of pepsin and trypsin on the detection of crude protein fibrinolytic activity of Armadillidium vulgare (porcellio plasmin) in order to obtain the proteins or peptides which have the smaller molecular weight but higher titer during the pepsin and trypsin degradation. Methods To study the effect of pepsin and trypsin deactivation on pH value, temperature, metal ions, enzyme inhibitor, surfactant, and responsing fibrinolytic, the fiber fibrin plate assay was usede. The better enzyme deactivation process was obtained and used for studying the effect on the fibrinolytic activity of urokinase, lumbrokinase, and porcellio plasmin. Results All the pH value, temperature, metal ions, enzyme inhibitor, and surfactant have had an impact on pepsin and trypsin fibrinolytic activity. Among them the optimum deactivation of pepsin was pH 6.0–8.0, while the optimum deactivation of trypsin was mixed preparation with TLCK at the concentration of 25 mg/mL and EDTA at the concentration of 1 mmol/L. Conclusion This study has obtained the better enzyme deactivation process which could be used for the detection of fibrinolytic activity of pepsin and trypsin degradation product by fiber fibrin plate assay; the operation is simple, and the repeatability and stability are good.

【Keywords】 pepsin; trypsin; fibrinolytic activity; fiber fibrin plate assay; enzyme deactivation; urokinase; lumbrokinase; porcellio plasmin;

【DOI】

【Funds】 National Natural Science Foundation of China (81274096, 81303230) Innovation Fund of Industry-University-Research Cooperation of Jiangsu Science and Technology Department-Prospective Research Project (BY2012036) Science and Technology Supportive Plan of Jiangsu—Social Development Project (BE-2012763) China Postdoctoral Science Foundation (2013M541704) Jiangsu Postdoctoral Science Foundation (1301133C)

Download this article

    References

    [1]Han YL, Li ZW. Journal of Chinese Medicinal Materials, 2006, 29 (8): 765-767 (in Chinese).

    [2]Xu WB, Wang YR, Huang NT. Journal of Beijing University of Traditional Chinese Medicine, 2010, 33(2): 127-129 (in Chinese).

    [3]Qing Q B, Jin X C, Yi L F, et al. Purification and characterization of a new serine protease with fibrinolytic activity from the Marine Invertebrate, Urechis unicinctus[J]. Appl Biochem Biotechnol, 2013, 170(3): 525-540.

    [4] Zhang HZ, Wen YL. 动物活性成分化学[M]. Tianjin: Tianjin Science and Technology Press, 1995 (in Chinese).

    [5] Wang XC. Chinese Journal of Zoology, 2002, 37(3): 88-91 (in Chinese).

    [6] Wang JH. Modern Journal of Integrated Chinese Traditional and Western Medicine, 2003, 12(15): 1662-1663 (in Chinese).

    [7] Liu YQ, Hong L, Wu HM, et al. Journal of Tropical Medicine, 2003, 3(4): 484-488 (in Chinese).

    [8]Wang B J, Won S J, Yu Z R, et al. Free radical scavenging and apoptotic effects of Cordyceps sinensis fractioned by supercritical carbon dioxide[J]. Food Chem Toxicol, 2005, 43(4): 543-552.

    [9]Wang F, Wang C, Li M, et al. Purification characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia foetida[J]. Biotechnol Lett, 2003, 25: 1105-1109.

    [10]Zhao R, Ji J G, Tong Y P, et al. Isolation and Identification of proteins with anti-tumor and fibrinolysogen kinase activities from Eisenia foetida[J]. Acta Bioch Bioph Sin, 2002, 34(5): 576-582.

    [11]Tian Z, Li B, Guo L W, et al. Purification and biochemical characterization of a novel fibrinolytic enzyme, PSLTro01, from a medicinal animal Porcellio scaber Latreille[J]. Int J Biol Macromol, 2015, 80: 536-546.

    [12] Tong GL. 实用生物化学与分子生物学词典 [M]. Beijing: Science Press, 2003 (in Chinese).

    [13]Rawlings N D, Barrett A J. Families of serine peptidases[J]. Methods Enzymol, 1994, 244: 19-61.

    [14]Mann M, Hendrickson R C, Pandey A. Analysis of proteins and proteomes by mass spectrometry[J]. AnnuRev Biochem, 2001, 70: 437-473.

    [15]Uesugi Y, Usuki H, Iwabuchi M, et al. Highly potent fibrinolytic serine protease from Streptomyces[J]. Enzyme Microb Technol, 2011, 48(1): 7-12.

    [16]Hellebrekers B W J, Trimbos-Kemper T C M, Trimbos J B M Z, et al. Use of fibrinolytic agents in the prevention of postoperative adhesion formation[J]. Fertil Steril, 2000, 74(2): 203-212.

    [17]Ktari N, Ben-Khaled H, Nasri R, et al. Trypsin from zebra blenny(Salaria basilisca)viscera: Purification, characterisation and potential application as a detergent additive[J]. Food Chem, 2012, 130(3): 467-474.

    [18]Ana G V, Juan C R, Elisa M V, et al. Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization[J]. Food Chem, 2013, 141(2): 940-945.

    [19]Khangembam B K, Chakrabarti R. Trypsin from the digestive system of carp Cirrhinus mrigala: Purification, characterization and its potential application[J]. FoodChem, 2015, 175: 386-394.

    [20]Klomklao S, Kishimura H, Yabe M, et al. Purification and characterization of two pepsins from the stomach of pectoral rattail(Coryphaenoides pectoralis)[J]. Compd Biochem Physiol Part B: Biochem Mol Biol, 2007, 147(4): 682-689.

    [21]Meridor D, Gedanken A. Enhanced activity of immobilized pepsin nanoparticles coated on solid substrates compared to free pepsin[J]. Enzyme MicrobTechnol, 2014, 67: 67-76.

    [22] 中国药典[S]. Volume One. 2010 (in Chinese).

    [23]Astrup T, Müllertz S. The fibrin plate method for estimating fibrinolytic activity[J]. Arch Biochem Biophys, 1952, 40(2): 346-351.

This Article

ISSN:0253-2670

CN: 12-1108/R

Vol 47, No. 01, Pages 46-56

January 2016

Downloads:1

Share
Article Outline

Abstract

  • 1 Instruments and materials
  • 2 Methods and result
  • 3 Discussion
  • References