Purification and characterization of novel fibrin(ogen)olytic protease from Curcuma aromatica Salisb.: Role in hemostasis.
BACKGROUND
The proteases from turmeric species have procoagulant and fibrinogenolytic activity. This provides a scientific basis for traditional use of turmeric to stop bleeding and promote wound healing processes.


OBJECTIVE
Our previous studies revealed that fibrinogenolytic action of crude enzyme fraction of Curcuma aromatica Salisb., was found to be more influential than those of Curcuma longa L., Curcuma caesia Roxb., Curcuma amada Roxb. and Curcuma zedoria (Christm.) Roscoe. Hence, the purpose of this study is to purify and characterize protease from C. aromatica and to explore its role in wound healing process.


METHODS
The protease was purified by Sephadex G-50 gel permeation chromatography. Peak with potent proteolytic activity was subjected to rechromatography and then checked for homogeneity by SDS-PAGE and native PAGE. Furthermore purity of the peak was assessed by RP-HPLC and MALDI-TOF. The biochemical properties, type of protease, kinetic studies, fibrinogenolytic, coagulant and fibrinolytic activities were carried out.


RESULTS
The two proteolytic peaks were fractionated in gel permeation chromatography. Among these, the peak-II showed potent proteolytic activity with specific activity of 10units/mg/min and named as C. aromatica protease-II (CAP-II). This protein resolved into a single sharp band both in SDS-PAGE (reducing and non-reducing) as well as in native (acidic) PAGE. It is a monomeric protein, showing sharp peak in RP-HPLC and its relative molecular mass was found to be 12.378kDa. The caseinolytic and fibrinolytic activity of CAP-II was completely inhibited by phenylmethylsulfonylfluoride (PMSF). The CAP-II exhibited optimum temperature of 45°C and optimum pH of 7.5. The Km and Vmax of CAP-II was found to be 1.616µg and 1.62units/mg/min respectively. The CAP-II showed hydrolysis of all three subunits of fibrinogen in the order Aα>Bß>γ. The CAP-II exhibited strong procoagulant activity by reducing the human plasma clotting time. It also showed fibrinolytic activity by complete hydrolysis of α-polymer and γ-γ dimer present in fibrin.


CONCLUSIONS
The CAP-II is a novel serine protease from C. aromatica, which has been demonstrated to stop bleeding and initiate wound healing through its procoagulant and fibrin(ogen)olytic activities. Our study demonstrates the possible role of CAP-II, as therapeutic enzyme to stop bleeding at the time of wounding.
Department of Biotechnology, JSS Science and Technology University, JSS Technical Institutions Campus, Mysuru-570 006, Karnataka, India; JSS Research Foundation, JSS Technical Institutions Campus, Mysuru-570 006, Karnataka, India; Section of Plant Breeding and Genetics, Coffee Research Sub-Station, Chettalli-571248, Kodagu, Karnataka, India.
B R
BR
Shivalingu
Department of Biotechnology, JSS Science and Technology University, JSS Technical Institutions Campus, Mysuru-570 006, Karnataka, India.
H K
HK
Vivek
Department of Studies in Chemistry, University of Mysore, Mysuru-570 006, Karnataka, India.
B S
BS
Priya
School of Ecology and Conservation, University of Agricultural Sciences, GKVK, Bengaluru-560065, Karnataka, India.
K N
KN
Soujanya
Department of Biotechnology, JSS Science and Technology University, JSS Technical Institutions Campus, Mysuru-570 006, Karnataka, India. Electronic address: nanju_chem@yahoo.com.
S Nanjunda
SN
Swamy
D002851:Chromatography, High Pressure Liquid; D003029:Coagulants / Q000494:pharmacology; D030024:Curcuma / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006487:Hemostasis / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D057057:Serine Proteases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D014945:Wound Healing / Q000187:drug effects
D003029:Coagulants; D005337:Fibrin; D005340:Fibrinogen; D057057:Serine Proteases
Curcuma aromatica Salisb.; Curcuma aromatica protease-II(CAP-II); Fibrinolysis; Hemostasis; Procoagulant