title: Characterization of a fibrinolytic enzyme (ussurenase) from Agkistrodon blomhoffii ussurensis snake venom: insights into the effects of Ca2+ on function and structure.
abstract: Fibrino(geno)lytic enzymes from snake venoms have been identified as high quality therapeutic agents for treatment of blood clots and strokes. They act on fibrinogen and fibrin, leading to defibrinogenation of blood, lysis of fibrin, and a consequent decrease in blood viscosity. In this work, a fibrinolytic enzyme (ussurenase) from China Agkistrodon blomhoffii Ussurensis snake venom, was purified to homogeneity, identified as a stable 23,367.8 Da monomeric protein, and was identified as a new kind of snake venom metalloproteinase. Ussurenase reacts optimally with fibrin clots at pH 7.5-8.3 and a temperature of 33-41 degrees C. Although many fibrinolytic enzymes are known to be zinc-dependent, measurements from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveal that ussurenase is a Ca2+-containing protein with a molar ratio of 1:1 ([Ca2+]:[enzyme]). Ca2+ is crucial to the fibrin clot hydrolysis by ussurenase but also plays an important role in maintaining the structural integrity of the enzyme. The addition of Ca2+ to the apoenzyme induces a conformational change making the environments surrounding the Trp residues of the enzyme more hydrophobic. The presence of Ca2+ also increases the structural stability of ussurenase, so that higher concentrations of the denaturant guanidine hydrochloride are required to denature the native ussurenase compared to the apo-form. UV absorption and CD spectroscopy experiments show that Ca2+ increases the thermostability and changes the secondary structure of ussurenase. All these data suggest that Ca2+ is crucial for the correct folding and activity of ussurenase.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, Dalian, 116027, China. ming-zhong.sun@case.edu
authors.forename: Ming-Zhong
authors.initials: MZ
authors.lastname: Sun
authors.forename: Shuqing
authors.initials: S
authors.lastname: Liu
authors.forename: Frederick T
authors.initials: FT
authors.lastname: Greenaway
mesh_terms: D017836:Agkistrodon / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000201:enzymology*; D005342:Fibrinolysis; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D008670:Metals; D008666:Metalloendopeptidases; C058789:fibrolase; D002118:Calcium