A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): purification and characterization.
A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N(alpha)-tosyl-l-arginine-methyl ester (TAME) at approximately pH 7.5 and at 51 degrees C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn(2+)-containing protein with a stoichiometry of 1:1 [Zn(2+)]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn(2+) plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca(2+), Mg(2+), Cu(2+), Ni(2+), Mn(2+), and Co(2+), increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.
Liu
Shuqing
S
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, Liaoning 116027, China.
Sun
Ming-Zhong
MZ
Greenaway
Frederick T
FT
D017836:Agkistrodon; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D010960:Plasminogen Activators / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020836:Protein Structure, Quaternary; D040901:Proteomics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D015032:Zinc / Q000032:analysis / Q000494:pharmacology
D003435:Crotalid Venoms; D008670:Metals; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases; D010960:Plasminogen Activators; C514126:plasminogen activator, Agkistrodon blomhoffii Ussurensis; D015032:Zinc