Metal-ion- and pH-induced conformational changes of acutolysin D from Agkistrodon acutus venom probed by fluorescent spectroscopy.
Acutolysin D isolated from the venom of Agkistrodon acutus is a protein of 44 kDa with marked hemorrhagic and proteolytic activities. The metal-ion- and pH-induced conformational changes of acutolysin D have been studied by following fluorescence and activity measurements. Here we provide evidence for the fact that native holo-acutolysin D adopts two different conformations, native state a, stable in the weak acidic pH range from 5.5 to 7.0 with low activity, and native state b, stable in the weak alkaline pH range from 8.0 to 9.0 with high activity. Holo-acutolysin D has an optimum pH of 9.0 for caseinolytic activity and a maximum fluorescence at pH 9.0. The protein adopts the most stable conformation at pH 9.0. The addition of 1 mM Zn(2+) shifts both the alkali-induced unfolding transition curve and the alkali-induced inactivation curve toward higher pH value but has little effect on the acid-induced unfolding transition curve. No obvious effects on the pH-induced unfolding transition curve and the pH-dependent activity curve have been observed after the addition of 1 mM Ca(2+) to holo-acutolysin D. The results indicate that Zn(2+) is essential for its CA, while Ca(2+) is not essential for its CA. Removal of Ca(2+) and Zn(2+) from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. The kinetic results of the demetalization of holo-acutolysin D show that the demetalization rate constant K(1) for a slower reaction linearly decreases with the pH increase from 5.0 to 9.0, while K(2) for the faster reaction linearly increases with the pH change from 5.0 to 7.0. It is also evident from the present work that the free Zn(2+)-induced inactivation in the pH range from 8.0 to 9.0 should be attributed to the effect of Zn(OH)(2) precipitation on the protein.
Xu
Xiao-Long
XL
Department of Chemistry, University of Science and Technology of China, Hefei, 230026, People's Republic of China.
Liu
Xiang-Hu
XH
Wu
Bin
B
Liu
Yi
Y
Liu
Wen-Qi
WQ
Xie
Yong-Shu
YS
Liu
Qing-Liang
QL
D017836:Agkistrodon; D000818:Animals; D002412:Cations; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D008670:Metals; D011487:Protein Conformation; D012910:Snake Venoms / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
D002412:Cations; D003435:Crotalid Venoms; D008670:Metals; D012910:Snake Venoms; C488502:acutolysin D, Agkistrodon; D014443:Tyrosine; D014364:Tryptophan; D008666:Metalloendopeptidases