Metal ions- and pH-induced conformational changes of acutolysin A from Agkistrodon acutus venom probed by fluorescent spectroscopy.
Acutolysin A isolated from the venom of Agkistrodon acutus is a protein of 22 kDa with marked haemorrhagic and proteolytic activities. The metal ions- and pH-induced conformational changes of acutolysin A have been studied by following fluorescence and activity measurements. Here, we provide evidence for the fact that native holo-acutolysin A adopts two subtly different conformations, native state a (Na) stable in the weak acidic pH range from 6.0 to 7.0 with low activity and native state b (Nb) stable in the weak alkaline pH range from 7.5 to 9.0 with high activity. Holo-acutolysin A has an optimum pH of 8.5 for caseinolytic activity, and the protein adopts the most stable conformation with the maximum fluorescence at pH 8.5. The Ca2+ and Zn2+ ions have significant effects on both the pH-induced denaturing transition curve and the pH-dependent activity curve. Addition of 1 mM Ca2+ to holo-acutolysin A shifts both the acid-induced denaturing transition curve and the end zone of acid-induced inactivation curve towards lower pH value, and shifts both the alkali-induced denaturing transition curve and the end zone of alkali-induced inactivation curve towards higher pH value. Addition of 1 mM Zn2+ also shifts both the alkali-induced denaturing transition curve and the end zone of alkali-induced inactivation curve towards higher pH value and shifts the acid-induced denaturing transition curve to lower pH value, but has little effect on the acid-induced inactivation. Removal of Ca2+ and Zn2+ from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. It is also evident from the present work that the free Zn2+ -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
Xiaolong
X
Department of Chemistry, University of Science and Technology of China, Hefei 230026, People's Republic of China. xuxl@ustc.edu.cn
Chen
Jiexia
J
Zhang
Liyun
L
Liu
Qingliang
Q
D017836:Agkistrodon; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002412:Cations; D003435:Crotalid Venoms / Q000737:chemistry*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000737:chemistry*
D002412:Cations; D003435:Crotalid Venoms; D008666:Metalloendopeptidases; C115407:acutolysin A; D015032:Zinc; D002118:Calcium