New fluorescent organophosphates as probes for studying aging-induced conformational changes in inhibited acetylcholinesterase.
Aging of organophosphoryl-acetylcholinesterase (AChE) conjugates, involving dealkylation of the bound organophosphoryl group, renders AChE resistant to reactivation by 2-pyridinealdoxime methiodide (2-PAM). The fluorescent organophosphates 1-pyrenebutyl ethylphosphorochloridate (PBEPC) and 1-pyrenebutylphosphorodichloridate (PBPDC) react stoichiometrically with purified electric eel AChE. PBEPC forms a non-aged AChE conjugate readily reactivated by 2-PAM; PBPDC forms an aged conjugate which cannot be reactivated. There is no difference in the wavelengths of excitation and emission maxima between the aged and non-aged AChE conjugates. However, the fluorescence quantum yield of pyrene in the non-aged conjugate is reduced by ca. 50% compared to the aged conjugate and from the shortening of the fluorescence decay time in the non-aged conjugate, it is concluded that the quenching is primarily dynamic. It is suggested that in the aged conjugate the organophosphoryl moiety is less accessible to the external medium than in the non-aged conjugate.
Amitai
G
G
Israel Institute for Biological Research, Ness-Ziona, Israel.
Ashani
Y
Y
Gafni
A
A
Silman
I
I