Characterization by NMR and Fluorescence Spectroscopy of Differences in the Conformation of Non-Aged and Aged Organophosphoryl Conjugates of AChE
Abstract
The objective of this project is to characterize, by nmr and by fluorescence spectroscopy, differences in the conformations of non-aged and aged organophosphoryl conjugates of acetylcholinesterase (AChE) and chymotrypsin (Cht). In the present study, O-l-pyrenebutyl O-ethylphosphorofluoridate (PBEPF) and O-l-pyrenebutyl phosphorodichloridate (PBPDC) were used to obtain the non- aged and aged conjugates of Cht respectively. 31P-nmr spectroscopy and reactivation studies support the hypothesis that the aged form, pyrenebutyl-O- P(O) (OH)Cht, contains a P-O(-) function. Significant conformational differences between the aged conjugate and the non-aged conjugate, pyrenebutyl-O-P(O) (oc2H5)Cht, were indicated by the optical spectroscopic results (steady-state and time-resolved fluorescence, circular dichroism (CD) and circularly polarized luminiscence (CPL) measurements). The interaction of the fluorophore with the protein backbone appears to be stronger for the aged conjugate than for the non- aged phosphorylated enzyme. Two conclusions have been derived so far: (a) The dihalide organophosphates employed are suitable for preparing aged organophosphoryl conjugates of Cht (and presumably for other serine hydrolases) which can be compared with the corresponding non-aged conjugates. (b) The spectroscopic data provide a partial interpretation for the known resistance of aged conjugates to reactivation.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 10, 1984
- Accession Number
- ADB092850
Entities
People
- I. Silman
- Y. Ashani
Organizations
- Israel Institute for Biological Research