Characterization by NMR and Fluorescence Spectroscopy of Differences in the Conformation of Non-Aged and Aged Organophosphoryl Conjugates of AChE
Annual summary rept. 1 Sep 1983-31 Aug 1984
ISRAEL INST FOR BIOLOGICAL RESEARCH NESS ZIONA
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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- PO OHCht, contains a P-O- function. Significant conformational differences between the aged conjugate and the non-aged conjugate, pyrenebutyl-O-PO oc2H5Cht, 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.
- Organic Chemistry
- Atomic and Molecular Physics and Spectroscopy