Conformational Differences between Aged and Nonaged Pyrenebutyl-Containing Organophosphoryl Conjugates of Chymotrypsin As Detected by Optical Spectroscopy

N. Steinberg, A. C.M. van der Drift, J. Grunwald, Y. Segall, E. Shirin, E. Haas, Y. Ashani, I. Silman

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

Homologous aged and nonaged fluorescent organophosphorus conjugates of α-chymotrypsin (Cht) were used in a comparative spectroscopic study of the conformation of their active sites, employingthe pyrene group as the fluorescent probe. Steady-state fluorescence measurements showed that the quantumyield of the pyrene probe which is stoichiometrically attached to the active site is ca. 20% lower in the aged conjugate, pyrenebutyl-O-P(O)(O~)-Cht (PBP-Cht), than in the nonaged conjugate, pyrenebutyl-O-P-(O-)(OC2H5)-Cht (PBEP-Cht). furthermore, fluorescence decay data indicate that quenching is dynamic and is not caused by oxygen. These data, together with collisional quenching data, imply that quenching originates in an internal interaction of the fluorophore with a group within the protein. Thus, of the pyrene moiety with the polypeptide chain is significantly stronger in the aged than in the nonaged conjugate, implying a different orientation of the fluorophore with respect to the protein. Circular dichroism measurements, which reflect the asymmetry of the bound pyrene in the ground state, as well as circularly polarized luminescence studies, which reflect its asymmetry in the excited state, also show that the relative configuration of the pyrene moiety and the polypeptide chain is significantly altered upon aging. Aged conjugates obtained by use of various fluorescent organophosphates [pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(P-nitrophenoxy)Cl, pyrenebutyl-O-P(O)(P-nitrophenoxy)2] exhibit similar spectroscopic features, thus substantiating the hypothesis that instantaneous aging, by use of pyrenebutyl-O-P(O) Cl2, and dynamic aging, by gradual removal of an aryloxy group, yield a similar product. This finding provides strong support for the formation of a P-O-moiety in the aged conjugates, since the only expected common product of the two processes is PB-O-P(O)(O-)-Cht. Formation of excimers of the pyrene-containing organophosphorylchymotrypsin conjugates at concentrations above 3 × 10-6 M is also reported.

Original languageEnglish
Pages (from-to)1248-1253
Number of pages6
JournalBiochemistry
Volume28
Issue number3
DOIs
StatePublished - 7 Feb 1989

Fingerprint

Dive into the research topics of 'Conformational Differences between Aged and Nonaged Pyrenebutyl-Containing Organophosphoryl Conjugates of Chymotrypsin As Detected by Optical Spectroscopy'. Together they form a unique fingerprint.

Cite this