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Proceedings Paper

Transient Effects In The Acrylamide Quenching Of Single Tryptophan Proteins, Observed Using Frequency-Domain Fluorometry
Author(s): Joseph R Lakowicz; Michael L Johnson; Ignacy Gryczynski; Henryk Szmacinski; N Joshi; G Laczko
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Paper Abstract

We used GHz frequency-domain fluorometry to examine the tryptophan intensity decays of NATA (M-acetyl-L-tryptophanamide), gly-trp-gly, and the single tryptophan proteins ACTH, E. nuclease and ribonuclease T, (Rhase 1%). In all cases the intensity decays became more heterogeneous in the presence of quenching, which we attribute to a time-dependent rate constant for quenching (transient effects). The frequency-domain data were analyzed using the Smoluchowski model (exp(-t/t 2b/T)) and the radiation boundary condition (RBC) model. In contrast to the IT model, the RBC model does not assume the fluorophore-quencher pair is immediately deactivated, but rather assumes a rate constant for deactivation of the pair (K) as well as a mutual diffusion coefficient (D). The RBC model provides dramatically improved fits to the data. The values of both D and decreases progressively in the order listed above, which is with decreasing exposure to the aqueous phase. Because the RBC model may not be strictly correct in homogeneous solution, and probably less so in the hindered anisotropic environment of the proteins, the recovered values of D and IC should be regarded as apparent values. The recovered intensity decays can be compared with molecular dynamic calculations of quencher trajectories in proteins.

Paper Details

Date Published: 24 June 1988
PDF: 8 pages
Proc. SPIE 0909, Time-Resolved Laser Spectroscopy in Biochemistry, (24 June 1988); doi: 10.1117/12.945383
Show Author Affiliations
Joseph R Lakowicz, University of Maryland at Baltimore (United States)
Michael L Johnson, University of Virginia (United States)
Ignacy Gryczynski, University of Maryland at Baltimore (United States)
Henryk Szmacinski, University of Maryland at Baltimore (United States)
N Joshi, University of Maryland at Baltimore (United States)
G Laczko, University of Maryland at Baltimore (United States)

Published in SPIE Proceedings Vol. 0909:
Time-Resolved Laser Spectroscopy in Biochemistry
Joseph R. Lakowicz, Editor(s)

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