Transient Effects In The Acrylamide Quenching Of Single Tryptophan Proteins, Observed Using Frequency-Domain Fluorometry

Joseph R Lakowicz; Michael L Johnson; Ignacy Gryczynski; Henryk Szmacinski; N Joshi; G Laczko

doi:10.1117/12.945383

24 June 1988 Transient Effects In The Acrylamide Quenching Of Single Tryptophan Proteins, Observed Using Frequency-Domain Fluorometry

Joseph R Lakowicz, Michael L Johnson, Ignacy Gryczynski, Henryk Szmacinski, N Joshi, G Laczko

Author Affiliations +

Proceedings Volume 0909, Time-Resolved Laser Spectroscopy in Biochemistry; (1988) https://doi.org/10.1117/12.945383
Event: 1988 Los Angeles Symposium: O-E/LASE '88, 1988, Los Angeles, CA, United States

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.

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Joseph R Lakowicz, Michael L Johnson, Ignacy Gryczynski, Henryk Szmacinski, N Joshi, and G Laczko "Transient Effects In The Acrylamide Quenching Of Single Tryptophan Proteins, Observed Using Frequency-Domain Fluorometry", Proc. SPIE 0909, Time-Resolved Laser Spectroscopy in Biochemistry, (24 June 1988); https://doi.org/10.1117/12.945383

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