J. Biol. Chem., Vol. 262, Issue 23, 10907-10910, Aug, 1987
Diffusion coefficients of quenchers in proteins from transient effects in the intensity decays
JR Lakowicz, NB Joshi, ML Johnson, H Szmacinski and I Gryczynski
We used 2-GHz frequency-domain fluorometry to examine the intensity decays
of N-acetyl-L-tryptophamide (NATA) and the protein staphylococcal nuclease
in the presence and absence of quenching by oxygen or acrylamide. When
analyzed with a multiexponential model, the decays of NATA and nuclease
both become more heterogeneous in the presence of quenching. We attribute
the increased complexity to transient effects in quenching or equivalently
a time-dependent rate constant for quenching. The frequency-domain data
were analyzed using the Smoluchowski model (exp(-t/tau-2b square root t))
and the radiation model, which is known to correct some flaws in the more
approximate Smoluchowski model. The radiation model provides improved fits
to the data, as evidenced by average 10-fold decreases in chi R2. The
radiation model also provides an estimate of the sum of the diffusion
coefficients and the specific rate constant for quenching. The apparent
diffusion coefficients for acrylamide and oxygen in nuclease, as seen by
its single tryptophan (residue 140) are 15- and 11-fold lower than in
water, respectively. The apparent values of the oxygen diffusion
coefficient in water, as seen by NATA, are 2- to 3-fold larger than
expected from earlier steady-state measurements. The ability to recover the
detailed form of the intensity decays by the frequency-domain method should
allow comparison of experimental results with calculated trajectories of
quenchers in proteins.
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