Diffusion coefficients of quenchers in proteins from transient effects in the intensity decays

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.