Experimental Results

The experiments are conducted by first simultaneously deoxygenating 250 ml of buffer and 10 ml of concentrated protein physically separated from each other in the same cuvette. After allowing for the samples to equilibrate to the appropriate temperature the buffer and protein are mixed together and the subsequent phosphorescence of the protein is monitored as a function of time. The final protein concentration is between 0.1 and 0.2 mg/ml and the buffer used for all proteins was 100 mM TRIS with an additional 1 mM Na₂HPO₄, 0.1 mM MgCl, 0.1 mM ZnCl. The H₂O buffer was at pH 8.0 (20 °C) and the D₂O buffer was at pD 8.0 (20 °C).

Wild-type (WT), Q320L and Q320G all exhibit monoexponential RTP decays when in equilibrium in H₂O buffer or when exchanged into H₂O buffer through the process described above.

Upon exchange into D₂O buffer, however, the RTP decays of all proteins are initially biphashic [Figure 3] and then become more monoexponential over time; the average RTP lifetime is always greater than that of the protein in H₂O buffer. Analysis of the two observed lifetime components reveals that the shorter component is very close to the monoexponential RTP lifetime of the protein in H₂O buffer [Figure 3] and the longer component is similar to the asymptote of the average RTP lifetime. The relative populations of these two components change as a function of time after the mixing and follow kinetics similar to those of the average RTP lifetimes.

D₂O Exchange With WT and Glutamine Mutants