H/D Exchange Increases the RTP Lifetime of Zn-AP
WT AP with a saturating concentration of Zn has a long RTP
lifetime of about 1.1-1.2 seconds at 20°C. Thus, our expectation
was that this protein would have a slow H/D exchange rate. However,
upon mixing into a D2O buffer, the RTP lifetime was observed
to increase immediately. Even when the temperature was reduced to 3°C,
the H/D exchange appears to occur within the dead time of our mixing procedure.
A verification of this rapid exchange is the fact that the protein in a
roughly equal mixture of H2O and D2O shows a single,
intermediate RTP lifetime. By contrast, holo AP has a biphasic decay.
Figure 9: Lifetime distributions by MEM analysis of the RTP decay
curves of apo AP after incubation with 100 µM ZnCl2.
The AP was mixed into 100 mM pH or pD 8.0 EPPS
plus 100 µM ZnCl2 at 20°C.
Conclusions