H/D Exchange Increases the apo AP RTP Lifetime

To assess the effects of metal binding on the AP RTP lifetime and H/D exchange rate, we started studies of apo AP.  The apo protein is prepared by dialyzing against a 2 M ammonium sulfate buffer, and then exchanging into a metal-free EPPS buffer.  As expected, mixing the apo AP into a D2O buffer substantially increases the RTP lifetime.  When we attempted to monitor the kinetics, however, the H/D exchange seemed to occur within the dead time of our mixing both at 20°C and 3°C.  Analysis of the RTP lifetime of apo AP in a roughly equal mixture of H2O and D2O shows a single, intermediate lifetime.  This suggests that the exchange occurs on a time scale much shorter than the RTP lifetime.




Figure 8a, 8b: Lifetime distributions by MEM analysis of the RTP decay curves of apo AP.  The apo AP was mixed into 100 mM pH or pD 8.0 EPPS at 20°C.

H/D Exchange Increases the RTP Lifetime of Zn-AP