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