Biomedical Engineering Reference
In-Depth Information
among others, in concentrations ranging from 1 to 50,000 ppm. A control was set up for
each sample, consisting of an equivalent volume of water added to the protein solution as
a substitute for the chemical of interest. Figure 14.9 shows typical M- and O-state datasets
derived from the wild-type protein, as a function of increasing concentration of a variety
of amine compounds. The raw O-state data shown in Figure 14.9 was collected at 650 nm
by the flash-photolysis prototype, and is typical for wild-type BR. Data collected for the M
state appears very similar, except the protein is monitored at 450 nm and the time con-
stants are generally shorter. Trends typical for the M-state lifetime as a function of several
chemical additives are also shown. The ability of the chemical to modulate the protein's
photokinetic response is clearly illustrated, and similar trends were seen for each of the
1.7
Wild-type BR O-state photokinetics with
varying concentrations of 1,2-diaminopropane
1.5
1.4
0 ppm
3 ppm
10 ppm
20 ppm
50 ppm
100 ppm
250 ppm
500 ppm
1000 ppm
1.2
1.1
0.9
0.8
0.6
0.5
0.3
0.1
0.0
0
4
8
12
16
20
24
(a)
Time (ms)
8
Variation of wild-type BR M-state decay time constant
as a function of chemical additive concentration
7
6
TC-H20
TC-1,4DAB
TC-1,2DAP
TC-TEA
TC-TEMED
5
4
3
2
1
0
0
1
2
3
4
5
6
(b)
Log (concentration/
µ
M)
FIGURE 14.9
(a) Wild-type BR photokinetics as a function of the concentration of 1,2-diaminopropane (1,2-DAP). A series of
concentrations of 1,2-DAP were prepared in a cuvette containing wild-type BR (~1.5 OD), and flash-photolysis
experiments were performed on the resulting samples to monitor the photokinetic response of the O state.
Similar photokinetic traces were collected for the M state. The rise and decay constants were determined for both
states at each concentration of 1,2-DAP. (b) The data shown in the lower panel were compiled in a plot as for the
wild-type M state, where the M decay time constant is plotted as a function of concentration for a variety of
chemicals: 1,4-diaminobutane (1,4-DAB), 1,2-DAP, triethylamine (TEA), and
N
,
N
,
N
′
,
N
′
-tetramethylethylenedi-
amine (TEMED). Water was used as a control (solid line).
