Biomedical Engineering Reference
In-Depth Information
l , correlates well with increasing
ferrocyanide concentrations. Figure 15.3B shows a quantitative test, in which 1/
shortening of the two exponential time constants,
s and
p (instead
l ) is plotted against the ferrocyanide concentration. Likewise, if an AC signal
is generated by the interfacial proton-transfer mechanism, the law of mass action predicts
that the signal is sensitive to pH because H (or, rather, hydronium ion) is one of the reac-
tants. Since the R 2 signal is generated during the metarhodopsin I to metarhodopsin II
reaction, the R 2 signal is expected to be pH-sensitive. Yet most, if not all, previous investi-
gations did not reveal this expected pH dependence [54]. This constitutes an apparent par-
adox that needs an explanation.
Since bR is a proton pump, interfacial proton transfer at the two surfaces of the purple
membrane is an obligatory process. However, the ERP-like photosignal from bR was also
found to be pH-insensitive [10,56]. The apparent paradox for both rhodopsin and bR can
be explained by the equivalent circuit analysis presented above. As mentioned earlier, the
relaxation of an open-circuit photovoltage reflects mainly the membrane RC relaxation
and is therefore a poor indicator of the intrinsic photokinetic relaxation (Figure 15.4). Since
most previous measurements of the ERP and the ERP-like photosignals in reconstituted
bR membranes were carried out under open-circuit conditions, the apparent lack of pH
dependence is thus more expected than surprising [27].
We subsequently measured the AC photoelectric signal of a reconstituted bR membrane
under near-short-circuit conditions. It was then found that the B 2 component is highly sen-
sitive to pH changes: it is reversibly inhibited by low pH [55]. Comparison of the near-
short-circuit data with the corresponding open-circuit data reveals two important
differences. The B 1 component is smaller than the B 2 component under open-circuit con-
ditions but the opposite is true under near-short-circuit conditions. Furthermore, the
apparent relaxation of the two components is considerably faster under near-short-circuit
conditions than under open-circuit conditions. That is, the time course of the AC photo-
signal varies with the change of measurement conditions. Conventional approaches offer
no satisfactory explanation of these kinetic features. In contrast, the equivalent circuit
analysis presented above predicts these features [27]. Similar observations in a reconsti-
tuted rhodopsin membrane were reported by Ostrovsky and coworkers [57,58], when the
membrane was short-circuited by shunting. The latter observation suggests that similar
photoelectric phenomena appear in reconstituted bR and rhodopsin membranes.
of 1/
s or 1/
15.4.2
Component Analysis
Semiquantitative investigations of the AC photosignal in reconstituted bR membranes indi-
cate that it is a composite signal comprising at least two separate chemical processes: one
for B 1 and another for B 2 [55]. The test of the prediction of the equivalent circuit as applied
to bR data requires an unequivocal decomposition of the AC signal into several (pure) com-
ponents for the following reasons. The equivalent circuit was derived for a single chemical
relaxation process, such as the case of Mg porphyrin membrane. For a single relaxation
chemical process, there are no freely adjustable parameters in the equivalent circuit because
all input parameters for the computation can be determined experimentally. If, however,
there are two or more chemical processes, two or more separate sets of parameters must be
used, one set for each process. These parameters can no longer be uniquely determined by
direct experimental measurements. Investigators often turned to curve fitting by trial and
error to get the best fit. This procedure appeared to be less satisfactory than for the case of
single relaxation because it is tantamount to the introduction of one or more adjustable
parameters, which determine how these signal components are to be decomposed. An
agreement between the equivalent circuit and the measured(composite) signal would be
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