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Fig. 11.20 The fluctuations of the fluorescence intensity of the cholesterol oxidase (COx)
molecule in its oxidized and reduced states adopted from Fig. 1b in Lu et al. (1998). It is postulated
here that the oxidized form of COx fluctuates between the fluorescence levels A and B, while the
reduced form of the enzyme fluctuates between the florescence levels C and D. The four
lines
decline with time more or less in parallel, most likely due to light-induced structural damages
suffered by the protein surrounding the fluorophore, which in turn alter the energy levels of the
fluorophore, affecting the fluorescence efficiencies of both the oxidized and reduced form of COx.
The average fluorescence intensity of the oxidized form of COx decreases from about 120 ct/ch
(counts per channel) to 90 ct/ch in 22 s, while that of the reduced form decreases from about 60 to
40 ct/ch (in the same time period) which is near the background fluorescence. In addition to the
fluorescence fluctuations occurring between A and B and between C and D, there are other
fluorescence intensity transitions taking place from B to C and from C to B. See the region
indicated by E (which is partially hidden) between B and C at around 8 s. Since there are four
fluorescence states of COx, there are 4
4
¼
16 possible transitions as shown in Table
11.7
. Due
to the diagonal symmetry of the table, there are only six distinct transitions whose probabilities of
occurrences need not be equal. The relative transition probabilities predicted on the basis of the
Table 11.6 The generalized Franck-Condon-principle-based explanations for the single-mole-
cule measurements of the cholesterol oxidase enzymic activities reported by Lu et al. (1998). A, B,
C, and D refer to the fluorescence levels defined in Fig.
11.20
Single-molecule measurements
on cholesterol oxidase (see
Figs.
11.17
and
11.18
)
Explanations offered by the Franck-Condon-principle-
based mechanism of action of COx (see Fig.
11.19
)
1. There are two kinds of
fluorescence fluctuations -
one averaging around 100
ct/ch and the other around
50 ct/ch
The fluorescence intensity of both oxidized and reduced
forms of COx fluctuates. The time-averaged
fluorescence intensity of the oxidized form of the
COx molecule is approximately 100 ct/ch, while the
corresponding value for the reduced form is around
50 ct/ch
2. Each form of the COx
molecule fluctuates between a
high and low fluorescence
level, giving rise to four
fluorescence levels denoted as
A, B, C, and D in Fig.
11.20
The molecular species responsible for the various
fluorescence levels are postulated to be as follows:
A
¼
E-FAD
B
¼
E-FAD* and E-FAD*•S
C
¼
E-FADH
2
*•P and E-FADH
2
*
D
¼
E-FADH
2
3. There are two kinds of
fluorescence transitions - (1)
from level B to level C,
and (2) from level C to level
B, both taking place almost
instantaneously compared
to the on- and off-times
(Fig.
11.17
)
(1) The transition from B to C passes through and is
rate-determined by the free energy level of the
Franck-Condon state [E -FAD
{
•S
$
E-FADH
2
{
•P]
(2) The transition from C to B passes through and is
rate-determined by the free energy level of the
Franck-Condon state [E-FADH
2
{
•O
2
$
E-FAD
{
•H
2
O
2
]
