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10.1002/cbic.201800071
ChemBioChem
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E+/E+ ratios across independent experiments performed
under the same conditions. This may be due to conversion
of unreacted HE to E+ during filtration performed for NMR
measurement (see supporting information). Therefore, the
ratio of 2-OH-E+/E+ was determined using HPLC by directly
injecting the reaction mixture for analysis. As a control, HE
was incubated in potassium phosphate buffer (pH 7.8) in
presence of oxygen and absence of enzyme, which resulted
Next, we investigated the effect of different co-factors on
yield of the reaction. NADP+ in the Q/QR system was found
to generate O2•– with better efficiency, as indicated by a ratio
of 69/31 and 58/42 for
5/4 (Table 1, entry 4, 5). Although
NADH is preferred by NfsB_his over NADPH,[26] glucose
dehydrogenase used for regeneration of cofactors might be
•–
accountable for higher O2 generation in Q/QR system.
Since quinone is recycled in the system via a redox cycle (as
discussed earlier), the amount of quinone is also crucial for
in the formation of 2-OH-E+ and E+ in a ratio of 32/68 (
5/4)
(Table S1, entry 1, Supporting Information). Similar
•–
efficient O2 generation. By varying the amount of lawsone
observations have been reported by other laboratories which
(0.1, 0.2 and 0.4 equivalents) in the system, it was found that
0.2 equivalents of lawsone were sufficient for efficient O2
•–
•–
used
3
for O2•– detection.[35] Nevertheless, formation of O2
in the control experiment might be due to presence of metal
ions in buffer (Table S1, entry 4,5 & 6; Supporting
Information). In addition, when lawsone and NADPH
(generated in situ using NADP+, glucose and GDH) were
incubated with HE in the absence of NfsB_his, the ratio of
generation; this was indicated by ratios of 52/48, 67/33 and
53/47 for OH-E+/E+ (Table 1, entry 6, 7, 8). Use of 0.1
equivalents of
1 in the system lowers the efficiency of the
•–
system to generate O2 due to the requirement of an
increased number of cycles. On the other hand, addition of
0.4 equivalents of quinone probably led to inactivation of
enzymatic activity, due to the production of semiquinone
radical over a period of time. In addition, the system was also
5/4 remained the same, indicating the essential role of
quinone reductase in O2•– formation in the system.
•–
Optimization of reaction conditions using lawsone as
substrate for quinone reductase
screened over a range of pH (6.0 to 8.0). Maximum O2
formation was observed at pH 7.8 and 8.0 (Table 1, entry 12
•–
& 13). Thus, the optimum reaction conditions for O2
Enzyme catalyzed reaction was optimized for the amount of
quinone, glucose, GDH, NAD(P)+ and pH required for the
system to generate O2•–, using lawsone (
1) as a model
production requires 10 equivalents of glucose, 0.2
equivalents of NADP+, 0.2 equivalents of lawsone and pH 7.8
or 8.0.
substrate (Table 1). Glucose concentration was the first
variable followed in the reaction, since it is crucial for
NAD(P)H generation, which is utilized by NfsB_his for
conversion of quinone to hydroquinone. 5, 10 and 20
•–
Screening of quinones for O2 generation by Q/QR
system
equivalents of glucose to that of HE (
3
) were used in the
Since the amount of O2•– generated in the proposed system
depends not only on the efficiency of autoxidation of
hydroquinones, but also on the ability of the NfsB_his to
reduce quinone to hydroquinone, we tested a variety of
system and the reaction mixture was analyzed using HPLC.
The ratio of 2-OH-E+/E+ was found to be 64/36, 67/33, and
69/31, respectively (Table 1, entry 1, 2 & 3). Since the use of
10 or 20 equivalents of glucose did not show an appreciable
quinones (7-13) in the system for their efficacy to produce
•–
•–
difference in O2 formation, 10 equivalents of glucose were
O2
.
•–
used for all further experiments.
First, O2 was generated using xanthine (6)/xanthine
oxidase system. Under an oxygen atmosphere, a ratio of
85/15 was observed for 2-OH-E+ /E+ (Table 2, entry 1). This
system served as a reference for O2 production using the
Table 1. Optimization of reaction conditions using lawsone (1) as
substrate. [a]
•–
Q/QR system described here. When HE was incubated with
Q/QR system under optimized conditions (0.2 equivalents of
lawsone, 0.25 U of NfsB_his, 10 equivalents of glucose, 5 U
Entry Lawsone
Glucose
pH
[2-OH-E+]/[E+]
1
2
0.2
0.2
0.2
0.2
0.2
0.1
0.2
0.4
0.2
0.2
0.2
0.2
0.2
5
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
6.0
7.0
7.4
7.8
8.0
64/36
67/33
69/31
58/42[b]
67/33
52/48
67/33
53/47
46/54
53/47
63/37
67/33
67/33
10
20
10
10
10
10
10
10
10
10
10
10
of GDH, 0.2 equivalents of NADP+, pH 7.8), the ratio of
was found to be 67/33 (Table 2, entry 2). Likewise, seven
5/4
3
4
•–
5
different quinones (
under the same conditions. p-Benzoquinone (
) (Table 2, entry 3 & 4) showed much lower O2•– production
as indicated by ratios of 17/83 and 13/87, respectively. This
7
-
13) were tested for O2 generation
6
7
) and juglone
7
(8
8
9
•– [36]
is because of their ability to scavenge O2
confirmed by addition of and with X/XO (Table 2, entry 5
& 6). Plumbagin (
) (Table 2, entry 7) showed a 2-OH-E+ / E+
,
which was
10
11
12
13
7
8
9
ratio of 64/36. This could be due to its limited preference for
NfsB as a substrate,[26] which limits the rate of hydroquinone
[a] Each reaction (performed in dark at room temperature for 24 hours)
comprised: buffer (50 mM potassium phosphate, 100 µM DTPA buffer)
(6 mL), HE (1 mg, 3.17 μmol, 1 equivalent), NfsB_his (0.25 U) and
GDH (5 U). Amount of lawsone and glucose are shown in equivalents
to HE. An equal amount of NADP+ and lawsone were used. [b] Instead
of NADP+, NAD+ is used.
•–
formation and hence slows down O2 generation. 2-
hydroxyjuglone (10) and 3-hydroxyjuglone (11) (Table 2,
entry 8 & 9) were also tested in the system (see Supporting
Information for synthesis). Both showed values similar to
lawsone (Table 2, entry 3), as indicated by
and 66/34, respectively.
5/4 ratios of 67/33
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Singh, Shailesh Kumar
