Effects of Using Different Assay Buffers on the Activity

of Recombinant Human CYP3A4 Co-Expressed in E. coli

with Human NADPH P450 Reductase.
Michael W. Voice
Cypex Ltd., 6 Tom McDonald Avenue, Dundee, DD2 1NH, Scotland
Abstract
Bacterial membrane preparations containing recombinant human cytochrome P450s (CYP) co-expressed with human
NADPH P450 reductase (reductase) are widely used in the in vitro analysis of drug metabolism. Although the effect Fig 1. Effect of phosphate concentration on testosterone Fig 2. Effect of 20 mM MgCl2 on testosterone turnover by
of using different buffer components on the activity of CYPs has been investigated to some extent in systems using turnover by CYP3A4R Bactosomes. CYP3A4R Bactosomes in different assay buffers.

nmol 6β-hydroxytestosterone/nmol P450

purified CYP reconstituted in an artificial phospholipid mix together with purified reductase it has not been investigated 2000
70
in the bacterial membrane preparations described above. The assay buffer components used can vary widely be-

Turnover (nmol/min/nmol P450)

1750
tween laboratories so we have studied the effects of changing the buffer components on the activity of recombinant 60
human CYP3A4 co-expressed with human NADPH P450 reductase in E. coli. 1500
50
The CYP3A4 activity as measured by testosterone and nifedipine turnover was much lower in 50 mM HEPES, 50 mM 1250
Tris and 25 mM phosphate buffer than in 50 and 100 mM phosphate buffer. We found that the effect of including MgCl2 40
1000
in the assay was buffer dependent, increasing turnover of both substrates in 50 mM HEPES, 50 mM Tris and, to a Turnover (pmol/min/pmol P450)

30
much lesser extent, in 25 mM and 50 mM phosphate buffers. MgCl2 did not affect the turnover of either substrate in 750
100 mM phosphate buffer. Altering the ratio of reductase : CYP is known to affect the activity of CYP3A4 and, by using 20
500
different expression systems, we are able to produce “low” and “high” reductase membrane preparations. Using
these preparations, we demonstrated that the buffer dependent effects of MgCl2 are independent of the reductase : 250 10

CYP ratio. We also showed that the inhibitory effect of α-naphthoflavone is buffer and MgCl2 dependent. Significant
0 0
inhibition of testosterone turnover by CYP3A4 by 10 µM α-naphthoflavone was only seen in HEPES, Tris and the 0 5 10 15 20 25 30 1 x TSE 50 mM HEPES 25 mM 50 mM 100 mM
phosphate phosphate phosphate
lower concentrations of phosphate buffer in the presence of MgCl2. Very little inhibition was observed in the absence Time (min) Control
of MgCl2 in those buffers. 10 µM α-naphthoflavone was inhibitory in 100 mM phosphate buffer regardless of whether 25 mM 50 mM 100 mM 150 mM 20 mM MgCl2 Assay Buffer
phosphate phosphate phosphate phosphate
MgCl2 was present or not.
MgCl2 included at 5 mM in all reactions
Clearly the effects associated with using different assay buffer components must be borne in mind when designing in
vitro metabolism assay protocols and interpreting the results of those assays.
Fig 3a. Effect of MgCl2 on testosterone turnover by Fig 3b. Effect of MgCl2 on testosterone turnover by
Introduction
CYP3A4R Bactosomes. CYP3A4R Bactosomes.
New chemical entities (NCEs) are routinely screened in vitro for
metabolism by, and inhibition of, cytochrome P450s, the principal 1500
Turnover (nmol/min/nmol P450)

20

% Turnover in 0 mM MgCl2
enzyme family involved in the oxidation of xenobiotics. Although 1250

Cypex Ltd uses either 50 mM or 100 mM potassium phosphate buffer 15

1000
for all of the QC assays carried out on its recombinant P450 products
(Bactosomes) end users may have established their assays using 10
750

other buffer systems. There is evidence that the activity of

500
recombinant cytochrome P450s can vary depending on the buffer used 5

in the assay. For example Crespi, C. (1998) showed that the activity of 250

insect cell expressed recombinant human cytochrome P450s can be 0 0

affected by the concentration of the phosphate buffer used and 0 5 10 15 20 0 5 10 15 20

MgCl2 (mM) MgCl2 (mM)

Yamazaki, H. et al (1995) demonstrated that the activity of recombinant
human CYP3A4 used in reconstituted systems can be affected by the 50 mM phosphate 25 mM phosphate 50 mM HEPES 50 mM Tris, (1 x TSE)

choice of assay buffer. Here we have investigated the effects of using

different buffers and different concentrations of potassium phosphate Fig 4. Effect of 20 mM MgCl2 on nifedipine turnover nmol product / nmol P450 Fig 5. Effect of 20 mM MgCl2 on testosterone turnover by
buffer on the activity of recombinant human CYP3A4 co-expressed by CYP3A4R Bactosomes in different assay CYP3A4LR Bactosomes in different assay buffers.
with human NADPH P450 reductase in E. coli. buffers.
50 12

Materials & Methods

Turnover (nmol/min/nmol P450)

Turnover (nmol/min/nmol P450)

10
All chemicals were obtained from Sigma-Aldrich or VWR unless 40

otherwise indicated. Recombinant human CYP3A4 co-expressed in E. 8

30
coli with human NADPH P450 reductase (Bactosomes) and
Turnover (pmol/min/pmol) 6
ketoconazole were supplied by Cypex Ltd. 20
4
CYP3A4 activity was assayed by measuring testosterone or nifedipine
turnover at 37°C for 5 minutes in the buffer specified (total assay 10
2

volume 0.2 ml). The buffers used were, 25, 50, 100 and 150 mM 0 0
potassium phosphate pH 7.4, 50 mM potassium HEPES pH 7.4 and 1 x TSE 50 mM HEPES 25 mM
phosphate
50 mM
phosphate
100 mM
phosphate
1 x TSE
Control
50mM HEPES 25mM
phosphate
50mM
phosphate
100mM
phosphate

Bactosome storage buffer, 50 mM Tris acetate pH 7.6, 250 mM Control

20 mM MgCl2 Assay Buffer
20 mM MgCl2 Assay Buffer
sucrose, 0.25 mM EDTA (1 x TSE). The substrate concentration in the
assay was 32 µM (testosterone) or 100 µM (nifedipine). The reactions
were started by the addition of 40 µl 5x NADPH generating system (5 Fig 6. Effect of 20 mM MgCl2 on inhibition of testosterone Fig 7. Effect of 20 mM MgCl2 on inhibition of testosterone
mM NADPH, 25 mM glucose-6-phosphate, 5 U/ml turnover by CYP3A4LR Bactosomes by 10 µM turnover by CYP3A4LR Bactosomes by 0.5 µM
glucose-6-phosphate dehydrogenase). Reactions were stopped by α-naphthoflavone in different assay buffers. ketoconazole in different assay buffers.
the addition of 25 µl 1 M HCl . Samples were centrifuged at 13,000 rpm
% Turnover in 0 mM α-naphthoflavone

120 70
% Turnover in 0 mM ketoconazole

in a microfuge and the supernatants analysed by reverse phase HPLC 60

100
with UV detection. All assays were performed on at least three 50
different preparations of E. coli membranes. 80

40
60

Results 30

40
As the phosphate concentration in the assay is increased, the activity 20

of E. coli expressed CYP3A4 increases (Fig. 1), testosterone turnover 20

10

was 23 fold higher in 150 mM potassium phosphate than in 25 mM 0 0

1 x TSE 50 mM HEPES 25 mM 100 mM 1 x TSE 50 mM HEPES 25 mM 100 mM
potassium phosphate. This is similar to the increase in activity seen in Control
phosphate phosphate
Control
phosphate phosphate

20 mM MgCl2 20 mM MgCl2
insect cell expressed CYP3A4 with increasing phosphate Assay Buffer Assay Buffer

concentration (Crespi, C. (1998)). There is a concomitant decrease in

the time for which testosterone turnover is linear as the activity Conclusions
increases. Testosterone turnover by CYP3A4 in 50 mM HEPES buffer - The buffer used in the assay system to determine CYP3A4 activity can significantly affect the activity of the
and 50 mM Tris based buffer was significantly lower than that seen in enzyme; both testosterone and nifedipine turnover by CYP3A4 is highest in potassium phosphate buffer.
50 mM phosphate buffer (Fig. 2). In 25 and 50 mM phosphate, 50 mM
HEPES and 50 mM Tris buffers the inclusion of MgCl2 resulted in a - The activity of CYP3A4 increases with potassium phosphate concentration.
significant increase in the activity of CYP3A4 when either testosterone
- The stimulatory effect of MgCl2 is buffer dependent, it is greatest in assay systems using Tris, HEPES and low
or nifedipine were used as substrate (Figs. 2, 3 and 4). Surprisingly,
concentration potassium phosphate buffers. The inclusion of MgCl2 in the assay system using 100 mM
the inclusion of MgCl2 had no effect in 100 mM phosphate buffer. The
potassium phosphate has no effect on the activity of CYP3A4.
same was seen when using E. coli membranes containing CYP3A4
with a lower level of co-expressed NADPH P450 reductase (low - The inhibitory action of α-naphthoflavone on testosterone turnover by CYP3A4 is dependent on the presence
reductase CYP3A4 Bactosomes) (Fig. 5). The activating effect of of MgCl2 in Tris, HEPES and low concentration phosphate buffer. It is MgCl2 independent in high concentration
MgCl2 was greatest in Tris and HEPES buffer (Fig. 3b) with phosphate buffer.
testosterone turnover increasing over 12 fold on the addition of MgCl2.
The effect was less marked in 25 mM phosphate buffer (approx 5 fold - The inhibitory action of ketoconazole is MgCl2 independent regardless of the assay buffer used
increase in activity and 50 mM phosphate buffer (2.5 fold increase in - These buffer and MgCl2 effects must be borne in mind when developing in vitro assays using cytochrome
activity). P450s.
The effect of using different assay buffers on the inhibition of
testosterone turnover by CYP3A4 by α-naphthoflavone and
ketoconazole was investigated. In Tris, HEPES and 25 mM phosphate References
Yamazaki, H., Ueng, Y-F., Shimada, T. & Guengerich, F.P. (1995) Biochemistry 34 pp 8380 - 8389
buffers 10 µM α-naphthoflavone was only inhibitory in the presence of Roles of divalent metal ions in oxidations catalysed by recombinant cytochrome P450 3A4 and replacement
MgCl2. In 100 mM phosphate buffer α-naphthoflavone was inhibitory of NADPH-Cytochrome P450 reductase with other flavoproteins, ferredoxin, and oxygen surrogates.

regardless of whether MgCl2 was included or not (Fig. 5). 0.5 µM Crespi, C. L. (1998) International Symposium on Microsomes & Drug Oxidations, 20 - 24 July 1998. Poster
Ketoconazole was inhibitory to the same degree in all of the buffers 95
Effect of salt concentration on the activity of liver microsomal and cDNA-expressed human cytochromes
tested and in both the presence and absence of MgCl2. P450.