Cytochrome P450 3A4 from Homo sapiens

Enzyme Description

Species

Extremophile

EC Number

1.14.14.1 ↗

Sequence

Length: 503 amino acids

MALIPDLAMETWLLLAVSLVLLYLYGTHSHGLFKKLGIPGPTPLPFLGNILSYHKGFCMFDMECHKKYGKVWGFYDGQQPVLAITDPDMIKTVLVKECYSVFTNRRPFGPVGFMKSAISIAEDEEWKRLRSLLSPTFTSGKLKEMVPIIAQYGDVLVRNLRREAETGKPVTLKDVFGAYSMDVITSTSFGVNIDSLNNPQDPFVENTKKLLRFDFLDPFFLSITVFPFLIPILEVLNICVFPREVTNFLRKSVKRMKESRLEDTQKHRVDFLQLMIDSQNSKETESHKALSDLELVAQSIIFIFAGYETTSSVLSFIMYELATHPDVQQKLQEEIDAVLPNKAPPTYDTVLQMEYLDMVVNETLRLFPIAMRLERVCKKDVEINGMFIPKGVVVMIPSYALHRDPKYWTEPEKFLPERFSKKNKDNIDPYIYTPFGSGPRNCIGMRFALMNMKLALIRVLQNFSFKPCKETQIPLKLSLGGLLQPEKPVVLKVESRDGTVSGA

Amandine Chefson et al. (2007) — CYP3A4 Activity in the Presence of Organic Cosolvents, Ionic Liquids, or Water-Immiscible Organic Solvents

ChemBioChem · doi:10.1002/cbic.200700128 ↗ · Activity - Classical

50 measurements ▼

Database ID

UniProt: P08684 ↗

Sequence Annotation

Inferred - from protein name

Protein Source

Recombinant, host bacterium Escherichia coli DH5alpha

Experimental Data (50 measurements)

50 measurements — page 2 of 3

Property	Assay	Solvent	Solvent Volume	Aqueous Reference	Measured Value	Units	Solution	pH	Temperature	Substrate(s)	Product(s)	Cofactor(s)	Shaking	Comments
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Methanol	1%	100	84	%	1 M potassium phosphate buffer	7	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	12.5 mg sodium percarbonate in 300 µL assay solution	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Methanol	2.5%	100	63	%	1 M potassium phosphate buffer	7	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	12.5 mg sodium percarbonate in 300 µL assay solution	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Methanol	5%	100	34	%	1 M potassium phosphate buffer	7	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	12.5 mg sodium percarbonate in 300 µL assay solution	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Methanol	10%	100	8	%	1 M potassium phosphate buffer	7	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	12.5 mg sodium percarbonate in 300 µL assay solution	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Methanol	15%	100	1	%	1 M potassium phosphate buffer	7	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	12.5 mg sodium percarbonate in 300 µL assay solution	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Dimethyl Sulfoxide (DMSO)	10%	100	12	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetone	2.5%	100	51	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetone	5%	100	29	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetone	10%	100	10	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetone	15%	100	1	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetonitrile	1%	100	64	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetonitrile	2.5%	100	36	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetonitrile	5%	100	6	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetonitrile	10%	100	0	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetonitrile	15%	100	0	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Dimethyl Sulfoxide (DMSO)	1%	100	90	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Dimethyl Sulfoxide (DMSO)	2.5%	100	61	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Dimethyl Sulfoxide (DMSO)	5%	100	36	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Acetone	1%	100	79	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)
Activity - Classical	Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)	Dimethyl Sulfoxide (DMSO)	15%	100	4	%	0.1 M potassium phosphate buffer	7.4	37°C	115 µM Testosterone	6beta-hydroxy- testosterone	1 mM cumene hydroperoxide	Yes, "with shaking"	Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

10%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

2.5%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

10%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

15%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

2.5%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

10%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

15%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

2.5%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

15%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

2.5%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

10%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

15%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

2.5%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

10%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

15%

100

1 M potassium phosphate buffer

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

12.5 mg sodium percarbonate in 300 µL assay solution

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

10%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

2.5%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

10%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

15%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

2.5%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

10%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetonitrile

15%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

2.5%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Acetone

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Dimethyl Sulfoxide (DMSO)

15%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

2.5%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

10%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Tetrahydrofuran (THF)

15%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

2.5%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

10%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

—

Activity - Classical

Activity measured in the presence of organic solvent by HPLC (product formation after 1 hour at 37°C)

Methanol

15%

100

0.1 M potassium phosphate buffer

7.4

37°C

115 µM Testosterone

6beta-hydroxy- testosterone

1 mM cumene hydroperoxide

Yes, "with shaking"

Classical aqueous control (in %)

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Visualization : Activity — Classical

Amandine Chefson et al. (2007)

One bar per measurement. Colour = solvent, shade = solvent volume.

Vanessa González-Pérez et al. (2012) — Impact of Organic Solvents on Cytochrome P450 Probe Reactions: Filling the Gap with (S)-Warfarin and Midazolam Hydroxylation

Drug Metabolism and Disposition · doi:10.1124/dmd.112.047134 ↗ · Activity - Classical

18 measurements ▼

Database ID

UniProt: P08684 ↗

Sequence Annotation

Inferred - from protein name

Protein Source

Recombinant, host insect cells

Experimental Data (18 measurements)

18 measurements

Property	Assay	Solvent	Solvent Volume	Aqueous Reference	Measured Value	Units	Solution	pH	Temperature	Substrate(s)	Product(s)	Cofactor(s)	Shaking	Comments
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Dimethyl Sulfoxide (DMSO)	0.1%	100	103	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Isopropanol	2%	100	35	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Isopropanol	1%	100	60	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Isopropanol	0.1%	100	99	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Acetone	2%	100	17	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Acetone	1%	100	27	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Acetone	0.1%	100	64	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Dimethyl Sulfoxide (DMSO)	2%	100	52	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Dimethyl Sulfoxide (DMSO)	1%	100	88	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Acetonitrile	0.1%	100	113	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Ethanol	2%	100	82	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Ethanol	1%	100	102	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Ethanol	0.1%	100	112	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Methanol	2%	100	81	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Methanol	1%	100	90	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Methanol	0.1%	100	121	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Acetonitrile	2%	100	80	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)
Activity - Classical	Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent	Acetonitrile	1%	100	113	%	100 mM potassium phosphate buffer	7.4	37°C	4 µM Midazolam	1′-Hydroxymidazolam	1 mM NADPH	—	Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

0.1%

100

103

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Isopropanol

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Isopropanol

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Isopropanol

0.1%

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Acetone

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Acetone

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Acetone

0.1%

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Acetonitrile

0.1%

100

113

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Ethanol

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Ethanol

100

102

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Ethanol

0.1%

100

112

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Methanol

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Methanol

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Methanol

0.1%

100

121

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Acetonitrile

100

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Activity - Classical

Activity measured by HPLC-coupled mass spectrometry in the presence of organic solvent

Acetonitrile

100

113

100 mM potassium phosphate buffer

7.4

37°C

4 µM Midazolam

1′-Hydroxymidazolam

1 mM NADPH

—

Classical aqueous control (in %)

Visualization : Activity — Classical

Vanessa González-Pérez et al. (2012)

One bar per measurement. Colour = solvent, shade = solvent volume. — — — Reference value. Hover for details.

Dennis Rokitta et al. (2013) — The effect of organic solvents on enzyme kinetic parameters of human CYP3A4 and CYP1A2 in vitro

Toxicology Mechanisms and Methods · doi:10.3109/15376516.2013.806622 ↗ · Activity - Michaelis-Menten

48 measurements ▼

Database ID

UniProt: P08684 ↗

Sequence Annotation

Inferred - from protein name

Protein Source

Commercially purchased

Experimental Data (48 measurements)

48 measurements — page 1 of 3

Property	Assay	Solvent	Solvent Volume	Aqueous Reference	Measured Value	Units	Solution	pH	Temperature	Substrate(s)	Product(s)	Cofactor(s)	Shaking	Comments
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Methanol	1%	0.75	0.59	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Ethanol	2%	4.1	6.3	nmol/min/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Ethanol	3%	4.1	6.5	nmol/min/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Ethanol	4%	4.1	6.3	nmol/min/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Dimethyl Sulfoxide (DMSO)	1%	4.1	4.2	nmol/min/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Dimethyl Sulfoxide (DMSO)	2%	4.1	5.0	nmol/min/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Dimethyl Sulfoxide (DMSO)	3%	4.1	3.6	nmol/min/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Dimethyl Sulfoxide (DMSO)	4%	4.1	2.9	nmol/min/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Acetonitrile	1%	0.75	0.64	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Acetonitrile	2%	0.75	0.54	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Acetonitrile	3%	0.75	0.58	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Acetonitrile	4%	0.75	0.54	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Ethanol	1%	4.1	6.3	nmol/min/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Methanol	2%	0.75	0.7	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Methanol	3%	0.75	0.79	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Methanol	4%	0.75	0.65	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Ethanol	1%	0.75	1.4	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Ethanol	2%	0.75	1.15	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Ethanol	3%	0.75	1.23	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes
Activity - Michaelis-Menten	Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent	Ethanol	4%	0.75	1.26	nmol/min/μM/nmol CYP	100 mM potassium phosphate buffer	7.4	37°C	Midazolam	1′-Hydroxymidazolam	NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration	Yes, "shaking water bath"	Classical aqueous control (in nmol/min/μM/nmol CYP) \| Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

0.75

0.59

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

4.1

6.3

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

4.1

6.5

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

4.1

6.3

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

4.1

4.2

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

4.1

5.0

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

4.1

3.6

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

4.1

2.9

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

0.75

0.64

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

0.75

0.54

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

0.75

0.58

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

0.75

0.54

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

4.1

6.3

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

0.75

0.7

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

0.75

0.79

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

0.75

0.65

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

0.75

1.4

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

0.75

1.15

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

0.75

1.23

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

0.75

1.26

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

0.75

0.74

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

0.75

0.57

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

0.75

0.56

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax/kM (in nmol/min/μM/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

0.75

0.64

nmol/min/μM/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/μM/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

5.5

5.7

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

5.5

5.9

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

5.5

5.3

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

5.5

6.5

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

5.5

7.4

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

5.5

6.8

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

5.5

6.8

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

5.5

8.3

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

5.5

4.5

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

5.5

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

5.5

5.3

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Ethanol

5.5

5.0

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

5.5

5.3

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

5.5

8.7

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

5.5

6.4

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Km (in µM) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Dimethyl Sulfoxide (DMSO)

5.5

4.5

µM

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in µM) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

4.1

3.4

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

4.1

3.2

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

4.1

3.1

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Acetonitrile

4.1

3.0

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

4.1

4.4

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

4.1

4.8

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

4.1

5.4

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

—

Activity - Michaelis-Menten

Vmax (in nmol/min/nmol CYP) measured by LC/MS after incubation (20 minutes at 37°C) in the presence of organic solvent

Methanol

4.1

5.4

nmol/min/nmol CYP

100 mM potassium phosphate buffer

7.4

37°C

Midazolam

1′-Hydroxymidazolam

NADPH regenerating system (containing NADP+, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase), no clear concentration

Yes, "shaking water bath"

Classical aqueous control (in nmol/min/nmol CYP) | Enzymes in supersomes

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Visualization : Activity — Michaelis-Menten

Dennis Rokitta et al. (2013)

One bar per measurement. Colour = solvent, shade = solvent volume.

Structure

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Cytochrome P450 3A4 from Homo sapiens

Enzyme Description

Sequence

Experimental Data (50 measurements)

Visualization : Activity — Classical

Experimental Data (18 measurements)

Visualization : Activity — Classical

Experimental Data (48 measurements)

Visualization : Activity — Michaelis-Menten

Structure

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