Determination of enzymatic activities

Michael Arand

Institute of Pharmacology and Toxicology University of Zürich

Two principally different modes of enzyme activity determination

�  Continuous monitoring of product formation (rate assay)

�  Determination of product formation after a fixed incubation period, usually after some kind of product enrichment/isolation

�  Extraction

�  Thin layer chromatography (TLC)

�  High performance liquid chromatography (HPLC)

�  Gas chromatography (GC)

etc

Frequently used detection methods

�  Spectrophotometric product quantification

�  Fluorimetric product quantification

�  Radiometric product quantification

�  Mass spectrometric product quantification

Basics of spectrophotometric analyses

Lambert-Beer Law:

Eλ = lg = ελ x c x d I0 I1 _ ( )

Eλ = measured light absorption (extinction)

I0 = light intensity before cuvette

I1 = light intensity after cuvette

ελ = extinction coefficient of analyte [mol-1 x cm-1]

c = analyte concentration

d = path length of the light through analyte solution [cm]

Note: ελ is – under specified conditions (solvent, temperature) – an absolute value, ie an analyte-specific constant => spectrophotometric analysis allows absolute quantification

Basics of fluorimetric analyses

Underlying Law:

F = 2.3 x QF x I0 x ε x c x d

F = measured fluorescence

QF = quantum yield of fluorescence

Io = excitation light intensity

ε = extinction coefficient of analyte [mol-1 x cm-1]

c = analyte concentration

d = path length of the light through analyte solution [cm]

Note: Due to a number of technical reasons, fluorimetry usually is not giving absolute values but requires calibration to obtain quantitative measures. It is very sensitive to quenching effects

Principles of radiometric analyses

In brief: -  Relies on the availability of radioactively labelled substrates

-  Typically used radioisotopes: 3H (t1/2 = 12 y), 14C (t1/2 = 6000 y) -  Advantage

-  low background -  good sensitivity -  Potentially equal between substrate and metabolites =>

easy quantification -  Disadvantages:

-  Potential hazard associated with radioactivity (usually not very high)

-  Does not discriminate between different modifications of a compound, no detection if label is lost

Principles of LC-MS/MS analysis

1st Step: Reversed Phase Chromatography

2nd Step: MS/MS analysis

Electrospray Ionisation (ESI)

LC-MS

Ion Selection

Multiple Reaction Monitoring (MRM)

select ion fragment ion select fragment

50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 m/z, amu

0.0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 3.0e5 3.5e5 4.0e5 4.5e5

Intensity, cps

258

157 199 128 133 145 77 105 127 103 159 115 131 171 91 65 185

60 80 100 120 140 160 180 200 220 240 260

m/z, amu

0.0 2.0e5 4.0e5 6.0e5 8.0e5 1.0e6 1.2e6 1.4e6 1.6e6 1.8e6 2.0e6 2.2e6 2.4e6 2.6e6

2.8e6 3.0e6 3.2e6 3.4e6

Intensity, cps

272

171 128 147 215 115 91 159 141 173 102 198 121 78

NCH3

OH

CYP 2D6

NCH3

MeO

Example: Dextrometorphan Demethylation

Components of enzyme assays

• buffer • cofactors, as required • enzyme source (eg tissue homogenate) • other additives (eg detergents for UGTs) • substrate

Rate assay: example sEH hydrolysis of CMNPC

O

O O

OO

N

O

O O

OOH

N

sEH

OH

O

OH

N

O

O

CMNPC

unstable diol intermediate

unstable cyanohydrin intermediate

fluorescent aldehyde

spontaneous decomposition

spontaneous decomposition

CMNPC = cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate

Fig. 1 Detection principle of CMNPC turnover by sEH

non-fluorescent

fluorescent

0

50

100

150

200

250

300

0:00 2:24 4:48 7:12 9:36 12:00

time

fluorescence

Protein linearity analysis

Endpoint determination: example CYP hydroxylation of testosterone

�  buffer: HEPES

�  cofactor: NADPH-regenerating system

�  substrate: testosterone

�  enzyme source: rat liver microsomes (also contain additional required components, in particular CYP reductase

�  method: end point

�  incubation conditions: 10 min, 37°C

�  separation: HPLC

�  detection: UV

HPLC profile of testosterone metabolites

OD240

time [min]

.02

.04

10 20 30

6α2β

15β16β

6β 2α

7α

16α

A

T

I

Change in the metabolite pattern after pretreatment of the animals

with phenobarbitone

OD240

time [min]

.02

.04

10 20 30

OD240

time [min]

.02

.04

10 20 30

2β

15β

16β

6β

2α

16α A

Control

Phenobarbitone

Evaluation of the assay performance: The Z‘-factor

(particularly important for high-throuput screening)

Z‘ = 1 - 3 x (σB + σP)

⏐µB - µP⏐

Desired: Z‘ ≥ 0.6 (=> σB + σP ≤ 0.13 x ⏐µB - µP⏐)

... down to 0.3 sometimes accepted (=> σB + σP ≤ 0.23 x ⏐µB - µP⏐)

σB = standard deviation of the blank σP = standard deviation of the positive control µB = mean of the blank µP = mean of the positive control

0

2

4

6

8

10

1 2

σB

σP

µB - µP

Z‘ = 0.61

B P 0

2

4

6

8

10

1 2

σB

σP

µB - µP

Z‘ = 0.31

B P

Some general rules for the determination of enzyme activity

• if possible, use a rate assay • work at substrate saturating conditions (important exception: enzyme inhibitor analyses!) • avoid too high substrate turnover • assure linear correlation between amount of enzyme in the assay and product formation rate • assure linear correlation between incubation time and product formation rate