Download - Determination of enzymatic activities
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