Drug screening assays for phosphate-generating enzymes

Dr Nick Gee
CEO/CSO of Innova Biosciences

1. Methods of phosphate detection
2. Application to assays for phosphate generating enzymes
3. Enzyme activity & calculations
4. Malachite green assays
5. Improved malachite assays with greater stability/linear range
6. PiColorLock reagent – advantages for HTS
7. ATPase/GTPase assays

Do I need to worry about enzyme units? Or calculate units?
For drug screening work – No.
Relative activity +/- drug is main consideration.
When comparing enzyme activity in different samples – Yes.
When purchasing enzymes – not essential, but useful to know about
units and unit definitions so you get the best value if there is more
than one supplier.

The enzyme unit:
The amount of enzyme that converts 1umol of substrate into product
in one minute (under standard assay conditions)
If we have ‘x’ enzyme units in 10ul and ‘x’ units in 1ml we have
the same number of units, but different concentrations (i.e. units/ml)
Specific enzyme activity (purity)
The number of enzyme units per mg of enzyme. In the above example,
the specific activity in each case is the same (i.e. units/ml div. by mg/ml
= units/mg)

Calculating enzyme units:
1. Determine the number of umols of product formed
2. Divide by the assay time (in minutes) (umol per min i.e. enzyme units)
3. Multiply by 1000/assay volume in ul (units per ml of reaction mix)
4. Multiply by total assay volume/vol enzyme added (units per ml enzyme)
5. Multiply by the dilution factor if the enzyme was diluted before use
i.e. if 1/100 diluted, multiply by 100 (units per ml of undiluted enzyme)
Signal
Pi

Terms in the numerator and denominator often cancel out:
Activity = (AxC)/500B
where, A = concentration of Pi (mM) determined
from the standard curve
B = assay time in minutes
C = dilution factor of the enzyme
e.g. Innova ATPase assay kit:

8
P O-
O
OH
Substrate O
Substrate P
P O-
O
OH
OH
Pi
Abbreviations:

9
Substrate P Product OH Pi+
Specific Universal
Product
Enzyme
Three assay approaches:
1. Measure loss of substrate (poor; only ~10% converted)
2. Measure appearance of ‘specific’ product (okay)
3. Measure appearance of universal product (ideal)
Assay of phosphate-generating enzymes:

Substrate 32P Product OH 32Pi+
Assay types (i) radioactive
OH Pi+14C-Substrate P 14C-Product
e.g. Protein substrates
Pase
e.g. Sugar phosphate
Pase
Pros: high sensitivity, no interference from endogenous Pi
Cons: radioactivity, separation steps

Pi + Dye/Stop Read assay
Assay types (ii) colorimetric, end point assay
e.g. Malachite green assay
Pros: simple, non-radioactive
Cons: interference from Pi in samples, very acidic reagent, risk of
non-enzymatic hydrolysis of phosphorylated substrates
Colour
developmentReaction

Pi + Inosine Hypoxanthine + R1P
Assay types (iii) Coupled assays
NP
Uric acid
XDH
Tetrazolium
Formazan
Pros: continuous, no acidic reagents
Cons: Complex three-enzyme system, not suitable for identification
of inhibitors in HTS
(Red)

Malachite green assay format – 2 steps:
(i) Generate Pi
(ii) Stop and measure

Very simple assay, but there are things to watch out for…
Pi contamination
Non-enzymatic decay
Precipitation

Pure enz.
Crude extract > substrate must be hydrolysed only by the enzyme of interest
Or, the enzyme has to be pulled down from the sample before assay
Crude
(v. specific) Enz 1,2,3,4 ..
Setting up assays. Step 1, Q. Pure enzyme or crude extract?

16
Setting up assays. Step 2, prepare standard curve
Measure absorbance with known amounts of Pi
Absorbance
Pi
Ideally the standard curve
should be linear
Simple to do, no enzyme, no
substrate.

Setting up assays. Step 3, find linear range for the assay
Choose a reaction time that is convenient; 15 min is typical
Add enzyme, measure amount of product formed
All readings must
fall within the linear
range – if not dilute
enzyme a bit more.
Time (min)
Linear
Non linear
Absorbance

A & B - ‘Standard’ malachite green reagents
C - PiColorLock, modified malachite reagent
Precipitation
within 30-60
minutes is
quite common

PiColorlock Gold/ALS and standard malachite reagents
Standard curves:
range of linearity
& sensitivity vary

PiColorlock malachite dye-Pi complex is stable for many hours
Ideal for drug screening labs or in any situation where each
plate cannot be read immediately after colour development

Inhibitor profile for Li+ on IMPase (IMPA-1)
-7 -6 -5 -4 -3 -2 -1 0
0
50
100
IC50 187 uM
Log dilution (mM)
%Activity

Interferences in PiColorLock malachite assays
Component Conc. Effect
NaCl 250 mM None
KCl 250 mM None
MgCl2 25 mM None
DTT 0.25 mM Slight signal loss
bME 0.5 mM None
Tris 25 mM None
Hepes 25 mM None
Mes 25 mM None
Mops 25 mM None
BSA 0.1 mg/ml None
BSA 1 mg/ml Precipitation
DMSO 2.5% None
Detergents 0, >0.03% None*

Substrate P Product OH (10%) Pi+
Enzyme
+ Substrate P (90%)
Non-enzymatic hydrolysis – a major problem and its cure.
Many phosphorylated substrates are sensitive to acid e.g. ATP, GTP
Malachite reagents are very acidic > high backgrounds

Background signals with various phosphate detection
reagents using ATP substrate (i.e. no enzyme present)
Background
suppression
system in
PiColorLock

Watch out - Pi may be present before you even start!
Substrate (esp. ATP, GTP) may be contaminated with Pi.
Buffers – do not use phosphate assay buffer!
Tissue/cell extracts contain lots of
inorganic phosphate.
Need to desalt or dialyse your samples,
or use PiBind phosphate-scavenging beads.

Removal of 1 mM Pi from buffers
of varying pH with PiBindTM
resin
G
lycine
pH
2.3
Acetate
pH
5
M
ES
pH
6
M
O
PS
pH
7
H
epes
pH
7.5Tris
pH
8N
o
resin
Blank
(w
ater)
0
1
2
3
A650

ATP
Applications of malachite assays:
ADP Pi
ATPase
ATP AMP + PPi Pi
ATPase PPase
PPi 2Pi
PPase
-
GTP GDP Pi
GTPase
Peptide-P Peptide-OH Pi
Phosphatase
X-Pn
Pi
?Pase
X-Pn-1
Sugar phosphate Sugar Pi
SPase

Examples from the literature that use modified malachite reagent:
Type Name Area of interest/Notes
Nuclease/Helicase DNA2 DNA repair
Heat shock protein gp96 Systemic lupus erythromatosis
P-type ATPase PARK9 Parkinsonism
ATPase EG5 Spindle formation
DNA-stimulated ATPase SWSAP1 Recombination repair
GTPase/kinase PARK8 Parkinsonism
GTPase DRP-1 Meiosis
GTPase ARF-6 Regulation of PIP2 synthesis
Nuclear GTPase SLIP-GC Replication
Uridyl transferase NAGUT Tuberculosis, UTP > PPi > Pi
Phosphatase PNPK DNA repair, oligo-P substrate
Na/K ATPase human isoforms Muscle control

Reminder of things to think about...
Is my substrate contaminated with free phosphate?
How quickly can I read my assay plates?
Is my substrate sensitive to acid?
By considering these questions it is easy to determine (i) the best
set up for your assay, (ii) whether you need ‘standard’ or ‘modified’
malachite reagent and (iii) whether sample clean up is required.
Is my enzyme pure or crude?
Does my enzyme contain any free phosphate?
Is my substrate hydrolysed by more than one enzyme?

Summary of Innova products and help…..
• Modified malachite reagent – PiColorlock Gold
with background suppression system for acid labile substrates
• PiBind resin for cleaning up enzyme samples (buffers)
• ATPase/GTPase assay kits with ultra-pure ATP/GTP for low backgrounds
• Guide to enzyme units (free)
• Litre quantities available for HTS

References

Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
Please keep an eye out for our future webinars and other exciting news on our
website and social media channels:
www.innovabiosciences.com/innova/webinars.html
YouTube: www.youtube.com/InnovaBiosciences

Innova Biosciences Ltd.
Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova Biosciences

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