Preparative Biochemistry & Biotechnology
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lpbb20
The influence of UV light on the course of
fluorescent enzyme assays
A. Samborski, P. Jankowski & R. Ostaszewski
To cite this article: A. Samborski, P. Jankowski & R. Ostaszewski (2022): The influence of UV
light on the course of fluorescent enzyme assays, Preparative Biochemistry & Biotechnology, DOI:
10.1080/10826068.2022.2119573
To link to this article: https://doi.org/10.1080/10826068.2022.2119573
Published online: 15 Sep 2022.
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PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
https://doi.org/10.1080/10826068.2022.2119573
The influence of UV light on the course of fluorescent enzyme assays
A. Samborskia
, P. Jankowskia
, and R. Ostaszewskib
a
Institute of Physical Chemistry PAS, Warsaw, Poland; bInstitute of Organic Chemistry PAS, Warsaw, Poland
KEYWORDS
ABSTRACT
Experiments were carried out to illustrate the effect of UV light on the course of the enzymatic
reaction of the coumarin derivative. Only the pulsating light of the UV diode gives the correct
results for the determination of the kinetic constants of the enzymatic reaction. The enzyme concentration limit was found where the description of the M-M model breaks. It was shown that the
system determines the kinetic parameters of enzymatic reactions: Vmax—the maximum rate of
reaction and KM—the Michaelis constant. This method produces kinetic constants calculated from
the changes in enzyme product concentration using the Michaelis-Menten model. To verify the
results, we used a statistical analysis that checks the correctness of the model used.
Introduction
Coumarins are nontoxic compounds that widely occur naturally in bacteria, plants, and fungi.[1,2] They are present in
many natural sources such as essential oils, fruits, green tea,
and other foods.[2] Coumarins play a primary role in plant
nutrition and health.[3] Coumarin possesses an extensively
conjugated system with electron-rich and charge transfer
properties. The presence of the hydroxyl group in the
7-position of coumarin yields a highly fluorescent molecule
whose derivatives are widely used as a fluorescence substrate[4] for serine and cysteine proteases[5] probes.
Unfortunately, coumarin and its derivatives undergo photodimerization reaction (Scheme 1) upon UV light irradiation
(<300 nm) in methanol, ethanol, and aqueous solutions.[6]
Photodimerization reaction leads to the formation of four
types of dimers: anti-head-to-head 5, syn head-to-head 6,
syn head-to-tail 7, and anti-head-to-tail 8. This reaction is
reversible and proceeds spontaneously in water and organic
solvents. Coumarins are highly fluorescent compounds in
the visible light range. These properties enable the application of coumarin molecules in numerous different fields
such as laser dyes,[7] optical data storage,[8] drug delivery
systems,[9] or organic light-emitting diodes (OLEDs).[10]
We recently used the unique properties of coumarin
derivatives to design a self-immolative coumarin-based fluorescent probe to detect salinary hydrogen sulfide[11] and
small amounts of hydrogen peroxide.[12] We designed mixed
carbonates containing coumarin as efficient fluorogenic
probes for screening enzyme enantioselectivity[13] and selfimmolative coumarin fluorogenic probes for screening of
hydrolytic activity of enzymes.[14] The designed coumarin
peptidomimetics were cytotoxic agents on E. coli strains.[15]
This work was extended to create a modular microfluidic
CONTACT R. Ostaszewski
ryszard.ostaszewski@icho.edu.pl
Authors contributed equally to this work.
ß 2022 Taylor & Francis Group, LLC
Enzymes assay; UV light;
Michaelis-Menten model;
Lambert function W(x)
system to execute enzyme assays that determine the kinetic
parameters (Vmax and KM) of the enzymatic reactions.[16] As
a substrate, coumarin carbonate 1 was used, which upon
enzyme-catalyzed reaction was transformed into alcohol 2,
coumarin derivative 3 (7-hydroxy-4-methyl-2H-chromen-2one), and carbon dioxide. For calculating kinetic constants
from the concentration of enzymatic product 3 changes via
the Michaelis–Menten model, Lambert function W(x) was
used. Our results pointed out that fluorogenic carbonate is
an excellent probe for rapidly determining the kinetic
parameters KM and kcat of hydrolases, such as lipases
and esterases.
The detailed analysis of data obtained during experiments
determining the course of an enzyme-catalyzed reaction is
complex and requires special attention. The mathematical
model may help determine the Michaelis-Menten (M-M)
parameters from experimental data.[17] The M-M model
commonly used to determine kinetic parameters assumes
that a single reaction catalyzed by an enzyme occurs. For
enzymatic reactions of coumarin derivatives, their progress
monitored by fluorescence spectroscopy reveals that additional responses can occur. Under UV irradiation, coumarin’s photodimerization may proceed according to general
Scheme 1. This process will diminish coumarin’s concentration since dimers 5–8 are non-fluorescent compounds. For
the reaction depicted in Scheme 2, UV light can lead to the
dimers of 7-hydroxy coumarin 3 and diminish its concentration. The respective dimers formed may act as enzyme
inhibitors that complicate experimental data interpretation.
In our system, enzymatic reactions are tested using the fluorescence method with UV light. Coumarin, a component of
the reaction product here, is destroyed by photodimerization. We decided to investigate how significant this effect is
by carrying out several experiments to illustrate the
Institute of Organic Chemistry PAS, Warsaw, Poland.
2
A. SAMBORSKI ET AL.
Scheme 1. Photodimerization of coumarin 4.
Scheme 2. The enzymatic reaction of carbonate 1.
influence of UV light on the course of the enzymatic reaction. The enzymatic reaction product, we irradiated with
UV light in different ways.
We changed the sample’s exposure time from very short
to continuous irradiated. In this way, we determined the
optimal method of irradiation, which allowed us to minimize the impact of photodimerization on the determined
kinetic constants of the reaction. This method can also be
used in situations where we have photodimerization of other
compounds. In this paper, we also specified the applicability
range of the M-M theory, where the parameter was the concentration of the enzyme. To verify the obtained results, we
used a statistical analysis—the chi-square test, which checks
the correctness of the model used.
Experimental
Materials and methods
The fluorogenic probe 1 was synthesized according to the
procedure described in the literature.[18] We purchased commercial enzyme Lipase Candida Rugosa from Sigma-Aldrich
(batch number: BCBV3825, powder, 67 kDa, enzymatic
activity 6.2 U/mg). We prepared the enzyme stock solution
for the enzymatic assay by dissolving 1 mg of the enzyme in
10 mL of PBS buffer (pH 7.4) and then diluting with the
buffer to the desired concentration.
All enzyme experiments we performed by adding 100 mL
of substrate[1] solution (0.5 mM in CH3CN) to 5 mL of the
enzyme solution in PBS buffer. After shaking, 2 mL of the
reaction mixture, we transferred it to the cuvette placed in
the experimental set-up. We performed the measurement at
room temperature.
The measuring system consists of a box made of black
polypropylene with a holder for measuring cuvettes inside
(Figure 1). It was equipped with a UV diode (365 nm) illuminating the sample and a diode for calibrating the measurement of light detection. In addition, we kit equipment
with a temperature control system. We performed fluorescence measurement with a QEPro spectrophotometer
(Ocean Optics) connected to the measuring box using an
optical fiber. We created a computer program to control the
diode lighting and analyze the data obtained from the
spectrophotometer.
Method of calculation of the kinetic parameters vmax
and KM
To estimate the enzyme kinetic parameters Vmax and KM, we
measure the fluorescence intensity signal proportional to the
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
3
Figure 1. Scheme of the experimental setup for determining the kinetic parameters of the enzymatic reaction.
product concentration P(t). We use the known solution of
the differential M-M equation[19–23] in terms of the Lambert
function W ðxÞ :[24]
S0
S0 Vmax t
(1)
PðtÞ ¼ S0 KM W
exp
KM
KM
where S0 is the initial substrate concentration. To estimate the
parameters Vmax and KM, we created a numerical code to fit
our results to the M-M equation (16). In this code, we use the
nonlinear least-squares routines (Levenberg-Marquardt[25,26]),
with the initial estimation of Vmax and KM that we obtained
through linearization of the M-M equation:[27]
PðtÞ
KM
S0
ln
(2)
¼ Vmax t
S0 PðtÞ
t
We prepared a calibration curve based on five different
dilutions of the product solution with buffer to find the relation between the fluorescence intensity and the product concentration P(t).
Figure 2. The fluorescence intensity (450 nm) of the product vs time, under various modes of illumination of the UV diode.
Results and discussion
As described in the introduction, the enzymatic reaction product degrades under the influence of UV radiation. In our
experiment, the kinetic parameters of the enzymatic reaction
are determined by measuring the fluorescence of product 3
(excitation 365 nm). To check the possible effect of UV on the
experiment results, we performed a few experiments with different ways of exposing the product solution (7-hydroxy coumarin) to UV radiation. We established the time of the product
irradiated and the time between exposures: (1 s, 9 s), (2 s, 8 s),
(5 s, 5 s), (1 s, 5 s), (1 s, 1 s). In the end, we continuously irradiated the product. We showed the results in Figure 2.
Only for the exposition 1 s and break 9 s the fluorescence
intensity remained constant over time—blue line. For other
parameters of the expositions, the fluorescence intensity of
the product decreased with time, as the same for continuously light.
Figure 3. The intensity vs time for the LED pulsed and continuous light.
To make sure that the way the diode is turned on does
not affect the intensity of the emitted UV light, we compared the intensity of the diode illumination for the wavelength of 365 nm for continuous (red line) and pulsed light
(blue line): Figure 3.
4
A. SAMBORSKI ET AL.
Figure 4. The intensity vs. time for Csub¼0.5[mM], Cenz¼0.02[mg/mL]: the blue
line – the LED pulsed lighting on for 1 s with an interval of 10 s: Vmax ¼
9.1 102 ± 2.83 103 [mM/s], KM ¼ 31. ± 1.12 [mM], v2 ¼ 16.99, and v2crit ¼
154.54; the red line – the product was continuously irradiated by UV light: we
cannot find the optimal parameters Vmax and KM.
Figure 5. The experimental results (squares) and fitting curve (line) for the product
concentration of enzymatic reaction: Csub¼0.5[mM], Cenz ¼ f0.1, 0.08, 0.06, 0.04,
0.02, 0.01, 0.005g [mg/mL]. The parameters: v2 and the critical value v2crit determined, which describes the correct fit of the experimental data to the
assumed model.
Table 1. The kinetic parameters obtained from the reaction for the enzyme CRL with fluorogenic probe 1.
Enzyme concentration [mg/mL]
1.
8.
6.
4.
2.
1.
5.
1
10
102
102
102
102
102
103
Vmax
[mM/s]
1
2
2.89 10 ± 1.75 10
1.79 101 ± 3.69 103
1.8 101 ± 6 103
1.46 101 ± 3.65 103
9.1 102 ± 2.83 103
2.32 102 ± 5.79 104
4.54 103 ± 6.25 105
The results showed that the intensity values are similar
and constant in time. Therefore, we assumed that the
observed changes in the fluorescence intensity in the previous experiments were related to the degradation of the
product. Hence, the conclusion is that pulsed illumination
with the UV diode should be used when determining the
fluorescence intensity of the enzymatic reaction product. We
verified this by performing two measurements for the same
set of enzymatic reaction parameters Figure 4.
We can see that the product fluorescence intensities for
the continuous and pulsating lighting of the LED are different. For a constant, we get the lowered values. Using our
numerical code for the continuous lighting results, we cannot find the optimal parameters Vmax and KM. Opposite,
for the pulsating lighting, we got the convergence of the
procedure. Therefore, we should use pulse light of the UV
diode to correctly determine the kinetic constants of the
enzymatic reaction using fluorescence. We used these experimental data and fit them into the M-M equation. In the
next step, we decided to check whether the measurement
method we used was also effective for low enzyme concentrations. We made a series of measurements of enzymatic
reactions for the substrate and the enzyme using the pulse
UV LED. In the experiment, we established a constant substrate concentration (Csub ¼ 9.8 mM), while we changed the
KM
[mM]
kcat
[1/s]
v2
v2crit
2.69 10 ± 1.93
1.93 101 ± 5.04 101
2.32 101 ± 9.46 101
2.37 101 ± 7.2 101
3.1 101 ± 1.12
1.68 101 ± 5.48 101
1.65 ± 6.97 102
0.194
0.15
0.2
0.245
0.305
0.156
0.061
9.74
2.21
1.02 101
3.16
1.69 101
2.47 102
2.51 102
7.44 101
5.95 101
5.95 101
7.53 101
1.55 102
1.55 102
2.1 102
1
concentration of the enzyme: Cenz ¼ f0.1, 0.08, 0.06, 0.04,
0.02, 0.01, 0.005g [mg/mL]. Figure 5 and Table 1.
The graph shows the experimental data and the curves
obtained by fitting the M-M model.[16] For the concentration of enzyme 0.02 < Cenz<0.1 [mg/mL], we see that our
model corroborates with the experimental results and provides a reasonable estimate of the kinetic parameters: Vmax,
KM; (v2 < v2crit that is, the theoretical curve describes the
experimental results). For low enzyme concentrations
Cenz<0.02 [mg/mL], the graphs show a deviation from the
M-M model; (v2 > v2crit it means that the assumed theoretical curve badly describes our experiment, with a significance
level of 0.95, see Table 1). It suggests the existence of a limit
in the enzyme concentration where the description by the
M-M model collapses. (the hypothesis H0 that the M-M
model good describes the enzymatic reaction should be
rejected). The parameters: v2 and the critical value v2crit also
determined, which describe the correct fit of the experimental data to the assumed model.
Conclusions
Coumarins are naturally occurring fluorescent compounds.
The high fluorescence of coumarin solutions and biocompatibility make them valuable substrates for enzymatic
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
assays.[27] This is due to the fact that fluorescence changes
upon coumarin concentration allow for monitoring the
progress of chemical and enzymatic reactions. However,
under the influence of UV radiation, the photodimerization
of coumarins occurs. Our experiments have noticed that we
do not get the correct experimental results with continuous
UV light irradiation of the enzyme sample. To check the
influence of UV light on the course of the reaction, we
changed the time of the product irradiated and the time
between exposures. The fluorescence intensity remains constant over time for the exposition 1 s and breaks 9 s. For
other parameters of the expositions, the fluorescence intensity of the product decreased with time. Therefore for the
sample’s exposure 1 s, we can ignore the destructive effect of
photodimerization on the kinetic constants of the enzymatic
reaction. With pulsed lighting, we got results that we can
correctly interpret. We used this method to study CRL
enzymes. We showed that we could accurately determine
the parameters of the enzymatic reactions. We used the
Michaelis–Menten model to describe the enzymatic kinetics
by determining the reaction’s kinetic parameters Vmax—the
maximum rate of reaction and KM—the Michaelis constant.
The statistical analysis gives us information about the scope
of application of a given model to describe the phenomenon. Here we used the chi-square test (v2), which checks
the correctness of the model used. If v2 > v2crit hypothesis
H0 that the model describes correctly enzymatic reaction
should be rejected.[28] For the enzymatic concentration Cenz
< 1 102 [mg/mL], we obtained that v2 > v2crit which
means that the assumed theoretical curve badly describes
the experiment. It suggests that we found the enzyme concentration limit below which the description by the M-M
model collapses.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Funding
This work was supported from the National Science Centre, Poland
project OPUS No. 019/33/B/ST4/01118.
[15]
ORCID
A. Samborski
P. Jankowski
R. Ostaszewski
http://orcid.org/0000-0001-8088-3891
http://orcid.org/0000-0002-0426-9221
http://orcid.org/0000-0002-3032-196X
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