Amu ocam. « n , VoL 40. No. 4, pp. 371-379. 1996
Copyright O 1996 Britnh Occop«tioo»l Hygfcra Sadeqr
Per Ramon
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PuHShedby b
Srii-mr Ud. Printed in Oral Britain
0003-4S7»/9«J15.00+0.00
Pn: S0003-4878(96)00008-7
USE OF CHROMOGENIC SUBSTRATES IN PERSONAL
MONITORS FOR THE DETECTION OF PROTEASE IN
FACTORY AIR: A THEORETICAL MODEL
I. Nitescu,0 Z. Koochaki,* F. J. Rowellf and R. H. Cumming*
'North Ea*t Biotechnology Centre, School of Science and Technology, University of Teesside,
Middlesbrough TS1 3BA, U.K.; and fNorth East Biotechnology Centre, School of Health Sciences,
University of Sunderland, Sunderland SR2 7EE, U.K.
(Received In final form 10 November 1995)
Abstract—A model is presented which predicts the accumulation of an airborne protease enzyme in
an aerosol sampler. The model allows the prediction of the product accumulation as a result of the
catalysis by the enzyme of a chromogenic substrate. The first and second derivatives of the product
accumulation curve are rnimiatnH and used to evaluate the best parameter for the device. If the
device is to be used for the detection of a pulse of a high local concentration of protease then it is
necessary for the device to be able to measure the first derivative of the product accumulation
curve. Factors affecting the sensitivity of the device are discussed. Copyright © 1996 British
Occupational Hygiene Society.
v
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NOMENCLATURE
rate of substrate breakdown or rate of product formation
maximum value for y
saturation constant
substrate concentration
turnover number
overall efficiency of sampler
flow
rate of air to sampler
concentration of enzyme in the air entering sampler
concentration of enzyme in sampler
volume of liquid in sampler
product concentration in sampler
rate constant for spontaneous breakdown of substrate
concentration of enzyme in the air leaving sampler
/nnol m~ 3 min~'
/jmol m~ 3 min" 1
mmol m ~ 3
mmol m~ 3
/zmol kg" 1 min~'
—
m 3 min~'
kg ra~3
kgm~3
nv
mmol m~ 3
mmol m ~ 3 m i n ~ '
kg m ~ 3
INTRODUCTION
During the manufacture of proteolytic enzymes and their incorporation into
biological washing powders, the factory air is monitored for airborne protease dust.
This is because airborne particles of protease dust can cause allergies when inhaled
(Behizad et al., 1989). We are interested in exploiting the potential of chromogenic
substrates for providing a real-time sensing system. Thus, the air would be passed
directly through a solution of buffered chromogenic substrate and the resulting colour
change through the action of airborne protease would be monitored using a
spectrophotometer. It is clear that the requirements for the chromogenic substrate is
that it should be both sensitive and stable over the operational period of the sensor, so
that the sensor can still detect protease during the last moments of a shift (typically
8 h). Factors affecting the selection and suitability of a range of aerosol monitoring
systems for the detection of airborne microbes have been reviewed in Nevalainen et al.
371
372
I. Nitescu el al.
(1992). The difference between this type of system for collection of microbes and the
one discussed in this paper is that the detection of the hazardous aerosol takes place in
the sampler in real-time, without the necessity of removing samples.
This paper applies rate data for a chromogenic substrate which we have previously
evaluated (Nitescu et al., 1996). In addition to the application of aerosol collection
models, we suggest suitable criteria for the evaluation of the performance of the device.
THEORY
Principle of the use of chromogenic substrates in personal monitoring
An obvious way of using the chromogenic substrate in a personal monitor would
be by incorporating the buffered substrate in a liquid impinger or bubbler. Factory
air would be drawn via a pump through the collecting device and contact with the
buffer would allow the transfer of the protease dust to the buffered substrate.
Protease in the dust particles would dissolve in the buffer and react with the
chromogen substrate to produce a coloured product which can then be detected by a
spectrophotometric device built into the collector. The data could be stored and later
down loaded; although it is preferable if the device could give a signal if a high level
of protease was detected. The operator could then leave the work area immediately.
Since it is normal to detect very low levels of background protease in the factory
air, the sensor must be able to distinguish between the sudden release of a high
concentration of protease from the background level. It is also desirable that at the
end of the monitoring period (that is an 8-h shift), the sensor could give the total
exposure of the wearer during that period.
Enzyme assay
The principle of an enzyme assay is to measure the rate of catalytic activity in the
sample and relate it to a calibration curve of rate vs enzyme concentration for the
enzyme in question. Usually the assay conditions strive to use a high substrate
concentration so that the catalytic rate is not limited during the assay procedure.
This means that the enzymic rate is an estimate of the maximum rate of catalytic
activity. Thus, assuming Michaelis-Menten kinetics apply for the substrate:
v=
VmS
(1)
where v is the rate of enzymic action (for chromogen substrate, it is often measured
as the amount of product formed per unit time, dP/dt), Vm is the maximum rate, S is
the concentration of substrate and K, is the saturation constant. Thus, if S is very
high during the assay, it can be seen that the equation reduces to
v = Vm.
(2)
If the substrate concentration does change during the assay, then initial rates of
enzymic activity have to used. Equation (2) can be expanded to
(3)
where k3 is the turnover number for the enzyme.
Chromogcnic substrate* for protease detection
373
It can now be seen that a plot of v vs the enzyme concentration will give a straight
line of slope fc3. This was the basis of the calibration curves for the reaction with
different chromogenic substrates, reported earlier. When the assay is used as a real
time sensor, the time of the assay is not fixed but continuous from 0 to 8 h. Thus, we
must ensure that the substrate concentration will always be sufficiently high during
the period of operation so that the enzyme rate will continue at Vm and not be
limited.
Mass balance for protease in the sampler
The accumulation rate of protease in the sampler is calculated from the mass
balance
rate of protease entering = rate of protease + rate of protease + accumulation rate,
sampler from air
leaving sampler breakdown
If we assume that protease which has been collected is stable then the
accumulation rate (dE/dt) is given by:
K
dt ~ v '
>
where t\ is the collection efficiency of the sampler and is given by l-E^/Ei, F is the
flow rate of air drawn into the sampler (m~ 3 min" 1 ), Ea is the concentration of
enzyme in the air (kg m~ 3 ) and V is the volume of substrate solution in the sampler
(m3); giving units of dE/dt as kg enzyme m~ 3 min" 1 . For a constant concentration
of enzyme in the air (£a) this equation can be integrated to give an expression for the
concentration of enzyme in the sampler (£,) at time t:
Thus, the concentration of enzyme in the sampler increases linearly with time. Since
the sensor will have excess substrate the rate of product formation will be given by
Equation (2). Therefore we can substitute for E in Equation (3) with Et and express
the rate of enzymic action as the rate of product formation (dP/dt) in the sensor to
give:
Integration of Equation (6), assuming Ea is constant gives the concentration of
product, Pt, in the sampler at any time t:
Stability of the substrate
If the substrate is not stable, then the coloured product of spontaneous
breakdown would occur in addition to that due to enzyme activity. Thus, an extra
term would be needed to be added to Equations (6) and (7) to encompass this. If the
374
I. Nitescu et al.
breakdown of substrate was linear of the time (the simplest case), then the new
models are:
dP
Tt
and
where k& is the zero-order rate constant for substrate decay.
Predicted performance of the sensor to detect background levels of protease
It is desirable for the sensor to be able to report the total exposure to enzyme
during the 8-h shift, and also the concentration in the air at any time. The former is
important for monitoring the accumulative dose for an individual. Throughout these
simulations we have assumed that the efficiency of collection is 100%. In practice this
will depend on the collection device selected. All glass liquid impingers and cyclones
seem to give near 100% efficiency for the collection of microbes, but bubblers would
be less efficient (Jensen et al., 1992; Stewart, personal communication, 1995).
(a) Total dose over the 8-h shift. For the calculation of the total dose of enzyme
received by the collector (and the wearer) over the 8-h shift, Equations (6), (7) or (8)
can be used. All the variables will be known in these equations if the 8-h value for
dP/dt is used, which will enable EK to be directly estimated. This can be multiplied by
the total volume of air passing through the sampler to give the total dose received by
the sensor. Hence day-to-day monitoring could be achieved. If the substrate is
unstable, however, the absorbance at the end of the shift does not represent the
exposure level. This absorbance could be corrected by running a parallel collection
chamber with an absolute filter removing all particles and hence protease from the
air bubbling through it. Where a stream of air bubbling through the substrate does
not increase the rate of spontaneous breakdown, then a simple sealed chamber will
act as a control for spontaneous breakdown. The absorbance of the control would
have to be subtracted from the absorbance of the test.
(b) The detection of the airborne concentration in real-time. If the sensor measured
the absorbance of the collection fluid on a continuous basis, then for a constant
background concentration of protease in the air over an 8-h shift period, the
concentration of product will follow Equation (7). It should, thus, increase in
proportion to the square of /. In fact, the absorbance would be measured by the
sensor, but this is readily converted to P with a knowledge of the molar extinction
coefficient. The ability to measure the actual airborne concentration of enzyme
would enable an alarm system to be constructed which would alert the wearer to a
dangerous (pre-set) level. It is obvious that a plot of P vs t would not give this.
However, a plot of the product gradient (dP/dt) should have a linear relationship
with time if the background concentration is constant [Equation (8)]. The rate of
change of product concentration (dP/dt) could readily be determined by a simple
Chromogenic substrates for protease detection
375
routine of subtracting successive absorbance measurements and dividing by the time
interval. Further, it is evident that if the gradient of product gradient itself is plotted
against time, then a constant value will be noted, from which EK can be determined
[Equation (10)]:
d2p
dfl :
(10)
It should be noted that providing any spontaneous breakdown of the substrate
occurs at a constant rate, Equation (10) will be independent of the stability of the
substrate up to the point when so much substrate is broken down that Equation (2)
no longer holds.
RESULTS
Case 1: prediction of the model for background monitoring
To illustrate these concepts, graphs of E, P, dP/dt and d^/dr 2 are shown in Fig.
l(a)-(d), using 7^-succinyl-ala-ala-ala-^-nitroanilide as the chromogen substrate.
This substrate was identified as being both stable and sensitive from our earlier study
(Nitescu et ah, 1996). These data are for the action of Alcalase on the substrates and
are shown in Table 1. Alcalase is not a pure form of enzyme since it contains
stabilizers and prilling agents, hence the units of enzyme are given as Anson units
(AU) which are general units of protease activity and are obtainable for the product
from the manufacturer. For modelling purposes, the differential equations were
integrated using a simple Euler technique. This modelling technique was used as it
will also allow Ea to be changes during the time-course later, to simulate an
accidental release or pulse of protease. The integration programme was checked by
HI
20 1
200 n
15
150
10 -
100-
5-
50"
0-
(b)
0
2
4
6
2
Time (h)
1
0.75 -
4
6
Time(h)
9.07.5-
(c)
(d)
6.04.5
3.0 -
0.50 0.25-
1.5-
0
0.0
Time (h)
Time (h)
Fig. 1. Case 1: a constant background concentration of protease. Model predictions for the accumulation
of (a) enzyme and (b) product in the sensor. The first and second derivatives are shown in (c) and (d),
respectively.
376
I. Nitescu el al.
Table 1. Data for the application of the model based on
the detection of a constant background level of protease
(Case 1). AU=Anson unit of protease activity
50.25
0
*3
*rt
F
E.
1
2x
8x
io-5J
io-
1
/flnol product AU ' m i n - '
ftmo\ of product m - 3 m i n - r
m min
AUm3
(-)
calculating the 8-h values of the key parameters from the integrated equations. The
substrate was found to be stable over an 8-h period (Nitescu et al., 1996), and hence
kd = 0 for the simulation.
Figure l(a)-(d) shows the predicted time courses for the key parameters in the
model, using data from Table 1. It can be seen that the enzyme concentration
changes linearly with time, but the product accumulates in as non-linear fashion.
This is because the rate of reaction is increasing with time: the enzyme collected in
the early part of the sampling procedure is producing product while new protease is
being collected in the later stages of the collection period. Thus, dP/dt increases
linearly with time while the second derivative d2P/dt2 remains constant. The airborne
enzyme concentration (E) can be calculated from the value of the second derivative
using Equation (10), with a knowledge of the other parameters.
Cases 2 and 3: predicted performance of the sensor to detect a sudden release
of protease
A sudden release of protease can be modelled in its simplest form by assuming
the concentration of protease in the air instantly goes up to a new concentration
higher than the background level, and remains so for a fixed period. In practice of
course, the concentration would rise and fall in a more gradual manner.
For the purpose of this paper, the pulse can be modelled in two ways. The first
(case 2) is a release of aerosol containing the same amount of protease in 10 min as
was collected in the whole 8-h collection period of case 1, in the absence of any
background protease. The second pulse simulation (case 3) is to simulate the release
of the protease for a 10-min period but with a low constant background
concentration of protease being present as well. Figure 2(a)-(d) shows this effect
for a 10-min release of air containing 3.84 x 10~3 AU m~3, which is equivalent to
the total amount released in the first simulation (case 1). Table 2 gives details of the
values used in the model. It is evident that the gradient plots [Equations (8) and (10)]
show the effect of the pulse very clearly. Note that the accumulation curve of product
[Fig. 2(b)] is now linear, since no protease is collected after the pulse itself. The
concentration of enzyme is thus constant and so the rate of product accumulation is
constant. This is shown by a constant dP/d/ value in Fig. 2(c). The change in slope
for before and after the pulse is shown in d2P/dr2 [Fig. 2(d)]. Notice that the pulse
only gives a peak in the d 2 P/d^. The gradient plot dP/df does not give a peak. The
total amount of enzyme released in case 1 and case 2 was the same; hence the final Ea
is the same (Table 2). The model predicts that the final P would also be the same
(Table 3). Thus time-averaged data using one 8-h sampling procedure will give the
same exposure level as the pulse. However, it can be seen that the nature of the
Chromogenic substrates for protease detection
20 -i
15 -
200 -i
(a)
(b)
E
"o
10 UJ
E
a
5-
()
2
4
6
50-
2
8
Time (h)
1 0.75 -
1100 8 0 - (d)
E
60
4020X
a. 0
"TJ
0
2
(c)
imol
|OUI
E.
6
E
E
'E
4
Time(h)
In"2
'c
377
0.50 0.25 -
TJ
n"
TJ
1
()
2
4
'
6
1
8
Time (h)
4
6
Time (h)
8
Fig. 2. Case 2: a 10-min pulse of protease after 4-h monitoring, zero background concentration. Model
predictions for the accumulation of (a) enzyme and (b) product in the sensor. The first and second
derivatives are shown in (c) and (d), respectively.
Table 2. Model parameters for the pulse simulation
Case 2
£.
F
V
1
ki
0
3.84 x l O - 3
2xlO~3
5xlO~ 6
1
50.25
0
Case 3
AUm-3
AUm~3
m3 min- 1
m3
(—)
/imol AU""1 min"1
/zmol m min
8x10-'
3.84 xlO~ 3
2xlO-3
5x10-*
1
50.25
0
AUm~3
AUm"3
m3 min"1
m3
(—)
/anol AU" 1 min"1
^mol m~ 3 min"1
Table 3. The 8 h and ^peak values predicted for the key parameters of the from the models.
The figures in parenthesis are the absorbance values based on a molar extinction coefficient of
8800 I."1 mol" 1 cm" 1
E, A U m ~ 3
P, mmol m" 3
dP/dt mmol m" 3 min"'
^ 2 /anol m" 3 min" 2 (peak)
Case 1
background only
Case2
pulse only
Case3
pulse and
background
15.36
185.2(1.6)
0.77 (0.007)
1.6
15.35
181.4(1.6)
0.77 (0.007)
77,tO
30.5
368.3 (3.2)
1.55 (0.014)
78.8,t 1.6
exposure is very different. In the former case (case 1) the worker is exposed to a very
low level over the 8-h period, whilst in the latter case (case 2) the worker would be
exposed to a high concentration for a short time. These differences may be important
in the stimulation of an allergic response.
378
I. Nitescu et al.
30 25 -
350 -i
<
111
2
4
2
4
6
Time (h)
Time (h)
(d)
a.
•a
2
4
Time (h)
6
2
4
6
Time (h)
Fig. 3. Case 3: a 10-min pulie of protease after 4-h monitoring, with a constant background concentration.
Model predictions for the accumulation of (a) enzyme and (b) product in the sensor. The first and second
derivatives are shown in (c) and (d), respectively.
In case 3, when the pulse is released into a background concentration of protease,
it can be seen that the effect of pulse is most easily detected for the gradient plots
[Fig. 3(c) and (d)] where step changes occur. Once again, only d2P/dt2 gives a peak in
correspondence with the pulse of protease release.
Implications for development of the sensor
A sensor such as the one proposed here could be used to detect a sudden release
of protease into the atmosphere. The concentration would thus be high locally and
temporarily. The device would be monitoring the absorbance of product produced in
the collection vessel. It is evident from the modelling that if the gradient of the
absorbance increase was calculated by the device, then any change in the gradient
could trigger an alarm signal. Thus, the sensitivity of the device would be dependent
on the machine used to measure the absorbance. Table 3 shows the maximum values
(8 h or peak) of key parameters in the models proposed. The slight differences are
due to errors in the integration program. While there may be no problem in a
spectrophotometric device measuring a final absorbance of 1.6, as in these case
studies, the ability to measure an absorbance change (for dP/di) of 0.0067 min" 1 is
questionable. Obviously, a lower airborne protease concentration would give an
even lower gradient. More critical perhaps is whether the spectrophotometer will be
able to resolve a difference in the dP/dt gradients = (dV/dr) which will be necessary
if the device is going to trigger an alarm. The relationship between pulse enzyme
concentration and dP/dl 2 expressed as absorbance units is shown in Fig. 4. If the
spectrophotometer can measure absorbance with precision to the third decimal place
then it can be seen that the limit of detection would be about 0.006 AU m~ 3 . In
order to improve the sensitivity of the device,fc3and Fneed to be large and V small,
k3 is dependent on the substrate in terms of the catalytic rate of reaction and the
extinction coefficient of the chromophore (Nitescu et al., 1996).
Chromogenic substrates for protease detection
379
0.002-1
.§. 0.0015c
a
u 0.0010 o
o
c
IS
.a
O 0.0005
0}
0.002
0.004
0.OO6
0.008
0.01
-3,
Protease concentration (AU m )
Fig. 4. The relationship between airborne enzyme concentration and the second derivative expressed in
absorbance units.
Another factor to be considered is the temperature of the collection device. If the
temperature in the factory environment fluctuated, the rate of catalysis of the
chromogenic substrate would change. At present, we do not have information on the
effect of temperature on the rate of reaction; nor indeed the likely temperature
fluctuations which occur in the factory environment. The device could have
thermostatic control of the chamber temperature if it was found that temperature
fluctuation was a problem.
The result of a validation study of this model will be shown in a later paper.
CONCLUSIONS
It is possible to estimate airborne protease levels using a device which depends on
the measurement of the colour produced from a chromogenic substrate. The device
should have a facility for the calculation of the increase in absorbance with time for
an alarm signal to be triggered when the airborne level exceeds a threshold value.
Acknowledgements—This work was supported by the Department of Trade and Industry, Chemical and
Biotechnology Division.
REFERENCES
Behizad, M., Cumming, R. H., Rowell, F. J., Salusbury, T. T. and Stewart, I. W. (1989) Safety in
biotechnology: the use of biosensors for the detection of hazardous biochemkals in the air. Process
Biochem. 24, 126-132.
Jensen, P. A., Todd, W. F., Davis, G. N. and Scarpino, P. V. (1992) Evaluation of eight bioaerosol
samplers challenged with aerosols of free bacteria. Am. ind. Hyg. Ass. J. 53, 660-667.
Nevalainen, A., Pastuszka, J., Liehaber, F. and Willeke, K. (1992) Performance of aerosol samplers:
collection characteristics and sampler design considerations. Almas. Environ. 26A, 531-540.
Nitescu, I., Cumming, R. H. and Rowell, F. J. (1996) Suitability of chromogen substrates for the detection
of Alcalase in an aerosol sampling system. Ann. occvp. Hyg. 40, 361-369.