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Inactivation of the enzyme trypsin in aerosols
Inactivation of the Enzyme Trypsin in Aerosols T. T R O U W B O R S T , * J. C. DE J O N G AND K. C. W I N K L E R Laboratory of Microbiology, State University, Catharijnesingd 59, Utrecht, The Netherlands Received December 8, 1972; accepted February 15, 1973
Inactivation of the enzyme trypsin in aerosols is studied after spraying from solutions of NaC1, NaBr and Na2 SO4. At high enzyme concentrations, when the amount of enzyme at the particle surface is small compared to the quantity in the interior of the droplet, no inactivation is found. At concentrations low enough to allow every enzyme molecule to spread at the surface, rapid inactivation is found at high RH (90%). After spraying from 0.1 M NaC1 the time for reduction to 25% of the activity present at t = 1 rain was 25 rain. It is suggested that inactivation is due to surface inactivation. The inactivation could be counteracted by addition of another protein (casein) at a concentration where complete surface occupation is likely to occur. Diffusion to the surface is, however, not the rate limiting process for the inactivation Inactivation at low relative humidity is much slower than at high relative humidity and is also counteracted by addition of casein. To study these phenomena, low enzyme concentrations are needed, and a method is described which can detect a concentration of 10-~ gg of trypsin/liter of air. I. INTRODUCTION Inactivation of biologically active molecules plays an important role in the inactivation of bacteria and viruses in aerosols. Inactivation m a y also occur during spray-drying of proteins. To get more general information about the inactivation of biologically active molecules in aerosols, aerosol inactivation of the enzyme trypsin is studied. The properties of an aerosol sprayed from a solution will depend on the composition of the spray medium and on the relative humidity (RH) of the ambient air. When the vapor pressure of water in the air (P,) is much smaller than the vapor pressure of the saturated solution of salt in the spray medium (Psat), the water will be removed from the aerosol droplets, which then will solidify (1, 2). If P~ > P ~ t the aerosol particles will be droplets, with a salt concentration related to the vapor pres* Present address: Du Pont de Nemours, Dordrecht, The Netherlands.
ure in the aerosol room (3). I n the first case, inactivation will occur in the solid state, whereas in the second case inactivation m a y be caused by toxicity of the concentrated salt solution in the aerosol particles or b y other factors, specific for the aerosol system. An important property of this system is the relatively large surface/volume ratio. This suggests inactivation by a surface dependent process. Previous results have shown that viruses m a y be inactivated at the air/water interface (4-6), and much work has been done on denaturation of proteins at interfaces (7-13). Monolayer studies of enzyme inactivation at interfaces are often hampered b y the problem of determination of the residual enzyme activity in the monolayer (7, 8, 12). With aerosol systems this problem does not arise, provided that the enzyme concentration is low enough to permit every enzyme molecule to reach the surface. This requirement makes it necessary to use very low enzyme 198
Journal of Colloid and Inlerface Science, Vol. 45, No. 1, October 1973
Copyright ~ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
199
ENZYME INACTIVATION
concentrations, which seem to be too low to use conventional enzyme assay and aerosol collection methods. For this reason a special detection method is developed based on the following principles: the aerosol particles are collected with a modified slit sampler (14) by impinging the particles on a slide with an agar layer containing substrate (casein) for trypsin. After collection the agar layer is incubated and the amount of casein which is broken down is determined by measuring the optical density (OD) of the agar/casein layer after staining with a protein dye. The decrease in OD is a measure for the enzyme activity collected. By this method it was possible to detect 10-~ /zg enzyme in 1 liter of air. II. EXPERIMENTAL METHODS
Aerosol Equipment and Performance The aerosols were generated with a spray gun of the type FK-8, which converted 1 ml enzyme solution to an aerosol in about 4 sec. The aerosol was kept at 20°C in a double walled static system of 2000 liters, described previously (15, 16), and was homogenized by a fan. The relative humidity was measured with a LiC1 dewcell element (Foxboro) and recorded
continuously. In all experiments 1 ml solution was sprayed. Just before each experiment, trypsin (crystallized trypsin, for analytical purposes, Boehringer and Sn) was dissolved in the spray solution prepared from double distilled water, and the activity was determined by the method of Schwert and Takenaka (17) using the synthetic substrate benzoyl arginine ethyl ester (BAEE). The activity is expressed in BAEE units, defined as the amount of activity in a mixture of 2.5 ml enzyme solution and 0.5 ml substrate (0.5 mg/ml) in 0.2 M PO4 buffer (pH 7.4), giving an increase in OD at 253 nm of 0.003 extinction units/rain in a light path of 1 cm. In experiments with high enzyme concentrations (5000 #g/ml) and in experiments with radioactive tracers, the aerosols were collected with a raised Porton impinger (18) filled with 0.1 M NaC1, which works at a flow rate of 11.5 liters/rain and has a collection efficiency of 99.7~ for particles of about 1 #m (22). The enzyme activity in the impinger fluid was determined as indicated above. Radioactivity was determined with a scintillation counter (Tracer Lab) using Cerenkov radiation of the 32p label in the whole amount of fluid.
Aerosol
LJ SLit
CoLLection surface
\
,/
\
u
(3
°
°
J ~ J
O ¸
° Conveyer
belt
Air pump FIG. 1. Slit sampler for collection of the aerosols. The collection surface is located at 0.6 mm distance from the slit. Journal of Colloid and Int~faee Science, Vol. 45, No. 1, October 1973
200
TROUWBORST, DE JONG AND WINKLER
FIG. 2. Determination of the decrease in OD (A~) from the plotted OD of the casein layer. ~0 is the OD of the unproteolyzed casein layer.
Slit Sampler The aerosols with low enzyme activity (4 vg/ml in the spray solution) were collected with the special designed slit samplers (Fig. 1). The slit has a width of 0.033 cm, a height of 0.323 cm and a length of 2.75 cm. The collection surface (3.0)< 16.0 cm) was moving during collection at a distance of 0.6 ram, perpendicular to the slit. The speed of the slide could be varied by means of a gear box and was fixed at resp., 2.0 and 1.0 cm/min for samplers I and II. With a negative pressure of 20 cm water in the sampler box, the amount of aerosol collected was 27 liters/rain as determined with a flowmeter (Krohne). The collection efficiency of this type of slit sampler ~E Eo 0.8
0.6
0.4
0.2
IO log )Jg trypsin/zone
FIG. 3. Relation between decrease of OD and the a m o u n t of enzyme p u t in 0.1 M NaC1 on a 0.7 cm broad zone on the casein layer. T h e specific activity of the enzyme was 5.3 B A E E u n i t s / ~ g enzyme.
was reported to be 99o-/0for particles of about 1 vm (22). The samples were taken during 20 sec with the fast sampler (I) and during 40 sec with the slow sampler (II), thus collecting on the same area twice as much aerosol with sampler II. Collection was automated by a sequence timer. After collection slides were kept moving for a short distance to prevent overlapping of zones. Sampling started at 20 sec for sampler I and at 40 sec for sampler I] after start of spraying, and was repeated at 5 min intervals. After collection the agar/casein slides were placed in an airtight box and incubated at 37°C during 22 hr.
Preparations of the A gar/ Substrate Layer The optimal concentration of casein in the agar layer was shown to be 0.2% (3). At lower concentration the substrate becomes soon depleted at high enzyme concentrations, whereas at higher substrate concentration the relation between OD and concentration was no longer linear, resulting in a decreased sensitivity, A solution of 0.4% casein (BDH, light white soluble) in 200 ml 0.02 M phosphate buffer (pH 7.4) in 0.1 M NaC1 and a suspension of 0.4o70 Noble agar (Difco) in 200 ml distilled water were heated in a waterbath at 100°C during 50 Inin. The two solutions were mixed and kept at 56°C during 15 rain. The mixture (50 ml) was then poured in each of 4 trays with a size of 18 X 14 cm. After cooling 4 slides of 3.0 X 16.0 X 0.1 cm were put on the solid layer in each tray. Over these slides, 47.6 ml of a freshly prepared mixture as described above were smoothly spread out. The
dournal of CoUoid end lnter/a*e S,i¢.~.e, Vol. 45, N'o. 1, October 197,~
ENZYME INACTIVATION layers were dried during 40 rain at room temperature, whereafter the trays were closed and kept at 4°C. These layers could be used during 2-10 days after preparation.
Incubation, Fixation, Drying and Staining of the Slides
201
c._~e log co
4
After collection of the aerosol, the agar/ casein layers were incubated at 37°C during 22 hr in a closed box. Thereafter, the layers were carried through 700/0 (v/v) ethanol with 5% (v/v) acetic acid (1.5 hr) and 960-/0 (v/v) acetone during 1.5 hr for fixation and dried at 37°C for 2 hr. The casein was then stained with 1% (w/v) nigrosine (Ciba) in 1% (v/v) acetic acid with 5~o (w/v) NaCI during 2.5 hr and washed twice with 260-/0 (v/v) acetic acid for 45 min. The OD of the colored protein layer was determined with a densitometer (Vitatron). The difference in OD between the trypsine containing zone and the unproteolyzed area (E0), is called be (Fig. 2) and is a measure for the activities of the collected enzyme. No straightforward method is available to relate the value of Ae with absolute enzyme activity. An approximation is possible by spreading a known amount of enzyme in solution over the detection zone. The relation obtained in this way is shown in Fig. 3. This form of application differs, however, from the parcellated ~-o
,5
\ \ \\ \ \
is not \
o~pl.t~-\
\
\
,.,y co,re,\ \
"
surface
\
\ \
\
\
\ \
/\,o'LF% 2
s
4
Log enzyme conc. (~Jg/cc) Fie. 5. A m o u n t of enzyme needed to cover the air/ water interface completely, as a function of the concentrating factor c,/co and calculated for droplets with a radius of 1, 3, 10 and 30 ~ m before evaporation, c, is the salt concentration in the aerosol particle after evaporation to equilibrium and c0 is the salt concentration in the spray medium.
distribution of trypsin in salt crystals or droplets collected from aerosols. For this reason, the following method is used. A second sampler collected twice as much aerosol per unit surface as the first. The 50070 reduction time can now easily be defined as the time necessary for A e on the second sample to decrease to the same level as the initial A~ with the first sampler (Fig. 4). In the tables (results) the values of A~ at t = 1 min (&~l) is tabulated, together with the reduction times t ½, t }, t I, t ~ , representing the times required to inactivate 50, 750-/0, etc., of the enzyme present at t = 1 rain.
Calculation of Protein Concentrations Just Covering the Air/Water Surface of Aerosol Droplets •
~t118
FIG. 4. Schematic representation Of the determination of the reduction times.
The surface of the aerosol droplet after evaporation to equilibrium is determined by the salt concentration in the spray medium and
Journal o] Colloid and Interface Science, Vol. 45, No. 1, October 1973
202
TROUWBORST, DE JONG AND WINKLER TABLE I IIq'AeTIVA'rlOIqOF TRYPSIN IN AEROSOLSAT 90% RH" Spray medium (M)
Enzyme act (BAEE units)
Ael/EO
23.9 24.5 27.2 20.8 23.3 19.7 19.8 24.5 22.8
0.17 0.31 0.28 0.27 0.24 0.29 0.23 0.16 0.19
NaC1, 0.003 NaCI, 0.1
NaBr, 0.003 0.1 NazSO4, 0.003 0.1
Reduction time (min)
RE (%)
t 1/2
t 1/4
t l/S
t 1/16
2 3 6 11 15 5 4 2 7
5 10 24 25 27 17 11 5 17
23 36 58 40 39 35 26 31 36
45 60 -55 53 60 57 60 52
90.0 88.0 87.8 87.4 87.4 86.0 87.0 90.6 91.6
"Sprayed: 4 #g trypsin in 1 ml spray solution. *o: OD of the agar/caseine layer. A,a: decrease of OD for the sample at t = 1 min; - - : t > 60 min. is given a p p r o x i m a t e l y b y
per microgram of enzyme is represented b y ,
4 r ( c ° ) ' X r02,
[1]
-
cm2/ug.
g0 X ~
keel
where co represents the salt concentration in the s p r a y medium, c, is the salt concentration in the aerosol droplet after evaporation to equilibrium and r0 is the radius of the aerosol particle before evaporation. Ce m a y be calculated from the relation between v a p o r pressure and salt concentration (3, 19, 20). If the enzyme concentration in the s p r a y solution is given as a u g / m l , then the available surface &E 8o OJMNoCI R.H.=88%
T h e surface occupied b y a protein spread at an interface is generally accepted to be 1 m g / m 2. If every enzyme molecule m u s t be able to spread completely, then
,
~--
X
gO X ~
(,o), >
10 cm2/ug.
Eo
0.60 0.50
OAO
0,40
0.30
0.30 2
0
~
0
O.IMNaSr R.H.=87%
2
0
~
20
40 rain.
0,10 20
40 mln.
[3-]
\Ce/
This condition is graphically represented in Fig. 5. I n our experiments, with 0.1 M NaC1
0.50
.
[2]
\eel
C,a
0.60
0
X
-
60
60
FIO. 6. Decrease of enzymatic activity (A,) of trypsin after aerosolization of 4 #g enzyme from the indicated medium at about 90% RH. Journal of Colloid and Interface Science, Vol. 45, No. I, October 1973
ENZYME
203
INACTIVATION
TABLE II
to °ctt
g o-~ o
INACTIVATION OF TRYPSIN IN SALT SOLUTIONS AT 2 0 ° C • Salt solution (M)
Activity at t = 2 h r / activity at t = 0 min
NaC1, 2.6 NaBr, 2.6 Na~S04 2.6
02
0.95 0.95 0.77
1.0
1.5
"Trypsin conc. : I00 t~g/ml.
x x R.H.= 89 % • - - - - - 4 R.H= 30 %
as spray fluid, this condition was met, as the enzyme concentration was 4 #g/ml, the radius of the largest aerosol particle was 13/zm and the logarithm of the concentrating factor ce/co was lower than 2.
2.0
03 H Noel, 5000 ~.g enzyme/m t
2~'
,~'
~',
FIG. 7. I n a c t i v a t i o n of t r y p s i n in a e r o s o l s a f t e r s p r a y i n g f r o m 1 rnl 0.1 g N a C 1 w i t h 5 0 0 0 t~g t r y p s i n / r n l .
III. RESULTS
Samples are collected with a liquid impinger and the activity is determined in the irnpinger fluid with the synthetic substrate BAEE.
Inactivation after aerosolization from solutions of NaC1, NaBr, Na2SO4 at high relative humidity is shown in Table I, and some representative inactivation curves in Fig. 6 a and b. A rapid inactivation is found. This inactivation is not due to the high salt concentration in the droplets, as trypsin is stable in a solution of 2.6 M NaC1 (Table II), which has a vapor pressure corresponding with 90% R H (20). Table I I I shows that oxidation is not contributing to the inactivation, as the inactivation occurs at the same rate in an a t m o sphere of pure nitrogen. The possibility was considered that the inactivation at high relative humidities was due to a process of surface inactivation. When high concentrations of enzyme are used, the fraction which can be inactivated at the surface will be relatively low.
This is indeed the case at low and at high R H as one can be seen from Fig. 7. Other proteins would presumably give protection against surface inactivation. Casein indeed gives protection (Table IV), and this protection is dependent on the surface area. Less casein is needed when trypsin is sprayed from low salt concentrations, resulting in a smaller droplet surface after evaporation to equilibrium. Inactivation at low R H is shown in Table V, and some representative inactivation curves in Fig. 8 a, b, c and d. Inactivation is less than at high R H with regard to the initial loss (A e0 as well as for inactivation in time. The contribution of physical loss of aerosol particles by sedimentation and settling on the vessel wall was estimated by z2p as shown in
TABLE
III
AEROSOL INACTIVATION OF TRYPSIN IN A NITROGEN ATMOSPHEREa Spray medium (M)
Enzyme act (BAEE units)
N a C 1 , 0.1
27.1 23.4 25.8
Ael/~0
0.55 0.35 0.12
Reduction time (min) t 1/2
t 1/4
t 1/8
20 5 10
42 21 17
--31
R H (%) t 1/16 --54
28.0 32.3 86.0
a S p r a y e d : 4 # g e n z y m e ; - - : t > 60 m i n . Journal of Colloid and Interface Science, Vol. 45, No. 1, October 1973
204
TROUWBORST, DE JONG AND W I N K L E R TABLE IV INILUENCE OF CASEIN ON THE AF.ROSOL INACTIVATION OF TRYPSIN AT 90% RH ~ Spray medium
Enzyme act (BAEE units)
AEI/*o
Reduction time (min)
t 1/2
t 1/4
t 1/8
RH (%)
t 1/16 89,0 87.6
0203 M NaC1; 0.1 mg casein/ml
15.5 21.3
0.37 0.32
-47
0.1 M NaCl; 0.1 nag casein/ml
27.2 21.8
0.24 0.17
5 7
10 18
21 24
51 60
89.6 90.0
0.1 M NaC1; 0.3 mg casein/ml
25.8
0.30
14
59
--
--
88.0
0.1 M NaC1; 1.0 mg casein/ml
23.9 24.5 25.8
0.21 0.26 0.26
6 30 23
60 49 45
----
----
89.0 89.0 89.0
Sprayed: 4 # g enzyme; - - : t > 60 rain. Fig. 9. T h e t w o f o l d r e d u c t i o n t i m e for p h y s i c a l f a l l o u t w a s l a r g e r t h a n 60 r a i n m e a s u r e d , for 0.003 M N a C 1 a f t e r e s t a b l i s h i n g of t h e aerosol d u r i n g 1 rain. I n t h e case of 0.1 M N a C I i t w a s 30 m i n a t h i g h a n d 40 r a i n a t low r e l a t i v e h u m i d i t y . As, i n b o t h cases, p h y s i c a l loss is smaller than the decrease in collected enzyme a c t i v i t y , t h e e n z y m e is i n a c t i v a t e d a t low R H . T h i s m a y also b e c o n c l u d e d f r o m t h e i n a c t i v a t i o n r a t e a f t e r a d d i t i o n of p r o t e c t i n g s u b stances. As Table VI shows, casein protects
also a g a i n s t i n a c t i v a t i o n a t low R H a n d t h e measured reduction time now corresponds with t h a t for p h y s i c a l f a l l o u t . IV. CONCLUSIONS AND DISCUSSION A t low R H o n l y s l i g h t aerosol i n a c t i v a t i o n of t r y p s i n is f o u n d a f t e r e s t a b l i s h i n g of t h e aerosol d u r i n g 1 rain. A t h i g h R H m o r e r a p i d i n a c t i v a t i o n is f o u n d . T h i s is n o t d u e t o t h e h i g h s a l t c o n c e n t r a t i o n i n t h e d r o p l e t or to o x y d a t i o n , b u t m o s t likely t o s u r f a c e i n a c t i v a -
TABLE V AEROSOL INACTIVATION OF TRYPSIN IN AEROSOLS AT L O W RH a Spray medium (M)
Enzyme act (BAEE units)
Reduction time (min)
A~,/*0 t 1/2
t 1/4
t 1/8
RH (%) t 1/16
NaC1, 0.003
21.7 23.3 24.5
0.48 0.51 0.41
45 22 29
-45 --
----
----
29.4 34.6 26.2
NaC1, 0.1
22.8 24.5 22.8 21.3
0.58 0.50 0.47 0.38
10 6 10 8
27 20 39 28
60 40 -58
-----
34.0 28.0 30.8 42.2
NaBr, 0.003 0.1
18.9 25.8
0.49 0.48
55 9
-26
-57
---
31.7 31.4
Na2SO4, 0.003 0.1
22.3 29.7
0.52 0.52
45 6
-13
-24
---
30.6 2~.6
" Sprayed: 4tag enzyme; - - : t > 60 min. Journal of Colloid and Interface Science, Vol. 45, No. 1, October 1973
ENZYME INACTIVATION
205
a_!
a_._g_~ o
0.60 )
£o
o6o[
O.IMNaCl R.H.=34%
0.50
oooLMN;g
0 , 5 0 ~
0.40 0.50 0.20
0.20[
-
I OJO~-
°l°f°
Ib 2%
4o
do
2b
~o
do
mira
rain.
6o
0.80
0.60 I-
O.IM NaBr
R.H=51%
0'5Cf
050
0.40
0.40
0.30
0.30
0.2C
0.20
OJO
OJO
0.003 MNoBr
d I 20
410
I 60
2'0
rain.
~0
6~
rnin
FIG. 8. Decrease of enzymatic activity (AE) of trypsin after aerosolization of 4 ug enzyme from the indicated medium at about 30 7o R H. tion, as the inactivated fraction is negligible in aerosols with high enzyme concentrations. This is supported by protection with casein as the following calculation of protective concentration demonstrates. The protecting concentration of casein after spraying from 0.1 M NaC1 is found between 0.1 and 0.3 mg/ml. From Eq. [-33 we calculate that at 0.1 mg/ml the surface of droplets of 3.4 um radius then just may be covered, whereas at 0.3 mg/ml this radius is 1.1 ~m. Our particle size distribution shows, that the aerosol particles have a radius nearly corresponding with these sizes (3). The theory of protection by surface occupation thus agrees well with the experimental results. However, it
cannot be excluded that protection is also obtained by substrate binding or by a less specific type of interaction of casein with the enzyme. Absorption of the enzyme at the air/water interface could occur rapidly during the rapid process of evaporation by which process a large amount of enzyme becomes concentrated at the droplet surface. Inactivation in this short time will have a measure in the value of & ~1. After evaporation to equilibrium the rate of inactivation could be determined by diffusion. Considering the process of diffusion, assuming a uniform distribution of enzyme at a certain time t = 0, and if there is no back diffusion from the surface as is generally accepted for protein adsorption at air/water interfaces
Journal of Colloid and Interface Science, Vol. 45, No. 1, October 1933
206
TROUWBORST, DE JONG AND WINKLER to,
log recovery p52(%) 2.0 [
NO
r=
1~
2~
8,u
~.u.
-t..Q
-
1.0
I
3.0
-2.11
• .... _=-. . . .
0.51
• O,IM NaCI R.H.=29.8 % o O.IM NaCI R.H=89.0% = 0.00~ MNaCI R.H.=30.5% • O.O03M NoCI RH.=88,0%
-ZO
0.0 -3.0
,b"
z'o"
3'o" ,¢o"
~o"
FIG. 9. Physical loss of aerosol particles, measured as a decrease in collected amount of aerosolized 3,p. (13), the amount of enzyme adsorbed at the surface at time t(Qt) is given by Q,=
SRe XCe T"
-
-1.tO [oq t (see)
0'.0
FIo. 10. Amount of enzyme left inside the droplet at time ~, if every enzyme molecule diffused to the air/water interface would be adsorbed. Calculated for aerosol droplets with a radius of 1, 2, 4, 8pro. coefficient of trypsin. The whole amount of enzyme at time t - 0 is represented by
Z:--
No = ~%rR o~ X c~.
n=l n 2
n~r~Dt~l ' X exp( where fusion in the of the
-2:0
6b" t
~
/}
[-43
t - 0 represents the time where difstarts, ce is the concentration of enzyme aerosol droplet at t = 0, Re is the radius aerosol particle, and D is the diffusion
i-5-]
The amount of enzyme adsorbed at time t b y diffusion is given b y formula [-4"1, so that the fraction of enzyme not yet adsorbed and thus not inactivated is given by N, 6 N0-1r ~
~__l~exp
~
/1,
TABLE VI INFLUENCE OF CAS1NE ON THE AEROSOL INACTIVATION" OF TRYPSIN AT 30070 R I ~ a Spray m e d i u m (M)
E n z y m e act ( B A E E units)
Aex/eo
0.003 NaC1; 0.1 mg casein/ml
19.6 21.3
0.47 0.39
. .
0.1 NaC1; 0.1 mg casein/ml
25.8 22.2
0.49 0.48
26 29
Reduction time (min) t 1/2
= Sprayed: 4~g enzyme; - - : t ) 60 min. Journal of Colloid and Interface Science, Vol. 45, No. 1, October 1973
l 1/4
. .
. .
t 1/S
. .
R H (%)
t 1/16
28.6 32.4 30.0 30.3
J-6]
207
ENZYME INACTIVATION
where Nt represents the amount of active enzyme at time t and 32o the amount of active enzyme initially present with the assumption that every enzyme molecule which diffused to the surface, is adsorbed and inactivated. This relation is graphically represented in Fig. 10 for various droplet sizes. Our largest particles have a radius of 4.3 gm at equilibrium at 90% RH (3), so even for the largest particles, diffusion is not the rate limiting process for the inactivation. We found that the enzyme activity is reduced by a factor 16 in about 53 rain after t = 1 rain (Table I). This slow inactivation cannot be attributed to prevention of adsorbtion by the presence of previously adsorbed enzyme molecules at the air/water interfaces, because in experiments with 0.1 M NaCI only a small part of the surface is occupied when all the enzyme molecules would be fully spread. It is more likely that charge effects are involved. MacRitchie and Alexander (13) found a large influence of the electrokinetic (zeta) potential at the surface upon the rate of adsorption of various proteins. They suggested that adsorption of proteins would be maximal at their isoelectric point. This point is, in the case of trypsin, found at pH = 10.8 (25) and is far removed from the pH of the neutral solutions used in our experiments. The inactivation at low RH is more difficult to interpret. At low RH the aerosol particles will be solid, but might contain a small layer of adsorbed water (2). The inactivation could happen in this water layer also by a mechanism of surface inactivation. This is supported by protection in experiments with large quantities of enzyme and by addition of casein. However, other explanations are possible. The absolute value of collected enzyme ~ctivity is unknown in the aerosol experiments For reasons mentioned earlier. The values of A~/A0 obtained by putting a known amount of mzyme on the agar/casein surface in the form )f a fluid are, however, not very different from
the values obtained with aerosols, if we assume that in aerosol experiments with maximal protection no inactivation has occurred. An aerosol sample of 18 liter at t = 1 rain will contain 0.023 gg enzyme, as calculated for spraying 4 ug enzyme and corrected for physical loss. A~l/~0 amounts to 0.40 in an experiment with maximal protection (Table VI). We find in Fig. 3 for 0.023 ug enzyme a value for ~ ~1/~0 of 0.56, which does not deviate much from 0.40. Thus, under the assumptions noted above, both measuring systems are comparable. The collection method described is thus able to detect concentrations of enzyme as small as 10-5 ug/liter of air. ACKNOWLEDGMENT The authors wish to thank Miss Sjoukje Kuyper for her excellent technical assistance. REFERENCES 1. HIDALGO, A. F., fred. Eng. Chem. Fundam. 7, 79 (1968). 2. ORR, C., HURD, F. K., AND CORBETT, W. J., J. Colloid Sci. 13, 472 (1958). 3. TROUWBORST, T. Thesis, Utrecht, 1971. 4. ADAMS,M. H., J. Gen. Physiol. 31,417 (1948). 5. TEOUWBOkST, T., DE JONG, J. C., AND WINKLER, K. C., J. Gen. Virol. lS, 235 (1972). 6. TROUWBORST, T., AND WINKLER, K. C., J. Gen. Virol. 17, 1 (1972). 7. RAY, B. R., AND AUGENSTEIN, L. G., Y. Phys. Chem. 10, 1193 (1956). 8. JAM~S, L. K., AND AO~ENSrEIN, L. G., Advan. Enzymol. Rd. Subj. Biochem. 28, 1 (1966). 9. CHEES~AN,D. F., ANDDAVIES, J. T., in "Advances in Protein Chemistry" (M. L. Anson, K. Bailey, and J. T. Edsall, Eds.), Vol. 9. p. 439. Academic Press, New York, 1954. 10. HA~GUC~I, K., J. Biochem (Tokyo) 42, 449 (1955). 11. HAM-~GUCrII, K., J. Biochem. (Tokyo) 42, 705 (1955). 12. AUGENSTEIN, L. G., GmRol,G C. A., AND NIMS, L. F., d. Phys. Chem. 62, 1231 (1958). 13. MAcRII'CHIE, F., AND ALEXANDER, A. E., J. Colloid Sci. 18, 453, 458 (1963). 14. BOUI~-OILLON,R. B., Med. Res. Counc. (Gt. Brit.) Spec. Rep. Ser. 262 (1948).
Journal of Colloid and Interface Science. Vol. 45. No. 1, October 1973
DH 15. DE JONG, J. C., thesis, Utrecht, 1967. K. C., J. Hyg. 66, 16. DE JONG, J. C., AND WINIUER, 557 (1968). 17. SCHWERT, G. W., AND TAKENAKA, Y., B&him. Biophys. Acta 16, 570 (1955). 18. MAY, R. R., AND HARPER, G. J., Byit. J. Id Med. 14,287 (1957). 19. HODGMAN, C. D., “Handbook of Chemistry and Physics.” Chem. Rubber Pub. Co., Cleveland 1955.
Jouvnal
of Colioid aad Interface
Science, Vol. 45, No. 1. October
“Physikalisch-chemischeTaSpringer-Verlag, Berlin, 1931. ZENTNER, R. J., Buded. Rev. 30, 551 (1966). BOURDILLON, R. B., LIDWELL, 0. M., AND THOUS, J. C., 1. Infec. Dis. 41, 197 (1941). TIETZE, F., J. Biol. Chem. 204, 1 (1953). JACOBS, M. W., “Diffusion processes.” SprmgerVerlag, Berlin, 1967.
20. LANDOLT-BBRNSTEIN,
bellen.”
21. 22. 23. 24.
25. TER~NIELLO, L. RAM, J. S., BIER, M., AND NoRD, F. F., Arch. Biochem. Biophys. 57, 252 (19.55).
1973
Inactivation of the enzyme trypsin in aerosols is studied after spraying from solutions of NaC1, NaBr and Na2 SO4. At high enzyme concentrations, when the amount of enzyme at the particle surface is small compared to the quantity in the interior of the droplet, no inactivation is found. At concentrations low enough to allow every enzyme molecule to spread at the surface, rapid inactivation is found at high RH (90%). After spraying from 0.1 M NaC1 the time for reduction to 25% of the activity present at t = 1 rain was 25 rain. It is suggested that inactivation is due to surface inactivation. The inactivation could be counteracted by addition of another protein (casein) at a concentration where complete surface occupation is likely to occur. Diffusion to the surface is, however, not the rate limiting process for the inactivation Inactivation at low relative humidity is much slower than at high relative humidity and is also counteracted by addition of casein. To study these phenomena, low enzyme concentrations are needed, and a method is described which can detect a concentration of 10-~ gg of trypsin/liter of air. I. INTRODUCTION Inactivation of biologically active molecules plays an important role in the inactivation of bacteria and viruses in aerosols. Inactivation m a y also occur during spray-drying of proteins. To get more general information about the inactivation of biologically active molecules in aerosols, aerosol inactivation of the enzyme trypsin is studied. The properties of an aerosol sprayed from a solution will depend on the composition of the spray medium and on the relative humidity (RH) of the ambient air. When the vapor pressure of water in the air (P,) is much smaller than the vapor pressure of the saturated solution of salt in the spray medium (Psat), the water will be removed from the aerosol droplets, which then will solidify (1, 2). If P~ > P ~ t the aerosol particles will be droplets, with a salt concentration related to the vapor pres* Present address: Du Pont de Nemours, Dordrecht, The Netherlands.
ure in the aerosol room (3). I n the first case, inactivation will occur in the solid state, whereas in the second case inactivation m a y be caused by toxicity of the concentrated salt solution in the aerosol particles or b y other factors, specific for the aerosol system. An important property of this system is the relatively large surface/volume ratio. This suggests inactivation by a surface dependent process. Previous results have shown that viruses m a y be inactivated at the air/water interface (4-6), and much work has been done on denaturation of proteins at interfaces (7-13). Monolayer studies of enzyme inactivation at interfaces are often hampered b y the problem of determination of the residual enzyme activity in the monolayer (7, 8, 12). With aerosol systems this problem does not arise, provided that the enzyme concentration is low enough to permit every enzyme molecule to reach the surface. This requirement makes it necessary to use very low enzyme 198
Journal of Colloid and Inlerface Science, Vol. 45, No. 1, October 1973
Copyright ~ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
199
ENZYME INACTIVATION
concentrations, which seem to be too low to use conventional enzyme assay and aerosol collection methods. For this reason a special detection method is developed based on the following principles: the aerosol particles are collected with a modified slit sampler (14) by impinging the particles on a slide with an agar layer containing substrate (casein) for trypsin. After collection the agar layer is incubated and the amount of casein which is broken down is determined by measuring the optical density (OD) of the agar/casein layer after staining with a protein dye. The decrease in OD is a measure for the enzyme activity collected. By this method it was possible to detect 10-~ /zg enzyme in 1 liter of air. II. EXPERIMENTAL METHODS
Aerosol Equipment and Performance The aerosols were generated with a spray gun of the type FK-8, which converted 1 ml enzyme solution to an aerosol in about 4 sec. The aerosol was kept at 20°C in a double walled static system of 2000 liters, described previously (15, 16), and was homogenized by a fan. The relative humidity was measured with a LiC1 dewcell element (Foxboro) and recorded
continuously. In all experiments 1 ml solution was sprayed. Just before each experiment, trypsin (crystallized trypsin, for analytical purposes, Boehringer and Sn) was dissolved in the spray solution prepared from double distilled water, and the activity was determined by the method of Schwert and Takenaka (17) using the synthetic substrate benzoyl arginine ethyl ester (BAEE). The activity is expressed in BAEE units, defined as the amount of activity in a mixture of 2.5 ml enzyme solution and 0.5 ml substrate (0.5 mg/ml) in 0.2 M PO4 buffer (pH 7.4), giving an increase in OD at 253 nm of 0.003 extinction units/rain in a light path of 1 cm. In experiments with high enzyme concentrations (5000 #g/ml) and in experiments with radioactive tracers, the aerosols were collected with a raised Porton impinger (18) filled with 0.1 M NaC1, which works at a flow rate of 11.5 liters/rain and has a collection efficiency of 99.7~ for particles of about 1 #m (22). The enzyme activity in the impinger fluid was determined as indicated above. Radioactivity was determined with a scintillation counter (Tracer Lab) using Cerenkov radiation of the 32p label in the whole amount of fluid.
Aerosol
LJ SLit
CoLLection surface
\
,/
\
u
(3
°
°
J ~ J
O ¸
° Conveyer
belt
Air pump FIG. 1. Slit sampler for collection of the aerosols. The collection surface is located at 0.6 mm distance from the slit. Journal of Colloid and Int~faee Science, Vol. 45, No. 1, October 1973
200
TROUWBORST, DE JONG AND WINKLER
FIG. 2. Determination of the decrease in OD (A~) from the plotted OD of the casein layer. ~0 is the OD of the unproteolyzed casein layer.
Slit Sampler The aerosols with low enzyme activity (4 vg/ml in the spray solution) were collected with the special designed slit samplers (Fig. 1). The slit has a width of 0.033 cm, a height of 0.323 cm and a length of 2.75 cm. The collection surface (3.0)< 16.0 cm) was moving during collection at a distance of 0.6 ram, perpendicular to the slit. The speed of the slide could be varied by means of a gear box and was fixed at resp., 2.0 and 1.0 cm/min for samplers I and II. With a negative pressure of 20 cm water in the sampler box, the amount of aerosol collected was 27 liters/rain as determined with a flowmeter (Krohne). The collection efficiency of this type of slit sampler ~E Eo 0.8
0.6
0.4
0.2
IO log )Jg trypsin/zone
FIG. 3. Relation between decrease of OD and the a m o u n t of enzyme p u t in 0.1 M NaC1 on a 0.7 cm broad zone on the casein layer. T h e specific activity of the enzyme was 5.3 B A E E u n i t s / ~ g enzyme.
was reported to be 99o-/0for particles of about 1 vm (22). The samples were taken during 20 sec with the fast sampler (I) and during 40 sec with the slow sampler (II), thus collecting on the same area twice as much aerosol with sampler II. Collection was automated by a sequence timer. After collection slides were kept moving for a short distance to prevent overlapping of zones. Sampling started at 20 sec for sampler I and at 40 sec for sampler I] after start of spraying, and was repeated at 5 min intervals. After collection the agar/casein slides were placed in an airtight box and incubated at 37°C during 22 hr.
Preparations of the A gar/ Substrate Layer The optimal concentration of casein in the agar layer was shown to be 0.2% (3). At lower concentration the substrate becomes soon depleted at high enzyme concentrations, whereas at higher substrate concentration the relation between OD and concentration was no longer linear, resulting in a decreased sensitivity, A solution of 0.4% casein (BDH, light white soluble) in 200 ml 0.02 M phosphate buffer (pH 7.4) in 0.1 M NaC1 and a suspension of 0.4o70 Noble agar (Difco) in 200 ml distilled water were heated in a waterbath at 100°C during 50 Inin. The two solutions were mixed and kept at 56°C during 15 rain. The mixture (50 ml) was then poured in each of 4 trays with a size of 18 X 14 cm. After cooling 4 slides of 3.0 X 16.0 X 0.1 cm were put on the solid layer in each tray. Over these slides, 47.6 ml of a freshly prepared mixture as described above were smoothly spread out. The
dournal of CoUoid end lnter/a*e S,i¢.~.e, Vol. 45, N'o. 1, October 197,~
ENZYME INACTIVATION layers were dried during 40 rain at room temperature, whereafter the trays were closed and kept at 4°C. These layers could be used during 2-10 days after preparation.
Incubation, Fixation, Drying and Staining of the Slides
201
c._~e log co
4
After collection of the aerosol, the agar/ casein layers were incubated at 37°C during 22 hr in a closed box. Thereafter, the layers were carried through 700/0 (v/v) ethanol with 5% (v/v) acetic acid (1.5 hr) and 960-/0 (v/v) acetone during 1.5 hr for fixation and dried at 37°C for 2 hr. The casein was then stained with 1% (w/v) nigrosine (Ciba) in 1% (v/v) acetic acid with 5~o (w/v) NaCI during 2.5 hr and washed twice with 260-/0 (v/v) acetic acid for 45 min. The OD of the colored protein layer was determined with a densitometer (Vitatron). The difference in OD between the trypsine containing zone and the unproteolyzed area (E0), is called be (Fig. 2) and is a measure for the activities of the collected enzyme. No straightforward method is available to relate the value of Ae with absolute enzyme activity. An approximation is possible by spreading a known amount of enzyme in solution over the detection zone. The relation obtained in this way is shown in Fig. 3. This form of application differs, however, from the parcellated ~-o
,5
\ \ \\ \ \
is not \
o~pl.t~-\
\
\
,.,y co,re,\ \
"
surface
\
\ \
\
\
\ \
/\,o'LF% 2
s
4
Log enzyme conc. (~Jg/cc) Fie. 5. A m o u n t of enzyme needed to cover the air/ water interface completely, as a function of the concentrating factor c,/co and calculated for droplets with a radius of 1, 3, 10 and 30 ~ m before evaporation, c, is the salt concentration in the aerosol particle after evaporation to equilibrium and c0 is the salt concentration in the spray medium.
distribution of trypsin in salt crystals or droplets collected from aerosols. For this reason, the following method is used. A second sampler collected twice as much aerosol per unit surface as the first. The 50070 reduction time can now easily be defined as the time necessary for A e on the second sample to decrease to the same level as the initial A~ with the first sampler (Fig. 4). In the tables (results) the values of A~ at t = 1 min (&~l) is tabulated, together with the reduction times t ½, t }, t I, t ~ , representing the times required to inactivate 50, 750-/0, etc., of the enzyme present at t = 1 rain.
Calculation of Protein Concentrations Just Covering the Air/Water Surface of Aerosol Droplets •
~t118
FIG. 4. Schematic representation Of the determination of the reduction times.
The surface of the aerosol droplet after evaporation to equilibrium is determined by the salt concentration in the spray medium and
Journal o] Colloid and Interface Science, Vol. 45, No. 1, October 1973
202
TROUWBORST, DE JONG AND WINKLER TABLE I IIq'AeTIVA'rlOIqOF TRYPSIN IN AEROSOLSAT 90% RH" Spray medium (M)
Enzyme act (BAEE units)
Ael/EO
23.9 24.5 27.2 20.8 23.3 19.7 19.8 24.5 22.8
0.17 0.31 0.28 0.27 0.24 0.29 0.23 0.16 0.19
NaC1, 0.003 NaCI, 0.1
NaBr, 0.003 0.1 NazSO4, 0.003 0.1
Reduction time (min)
RE (%)
t 1/2
t 1/4
t l/S
t 1/16
2 3 6 11 15 5 4 2 7
5 10 24 25 27 17 11 5 17
23 36 58 40 39 35 26 31 36
45 60 -55 53 60 57 60 52
90.0 88.0 87.8 87.4 87.4 86.0 87.0 90.6 91.6
"Sprayed: 4 #g trypsin in 1 ml spray solution. *o: OD of the agar/caseine layer. A,a: decrease of OD for the sample at t = 1 min; - - : t > 60 min. is given a p p r o x i m a t e l y b y
per microgram of enzyme is represented b y ,
4 r ( c ° ) ' X r02,
[1]
-
cm2/ug.
g0 X ~
keel
where co represents the salt concentration in the s p r a y medium, c, is the salt concentration in the aerosol droplet after evaporation to equilibrium and r0 is the radius of the aerosol particle before evaporation. Ce m a y be calculated from the relation between v a p o r pressure and salt concentration (3, 19, 20). If the enzyme concentration in the s p r a y solution is given as a u g / m l , then the available surface &E 8o OJMNoCI R.H.=88%
T h e surface occupied b y a protein spread at an interface is generally accepted to be 1 m g / m 2. If every enzyme molecule m u s t be able to spread completely, then
,
~--
X
gO X ~
(,o), >
10 cm2/ug.
Eo
0.60 0.50
OAO
0,40
0.30
0.30 2
0
~
0
O.IMNaSr R.H.=87%
2
0
~
20
40 rain.
0,10 20
40 mln.
[3-]
\Ce/
This condition is graphically represented in Fig. 5. I n our experiments, with 0.1 M NaC1
0.50
.
[2]
\eel
C,a
0.60
0
X
-
60
60
FIO. 6. Decrease of enzymatic activity (A,) of trypsin after aerosolization of 4 #g enzyme from the indicated medium at about 90% RH. Journal of Colloid and Interface Science, Vol. 45, No. I, October 1973
ENZYME
203
INACTIVATION
TABLE II
to °ctt
g o-~ o
INACTIVATION OF TRYPSIN IN SALT SOLUTIONS AT 2 0 ° C • Salt solution (M)
Activity at t = 2 h r / activity at t = 0 min
NaC1, 2.6 NaBr, 2.6 Na~S04 2.6
02
0.95 0.95 0.77
1.0
1.5
"Trypsin conc. : I00 t~g/ml.
x x R.H.= 89 % • - - - - - 4 R.H= 30 %
as spray fluid, this condition was met, as the enzyme concentration was 4 #g/ml, the radius of the largest aerosol particle was 13/zm and the logarithm of the concentrating factor ce/co was lower than 2.
2.0
03 H Noel, 5000 ~.g enzyme/m t
2~'
,~'
~',
FIG. 7. I n a c t i v a t i o n of t r y p s i n in a e r o s o l s a f t e r s p r a y i n g f r o m 1 rnl 0.1 g N a C 1 w i t h 5 0 0 0 t~g t r y p s i n / r n l .
III. RESULTS
Samples are collected with a liquid impinger and the activity is determined in the irnpinger fluid with the synthetic substrate BAEE.
Inactivation after aerosolization from solutions of NaC1, NaBr, Na2SO4 at high relative humidity is shown in Table I, and some representative inactivation curves in Fig. 6 a and b. A rapid inactivation is found. This inactivation is not due to the high salt concentration in the droplets, as trypsin is stable in a solution of 2.6 M NaC1 (Table II), which has a vapor pressure corresponding with 90% R H (20). Table I I I shows that oxidation is not contributing to the inactivation, as the inactivation occurs at the same rate in an a t m o sphere of pure nitrogen. The possibility was considered that the inactivation at high relative humidities was due to a process of surface inactivation. When high concentrations of enzyme are used, the fraction which can be inactivated at the surface will be relatively low.
This is indeed the case at low and at high R H as one can be seen from Fig. 7. Other proteins would presumably give protection against surface inactivation. Casein indeed gives protection (Table IV), and this protection is dependent on the surface area. Less casein is needed when trypsin is sprayed from low salt concentrations, resulting in a smaller droplet surface after evaporation to equilibrium. Inactivation at low R H is shown in Table V, and some representative inactivation curves in Fig. 8 a, b, c and d. Inactivation is less than at high R H with regard to the initial loss (A e0 as well as for inactivation in time. The contribution of physical loss of aerosol particles by sedimentation and settling on the vessel wall was estimated by z2p as shown in
TABLE
III
AEROSOL INACTIVATION OF TRYPSIN IN A NITROGEN ATMOSPHEREa Spray medium (M)
Enzyme act (BAEE units)
N a C 1 , 0.1
27.1 23.4 25.8
Ael/~0
0.55 0.35 0.12
Reduction time (min) t 1/2
t 1/4
t 1/8
20 5 10
42 21 17
--31
R H (%) t 1/16 --54
28.0 32.3 86.0
a S p r a y e d : 4 # g e n z y m e ; - - : t > 60 m i n . Journal of Colloid and Interface Science, Vol. 45, No. 1, October 1973
204
TROUWBORST, DE JONG AND W I N K L E R TABLE IV INILUENCE OF CASEIN ON THE AF.ROSOL INACTIVATION OF TRYPSIN AT 90% RH ~ Spray medium
Enzyme act (BAEE units)
AEI/*o
Reduction time (min)
t 1/2
t 1/4
t 1/8
RH (%)
t 1/16 89,0 87.6
0203 M NaC1; 0.1 mg casein/ml
15.5 21.3
0.37 0.32
-47
0.1 M NaCl; 0.1 nag casein/ml
27.2 21.8
0.24 0.17
5 7
10 18
21 24
51 60
89.6 90.0
0.1 M NaC1; 0.3 mg casein/ml
25.8
0.30
14
59
--
--
88.0
0.1 M NaC1; 1.0 mg casein/ml
23.9 24.5 25.8
0.21 0.26 0.26
6 30 23
60 49 45
----
----
89.0 89.0 89.0
Sprayed: 4 # g enzyme; - - : t > 60 rain. Fig. 9. T h e t w o f o l d r e d u c t i o n t i m e for p h y s i c a l f a l l o u t w a s l a r g e r t h a n 60 r a i n m e a s u r e d , for 0.003 M N a C 1 a f t e r e s t a b l i s h i n g of t h e aerosol d u r i n g 1 rain. I n t h e case of 0.1 M N a C I i t w a s 30 m i n a t h i g h a n d 40 r a i n a t low r e l a t i v e h u m i d i t y . As, i n b o t h cases, p h y s i c a l loss is smaller than the decrease in collected enzyme a c t i v i t y , t h e e n z y m e is i n a c t i v a t e d a t low R H . T h i s m a y also b e c o n c l u d e d f r o m t h e i n a c t i v a t i o n r a t e a f t e r a d d i t i o n of p r o t e c t i n g s u b stances. As Table VI shows, casein protects
also a g a i n s t i n a c t i v a t i o n a t low R H a n d t h e measured reduction time now corresponds with t h a t for p h y s i c a l f a l l o u t . IV. CONCLUSIONS AND DISCUSSION A t low R H o n l y s l i g h t aerosol i n a c t i v a t i o n of t r y p s i n is f o u n d a f t e r e s t a b l i s h i n g of t h e aerosol d u r i n g 1 rain. A t h i g h R H m o r e r a p i d i n a c t i v a t i o n is f o u n d . T h i s is n o t d u e t o t h e h i g h s a l t c o n c e n t r a t i o n i n t h e d r o p l e t or to o x y d a t i o n , b u t m o s t likely t o s u r f a c e i n a c t i v a -
TABLE V AEROSOL INACTIVATION OF TRYPSIN IN AEROSOLS AT L O W RH a Spray medium (M)
Enzyme act (BAEE units)
Reduction time (min)
A~,/*0 t 1/2
t 1/4
t 1/8
RH (%) t 1/16
NaC1, 0.003
21.7 23.3 24.5
0.48 0.51 0.41
45 22 29
-45 --
----
----
29.4 34.6 26.2
NaC1, 0.1
22.8 24.5 22.8 21.3
0.58 0.50 0.47 0.38
10 6 10 8
27 20 39 28
60 40 -58
-----
34.0 28.0 30.8 42.2
NaBr, 0.003 0.1
18.9 25.8
0.49 0.48
55 9
-26
-57
---
31.7 31.4
Na2SO4, 0.003 0.1
22.3 29.7
0.52 0.52
45 6
-13
-24
---
30.6 2~.6
" Sprayed: 4tag enzyme; - - : t > 60 min. Journal of Colloid and Interface Science, Vol. 45, No. 1, October 1973
ENZYME INACTIVATION
205
a_!
a_._g_~ o
0.60 )
£o
o6o[
O.IMNaCl R.H.=34%
0.50
oooLMN;g
0 , 5 0 ~
0.40 0.50 0.20
0.20[
-
I OJO~-
°l°f°
Ib 2%
4o
do
2b
~o
do
mira
rain.
6o
0.80
0.60 I-
O.IM NaBr
R.H=51%
0'5Cf
050
0.40
0.40
0.30
0.30
0.2C
0.20
OJO
OJO
0.003 MNoBr
d I 20
410
I 60
2'0
rain.
~0
6~
rnin
FIG. 8. Decrease of enzymatic activity (AE) of trypsin after aerosolization of 4 ug enzyme from the indicated medium at about 30 7o R H. tion, as the inactivated fraction is negligible in aerosols with high enzyme concentrations. This is supported by protection with casein as the following calculation of protective concentration demonstrates. The protecting concentration of casein after spraying from 0.1 M NaC1 is found between 0.1 and 0.3 mg/ml. From Eq. [-33 we calculate that at 0.1 mg/ml the surface of droplets of 3.4 um radius then just may be covered, whereas at 0.3 mg/ml this radius is 1.1 ~m. Our particle size distribution shows, that the aerosol particles have a radius nearly corresponding with these sizes (3). The theory of protection by surface occupation thus agrees well with the experimental results. However, it
cannot be excluded that protection is also obtained by substrate binding or by a less specific type of interaction of casein with the enzyme. Absorption of the enzyme at the air/water interface could occur rapidly during the rapid process of evaporation by which process a large amount of enzyme becomes concentrated at the droplet surface. Inactivation in this short time will have a measure in the value of & ~1. After evaporation to equilibrium the rate of inactivation could be determined by diffusion. Considering the process of diffusion, assuming a uniform distribution of enzyme at a certain time t = 0, and if there is no back diffusion from the surface as is generally accepted for protein adsorption at air/water interfaces
Journal of Colloid and Interface Science, Vol. 45, No. 1, October 1933
206
TROUWBORST, DE JONG AND WINKLER to,
log recovery p52(%) 2.0 [
NO
r=
1~
2~
8,u
~.u.
-t..Q
-
1.0
I
3.0
-2.11
• .... _=-. . . .
0.51
• O,IM NaCI R.H.=29.8 % o O.IM NaCI R.H=89.0% = 0.00~ MNaCI R.H.=30.5% • O.O03M NoCI RH.=88,0%
-ZO
0.0 -3.0
,b"
z'o"
3'o" ,¢o"
~o"
FIG. 9. Physical loss of aerosol particles, measured as a decrease in collected amount of aerosolized 3,p. (13), the amount of enzyme adsorbed at the surface at time t(Qt) is given by Q,=
SRe XCe T"
-
-1.tO [oq t (see)
0'.0
FIo. 10. Amount of enzyme left inside the droplet at time ~, if every enzyme molecule diffused to the air/water interface would be adsorbed. Calculated for aerosol droplets with a radius of 1, 2, 4, 8pro. coefficient of trypsin. The whole amount of enzyme at time t - 0 is represented by
Z:--
No = ~%rR o~ X c~.
n=l n 2
n~r~Dt~l ' X exp( where fusion in the of the
-2:0
6b" t
~
/}
[-43
t - 0 represents the time where difstarts, ce is the concentration of enzyme aerosol droplet at t = 0, Re is the radius aerosol particle, and D is the diffusion
i-5-]
The amount of enzyme adsorbed at time t b y diffusion is given b y formula [-4"1, so that the fraction of enzyme not yet adsorbed and thus not inactivated is given by N, 6 N0-1r ~
~__l~exp
~
/1,
TABLE VI INFLUENCE OF CAS1NE ON THE AEROSOL INACTIVATION" OF TRYPSIN AT 30070 R I ~ a Spray m e d i u m (M)
E n z y m e act ( B A E E units)
Aex/eo
0.003 NaC1; 0.1 mg casein/ml
19.6 21.3
0.47 0.39
. .
0.1 NaC1; 0.1 mg casein/ml
25.8 22.2
0.49 0.48
26 29
Reduction time (min) t 1/2
= Sprayed: 4~g enzyme; - - : t ) 60 min. Journal of Colloid and Interface Science, Vol. 45, No. 1, October 1973
l 1/4
. .
. .
t 1/S
. .
R H (%)
t 1/16
28.6 32.4 30.0 30.3
J-6]
207
ENZYME INACTIVATION
where Nt represents the amount of active enzyme at time t and 32o the amount of active enzyme initially present with the assumption that every enzyme molecule which diffused to the surface, is adsorbed and inactivated. This relation is graphically represented in Fig. 10 for various droplet sizes. Our largest particles have a radius of 4.3 gm at equilibrium at 90% RH (3), so even for the largest particles, diffusion is not the rate limiting process for the inactivation. We found that the enzyme activity is reduced by a factor 16 in about 53 rain after t = 1 rain (Table I). This slow inactivation cannot be attributed to prevention of adsorbtion by the presence of previously adsorbed enzyme molecules at the air/water interfaces, because in experiments with 0.1 M NaCI only a small part of the surface is occupied when all the enzyme molecules would be fully spread. It is more likely that charge effects are involved. MacRitchie and Alexander (13) found a large influence of the electrokinetic (zeta) potential at the surface upon the rate of adsorption of various proteins. They suggested that adsorption of proteins would be maximal at their isoelectric point. This point is, in the case of trypsin, found at pH = 10.8 (25) and is far removed from the pH of the neutral solutions used in our experiments. The inactivation at low RH is more difficult to interpret. At low RH the aerosol particles will be solid, but might contain a small layer of adsorbed water (2). The inactivation could happen in this water layer also by a mechanism of surface inactivation. This is supported by protection in experiments with large quantities of enzyme and by addition of casein. However, other explanations are possible. The absolute value of collected enzyme ~ctivity is unknown in the aerosol experiments For reasons mentioned earlier. The values of A~/A0 obtained by putting a known amount of mzyme on the agar/casein surface in the form )f a fluid are, however, not very different from
the values obtained with aerosols, if we assume that in aerosol experiments with maximal protection no inactivation has occurred. An aerosol sample of 18 liter at t = 1 rain will contain 0.023 gg enzyme, as calculated for spraying 4 ug enzyme and corrected for physical loss. A~l/~0 amounts to 0.40 in an experiment with maximal protection (Table VI). We find in Fig. 3 for 0.023 ug enzyme a value for ~ ~1/~0 of 0.56, which does not deviate much from 0.40. Thus, under the assumptions noted above, both measuring systems are comparable. The collection method described is thus able to detect concentrations of enzyme as small as 10-5 ug/liter of air. ACKNOWLEDGMENT The authors wish to thank Miss Sjoukje Kuyper for her excellent technical assistance. REFERENCES 1. HIDALGO, A. F., fred. Eng. Chem. Fundam. 7, 79 (1968). 2. ORR, C., HURD, F. K., AND CORBETT, W. J., J. Colloid Sci. 13, 472 (1958). 3. TROUWBORST, T. Thesis, Utrecht, 1971. 4. ADAMS,M. H., J. Gen. Physiol. 31,417 (1948). 5. TEOUWBOkST, T., DE JONG, J. C., AND WINKLER, K. C., J. Gen. Virol. lS, 235 (1972). 6. TROUWBORST, T., AND WINKLER, K. C., J. Gen. Virol. 17, 1 (1972). 7. RAY, B. R., AND AUGENSTEIN, L. G., Y. Phys. Chem. 10, 1193 (1956). 8. JAM~S, L. K., AND AO~ENSrEIN, L. G., Advan. Enzymol. Rd. Subj. Biochem. 28, 1 (1966). 9. CHEES~AN,D. F., ANDDAVIES, J. T., in "Advances in Protein Chemistry" (M. L. Anson, K. Bailey, and J. T. Edsall, Eds.), Vol. 9. p. 439. Academic Press, New York, 1954. 10. HA~GUC~I, K., J. Biochem (Tokyo) 42, 449 (1955). 11. HAM-~GUCrII, K., J. Biochem. (Tokyo) 42, 705 (1955). 12. AUGENSTEIN, L. G., GmRol,G C. A., AND NIMS, L. F., d. Phys. Chem. 62, 1231 (1958). 13. MAcRII'CHIE, F., AND ALEXANDER, A. E., J. Colloid Sci. 18, 453, 458 (1963). 14. BOUI~-OILLON,R. B., Med. Res. Counc. (Gt. Brit.) Spec. Rep. Ser. 262 (1948).
Journal of Colloid and Interface Science. Vol. 45. No. 1, October 1973
DH 15. DE JONG, J. C., thesis, Utrecht, 1967. K. C., J. Hyg. 66, 16. DE JONG, J. C., AND WINIUER, 557 (1968). 17. SCHWERT, G. W., AND TAKENAKA, Y., B&him. Biophys. Acta 16, 570 (1955). 18. MAY, R. R., AND HARPER, G. J., Byit. J. Id Med. 14,287 (1957). 19. HODGMAN, C. D., “Handbook of Chemistry and Physics.” Chem. Rubber Pub. Co., Cleveland 1955.
Jouvnal
of Colioid aad Interface
Science, Vol. 45, No. 1. October
“Physikalisch-chemischeTaSpringer-Verlag, Berlin, 1931. ZENTNER, R. J., Buded. Rev. 30, 551 (1966). BOURDILLON, R. B., LIDWELL, 0. M., AND THOUS, J. C., 1. Infec. Dis. 41, 197 (1941). TIETZE, F., J. Biol. Chem. 204, 1 (1953). JACOBS, M. W., “Diffusion processes.” SprmgerVerlag, Berlin, 1967.
20. LANDOLT-BBRNSTEIN,
bellen.”
21. 22. 23. 24.
25. TER~NIELLO, L. RAM, J. S., BIER, M., AND NoRD, F. F., Arch. Biochem. Biophys. 57, 252 (19.55).
1973