water interface

water interface

Catalase Monolayers at the Air/Water Interface II. Time-Dependent Processes R O M A N MAKSYMIW 1 AND W A L T E R NITSCH 2 Technische Universitdt Mfinc...

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Catalase Monolayers at the Air/Water Interface II. Time-Dependent Processes R O M A N MAKSYMIW 1 AND W A L T E R NITSCH 2 Technische Universitdt Mfinchen, Institut fur Technische Chemie, Lichtenbergstr. 4, 8046 Garching, FRG Received September 3, 1990; accepted April 15, 1991 The present work is focussed on the behavior ofcatalase at the air/water interface with regard to timedependent processes. Catalase monolayers have been quantitatively established by the method of Trurnit. A pH-dependent process of molecular unfolding is observed. It is demonstrated that this charge-induced unfolding is a slow process running on time scales up to 60 min. The final molecular conformation ranges from compact to almost completely unfolded. The kinetics of the unfolding is shown to resemble a first-order process. Above a critical pressure 7re ~ 18 m N . m -~ , which characteristically occurs in the ~/A-diagrams, vast r-relaxations with characteristic times of 10-102 s are observed. Corresponding to a distortion of the molecules beyond their minimal molecular area A¢, these relaxations are related to the squeezing out of segments into a subsurface region. From the investigation of the desorption kinetics at constant ~r, the desorption is concluded to be a three-step process. An induction period is observed which points at a complex process being necessary for the destabilization of the monolayer. The existence of an adsorption barrier is concluded from time-dependent surface tension measurements at various temperatures. The corresponding activation energy is estimated to about 180 kJ- mol -~ . © 1991Academic Press, Inc.

INTRODUCTION

ers is essential for their use in biosensors (3, 4). Furthermore, the question of denaturation and unfolding, resp., has been pointed out to be crucial for protein crystallization at a fluid interface (5). Recently, X-ray scattering and diffraction have been shown to be powerful tools in the investigation of insoluble amphiphilic monolayers at the air/water interface (see, e.g., (6)). For the application of these advanced methods to macromolecular monolayers such as enzymatic ones, the question of long-term stability as well as interfacial coverage also seems to be substantial. Finally, the problem of enzyme denaturation at interfaces is vital for the field of biotechnology with regard to enzyme immobilization as well as enzyme recovery (see, e.g., (7)). It has been recently demonstrated for lipase monolayers (8) that the quantitative investigation of the enzymatic reaction at the interface is possible on the basis of detailed information about the interfacial coverage and molecular as well as monolayer stability.

From the interfacial behavior of macromolecules--and enzymes, in particular--several basic features arise which are essential with regard to biological systems as well as to biotechnology: interfacial coverage, molecular as well as monolayer stability, lateral ordering due to intermolecular interactions, and the question of enzyme denaturation at interfaces. These topics are intercorrelated and refer to different fields of interfacial macromolecular systems. For example, the number of enzyme molecules as well as their average molecular area are necessary to know for a quantitative investigation of the frequently discussed lipid/ protein monolayers at the air/water interface ( l , 2). Additionally, the question of enzyme activity conservation in such mixed monolayPart of thesis, T U Miinchen, 1990. Present address: Fraunhofer-Gesellschaft, Institut for Grenzfl~ichen- u n d Bioverfahrenstechnik, Stuttgart, FRG. 2 To w h o m correspondence should be addressed. 67

0021-9797/91 $3.00 Journal of Colloid and lnterface Science, Vol. 147, No. 1, November 1991

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

68

MAKSYMIW AND N1TSCH

In (9) (part one of the present paper), some of the aspects outlined above have been discussed for catalase monolayers. On the basis of film balance experiments, the possibility of the quantitative spreading of the enzyme molecules at the air/water interface was demonstrated. Consequently, the resulting pressure / area diagrams were analyzed in view of molecular data. Thereof, the conservation of a compact nondenatured molecular configuration at the interface turned out as the most important result and a mechanism of molecular stabilization was proposed which was referred to as "hydrophobic anchoring." In the present paper, corresponding timedependent processes are reported in view of the basic features faced above. EXPERIMENTAL All film balance experiments were carried out with a FW-1 by Lauda, FRG. The temperature was always T = 20°C. Unless otherwise stated, the compression rate was F = 45 cm 2. m i n - l . Chemicals as wellas the preparation of the enzyme and bulk solutions were as described in (9). The catalase concentration was in the range of 0.5 mg. m1-1 ~ c ~< 1.6 mg. m l - l , and the enzyme solution was confirmed to be pure on native as well as SDS polyacrylamidgel electrophoresis and on gelchromatography (Sephacryl S-300). The bulk solutions (66 mMNa-Phosphate, 1.7 MNaCI, 3 m M NaN3 with pH values of 7.2, 6.4, 5.8, 5.4, 4.6, 4.3 ) were always freshly prepared and filtered through Millipore disc filters (HATF, pore size 0.45 tam). The bulk solutions were confirmed to be pure by compression of the free surface area on the film balance yielding always a ~r-raise < 0.5 m N . m -I on complete compression (compression ratio 1:35). Purity of the bulk solutions was also confirmed by the recording of the surface tension a during up to 5 h in a K_riiss " K 10" tensiometer (DuNouy-Ring) yielding a constant a = 78.6 m N - m -1 at 20°C. Spreading of the catalase solution was carried out according to the method of Trurnit (10) and has been confirmed to be quantitative (9). The effect on Journal of Colloid and Interface Science, Vol. 147, No. l, November 1991

the 7r/A-diagrams of a waiting period At0, which was performed after each spreading procedure, was tested by varying Ato in the range of 0 min ~< At0 ~< 90 min. The total surface area was suitably expanded before each spreading so that there was no detectable ~rraise during the spreading procedure and the succeeding waiting period At0. Thus, always a completely expanded monolayer was established. For the investigation of the behavior of a compressed monolayer at constant total area, the compression was stopped at a pressure ~-, which has been variably chosen. Then, ~r(t) at constant total area was recorded. After reexpansion to the initial total area (zero surface pressure) and a regeneration period of approximately 45 min ("compression/expansion-cycle" described in (9)), the original ~r/A-curve could be reproduced by a further compression. Thus, the lack of molecular loss was confirmed. The waiting period At0 after spreading was always 60 min. Constant pressure experiments were carried out as follows: after a waiting period At0 = 60 min, the catalase monolayers were compressed up to a variable ~r = ~" and ~- was then maintained at a constant value for a variable time At,: 0 min ~< At~ ~< 120 min. Then the monolayer was reexpanded to the initial total area and the molecular loss which occurred at constant ~- during At, was investigated after a 45 min regeneration period ("compression/expansion-cycle" ). The surface tension o- of catalase solutions with various enzyme concentrations (3.3. l0 -3 m g . m l -l ~< c ~ 0.4 mg-m1-1) in 1.7 M NaC1, 0.066 M Phosphate, 0.003 M NAN3, pH 7.2 was measured with a Du-NouyRing tensiometer ( " K 10", Krfiss, F R G ) , as described in (9). Unless otherwise stated, the temperature was 20°C. For the investigation of a temperature effect on the adsorption behavior of catalase (enzyme concentration c = 6.8. I0-3 mg. ml -z ), the temperature of the bulk and the catalase solution were equilibrated at a variable value ( 15°C ~< T ~ 35°C) before injection of the enzyme solution.

CATALASE MONOLAYERS

IIN 4.6



5.0

30 A II

20

*

5.4

®

5.8



6.4



7.2



69

total area is shown for p H 7.2 as a function of time (a schematic plot of the relaxation process as well as a definition of the characteristic relaxation times are given in Fig. 4). F r o m Fig. 3 it is obvious that there are three ~--intervals, each yielding a different relaxation type:

RESULTS

(a) ~ ~< 10 r a N . m - l : a-(t) ~ constant, i.e., there is no ~r-relaxation. (b) 10 m N - m -I ~< ~- ~< 20 m N . m - ~ : a slow 7r decrease with the final amplitude A % = ~ -- ~rend smaller than 5 r a N . m -~ is observed. The final state ( r e"d) is established within a typical time of 103 s. Thus, the corresponding hysteresis is negligible for instantaneous reexpansion. (c) ~- >~ 20 m N . m - l : the typical timescale is 10-102 s and the final amplitude A°a- increases with increasing initial pressure ~- (for ~- = 42 m N . m -1 , the corresponding A°Tr is about 25 m N . m -1 !).

1. In Fig. 1, a critical pressure 7re is defined (insert). In (9), this critical pressure was interpreted in terms of quantitative enzyme spreading to refer to the onset of the closest arrangement of molecules in a monolayer at the interface. Figure 1 represents 7rc as function of the waiting period At0 which has been performed after the spreading procedure. Independent of Ato, 7re amounts to about 18 m N - m -~ for each realized pH-value. Figure 2 shows the molecular area A~ at 7re derived from 7r/A-diagrams, obtained by variation of the waiting period Ato between the end of spreading and the start of compression. As can be seen, there is one curve of Ac vs. At0 for each pH-value of bulkphase. In each case, A~ increases with increased At0, reaching a corresponding final value, A eno, at Ato ~ 20-60 min. As was already pointed out in (9), the final Acend does not depend on the enzyme concentration of the original enzyme solution. 2. z-relaxations occurring after the stop of a continuous compression up to various pressures ~-, have already been discussed in (9) with regard to the corresponding final states. In Fig. 3, now, the decrease of ~- at constant

As a characteristic relaxation time, r05 (time after which the amplitude A~- is half of its maximal value A % ) is shown in Fig. 4 for a p H 7.2 bulkphase as a function of the initial pressure ~ (semilogarithmic plot; definition of T05 see insert sketch). Obviously, for the initial pressure ~ exceeding 20 m N . m -1 , there is a drastic acceleration of the relaxation process: ro.s is of the order of magnitude 103 s for ~" ~<20 m N . m -~ and 10 s in the range ~>~ 20 m N . m -1 . In the range ~- >~ 20 m N . m -1 where the relaxation is running fast (e.g., ~- = 42 m N . m -~) the p H value does not show any effect on the relaxation time. Also, the effect of the compression rate F turns out to be small compared to the decrease by a factor of 10102 s of to.5 (Fig. 5). 3. Figure 6 shows the relative n u m b e r AN/ No of molecules desorbed out of the monolayer during the variable time At, of keeping the pressure at different constant values ~-. It can be clearly seen that for p H 7.2 there is an induction period At ° ~ 14 min during which no loss of molecules is observed under constant ~ = 42 m N . m -~ . For At~ > 14 rain, the

I

i

0

50

I

100 At0Iminl

FIG. 1. Critical surface pressure rc as function of the waiting period Ato for various pH values. 7re corresponds to the closest arrangement of molecules in the interface; insert: definition of re.

Journal of Colloid and Interface Science, Vol. 147, No. 1, November 1991

70

MAKSYMIW AND NITSCH

A~ tA:l 3.10" -A~, • pH4.6

f



2.10~



5.0

~r

5.4

®

5.8



6.4



7.2

1.10k

/

*

® .

OI

i

i

i

0

5

15

30

i

i

60

i

At0[min]

i

90

FIG. 2. Increase of the molecular area Ac (average area per molecule at 7re)by variation of the waiting period At0 for various pH values. A®: calculated molecular area for complete unfolding and a segmental area of 15 A2. Drawn curves are calculated according to Eq. [2].

40Fl(t) ['~--] 30

end

20 "~r . ~ r a n d_ . . . . . . . . . . . . . . . . . . .

10 -~q

I

I

I

o

200

~oo t [see]

FIG. 3. Time-dependent surface pressure ~rat constant total area after stop of compression at various pressures ~-, pH 7.2, 7re"d:corresponding final pressures.

Journal of Colloid and Interface Science, V o l .

147, N o . 1, N o v e m b e r

! 99 l

relative n u m b e r o f desorbed molecules increases with increasing Ate. F o r the same conditions, the m o l e c u l a r loss is r e m a r k a b l y increased by c o n v e c t i o n i n the u n d e r l y i n g b u l k phase, while there still r e m a i n s a n " i n d u c t i o n period." C o m p a r e d to ~" = 42 m N . m - l , there is n o desorptive loss for keeping the m o n o l a y e r at a c o n s t a n t value ~ = 33.6 m N - m -~ d u r i n g 30 min. I n contrast to p H 7.2, the r - c o n s t a n c y at ~- = 33.6 m N . m -~ as well as ~- = 42 m N . m -1 o n a p H 5.8 b u l k solution yields n o detectable loss d u r i n g At~ = 60 m i n , even with convective m o t i o n in the b u l k solution at ~= 42 m N . m -1 . F o r a p H 5.0 b u l k phase, a substantial desorptive loss is observed d u r i n g At, = 60 m i n at ~- = 42 m N - m -~ . Nevertheless, the relative n u m b e r ofdesorbed molecules ( ~ V / N o ~ 0.02) is r e m a r k a b l y smaller t h a n the c o r r e s p o n d i n g value for p H 7.2 (2tN/No 0.09).

CATALASE MONOLAYERS

71

• 1~7.2 ® 5.8 0 5.0

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(t)

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ti5 FIG. 5. Characteristic relaxation time z0.5 vs. compression rate F:, ~- = 42 mN. m -~ .

_

I

II

I

I

I

10

20

30

40

50 I-I (mN/m)

FIG. 4. Definition of the characteristic relaxation time r0.s (insert) and semilogarithmic plot of z0~ as a function of the initial pressure ~, compression up to ~-with constant = 45 c m 2 . m i n - ~ .

4. T h e a d s o r p t i o n b e h a v i o r o f c a t a l a s e in " s t a n d a r d p h o s p h a t e " buffer w i t h p H 7.2 is s h o w n for t h e first 180 m i n a f t e r t h e i n j e c t i o n o f t h e e n z y m e s o l u t i o n in Fig. 7 as t i m e - d e p e n d e n t d e c r e a s e o f t h e s u r f a c e t e n s i o n a for v a r i o u s e n z y m e c o n c e n t r a t i o n s . A s c a n be clearly seen, t h e initial v a l u e s o f ~ for d i f f e r e n t e n z y m e c o n c e n t r a t i o n s are t h e s a m e as for t h e p u r e buffer ( a = 78.6 r a N . m -~ ). F o r the l o w e r e n z y m e c o n c e n t r a t i o n s ( c ~< 10 -2 m g . m l -~ ) a d i s t i n c t i n d u c t i o n p e r i o d r,,0 (cf. Fig. 7, insert) c a n b e o b s e r v e d d u r i n g w h i c h n o d e c r e a s e o f t h e s u r f a c e t e n s i o n occurs. A f t e r 300 m i n , ( d a / d t ) is s m a l l e r t h a n 3 - 1 0 -2 m N . m - l . T h u s , as has a l r e a d y b e e n m e n t i o n e d in ( 9 ) , t h e c o r r e s p o n d i n g a - v a l u e s w e r e t a k e n as t h e

a s y m p t o t i c s u r f a c e t e n s i o n aad. A c c o r d i n g l y , t h e surface p r e s s u r e 7tad = a0 - aad c a u s e d b y a d s o r p t i o n is r e p r e s e n t e d in Fig. 8 as f u n c t i o n Ak

II

0.1~-

0.10 •



0.05-

mmm

oI

~c

s'o

lOO At= [mini

FIG. 6. Relative number AN/No of molecules desorbed during At0 at various constant ff for different pH values. The drawn curve has been calculated according to Eq. [3]. At°: desorptive induction period, m: pH 7.2, # = 42 mN. m ~; • : • + convection; O: pH 7.2, # = 33.6 mNm-l; ®: pH 5.8, ff = 33.6 + 42 mN - m ~+ convection; ~: pH 5.0, ~ = 42 mN • m-~. Journal of Colloid and Interface Science, Vol. 147, No. 1, N o v e m b e r 1991

72

M A K S Y M I W A N D NITSCH

8070-

%_

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I[1°,° t I 100

I 150 t

I 180

[min]

FIG. 7. Surface tension a vs. adsorption time for catalase solutions with different enzyme concentrations. Insert: definition of the adsorptive induction period r,,0.

of the enzyme concentration, yielding a m a x max ~ 24 m N . m - Z . Figure 9 shows the 7raa effect of temperature on the induction period r~,o (see Fig. 7) for ¢ = 6 . 8 . 1 0 - 3 mg. m1-1 in the range 15°C ~ T ~< 30°C: An increased imal

temperature of the enzyme solution yields a decreasing r.,0. DISCUSSION

1. Trurnit Technique for Spreading It has been clearly shown in (8) and (9) that quantitative deposition of molecules in a

~:= oo- o~e[-~-]

Into 20-

4-

2-

10-

o-

0

o

o'.s

I

,:s -tog C

FIG. 8. Adsorptive surface pressure 7tad as function of the enzyme concentration c. 7tad: corresponding surface tension after 180 rain. 7to: surface tension of pure bulk solution.

Journal of Colloid and Interface Science, Vol. 147,No. 1, November1991

I 0,0033

I 0.0034

I 0.0035

T'I[I/K] FIG. 9. Arrhenius-plot of the adsorptive induction period r.,o (Fig. 7, insert); enzyme concentration c = 6 . 8 . 1 0 -3 mg. ml-l.

CATALASE MONOLAYERS

73

monolayer arrangement is achieved at the the bulk to which the changes of the molecular surface of a bulk solution containing a high area in Fig. 2 could be attributed. Furtheramount of salt ( 1.7 M NaCI!) by the Trurnit more, the changes in A t ( t ) and the increase of method (the enzyme concentration was about the final values .4 ~nd, resp., do not refer to in0.9 mg. m1-1 , in the case of catalase mono- termolecular electrostatic repulsion, as has layers). Thus, the establishment ofmultilayer been already demonstrated in (9). Thus, the assemblies at the interface during spreading is changes of the molecular area--which occur definitely excluded. This is reasonable with only during the waiting period Ato--are efregard to spreading experiments carried out fects of intramolecular electrostatic repulsion with a catalase solution which had been caused by lowering the bulk pH below the isostained according to ( 11 ): during spreading of electric point of catalase, leading to a chargethe enzyme solution on pure water and on induced molecular unfolding. From Fig. 2 it bulk solution of the same electrolyte compo- is obvious that the degree of unfolding ranges sition as the catalase solution (0.17 M NaC1, from compact molecules in the case o f p H 7.2 in particular), a slow propagation of the dye- (with a molecular area of about 1780 ,~2 comfront is observed. In the course of the spread- parable to the cross sectional area of about ing, the propagation slowed down and finally 1960 A2 referring to a roughly dumbbell the dyefront stopped. This may be due to in- shaped molecule with a 50 A diameter (13, teraction of the spreading layer with the un- 14)) to almost completely unfolded ones for derlying liquid and to the slow diffusion of the decreasing pH (the final value A ~nd approxiprotein molecules in the interface. Conse- mates ,4~ which is calculated with an average quently, distinct blue floe-like aggregates con- interfacial area of 15 A2 ( 15, 16) for each of centrical to the glass rod are obtained presum- the 2024 amino acid residues (17)). As can ably corresponding to multilayer assemblies. be seen from Fig. 2, the kinetics of the process Therefore, this leads to molecular loss out of of unfolding can be resolved just by variation the interface, i.e., no quantitative deposition of the waiting period At0 between spreading can be achieved. However, if the deposition is and compression; the critical pressure 7re which carried out on the bulk solution with 1.7 M corresponds to the closest arrangement of NaC1, an abrupt spreading of the stained so- molecules in the monolayer is independent of lution over the whole surface occurs. The latter Ato (Fig. 1 ). Therefore, it is possible to comobservation is reasonable in terms of a spread- pare the molecular areas (average area per ing coefficient (12): The surface tension of the molecule at ~rc) with respect to At0 for each bulk phase is O"1 ~ 78.6 m N - m -1 (high NaC1 particular pH-value. The drawn curves in Fig. 2 are calculated content!), that of the enzyme solution is ~2 < 72 m N . m -~ because of protein adsorption in terms of a first-order process for each pH(cf. RESULTS, Adsorption). The interfacial value. Such a process can be understood as tension between the two liquids, ~1/2, vanishes follows: the initial state of the molecules in the because of miscibility of the two aqueous liq- interface is the same for all conditions because uids. Thus, the corresponding spreading coef- the spreading solution is the same in each case. ficient S = or1 - (a2 + at/2) is positive, yielding Thus, it seems to be justified to take the mothe spreading of the enzyme solution over the lecular area for pH 7.2 as that c o m m o n initial total surface area of the bulk solution. Thereof, value ('4c = 1780 A2). The slight increase of Ac for pH 7.2 could be related to molecular a monolayer is obtained. reorientation at the interface as well as to mi2. Kinetics o f Unfolding: 7r = 0 m N . rn -1 nor conformational changes which lead to the From the experimental section it is evident orientation of hydrophobic parts of the molthat there is no contaminating adsorption from ecules toward the unpolar medium. Such conJournal of Colloid and Interface Science, Vol. 147, No. I, N o v e m b e r 1991

74

MAKSYMIW

formational changes may be attributed to what was proposed in (9) to be the establishment of a "hydrophobic anchor." Roughly below the isoelectric point of catalase (pH 5.8 ) ( 18 ), molecular segments are charged by protonating. Intramolecular electrostatic repulsion leads to the unfolding of the molecules, the final degree of unfolding being determined by the amount of charges on each molecule. The time-dependent distribution of unfolded and not-yet-unfolded molecules could be understood by assuming a thermal activation bartier, which has to be overcome for destabilizing the compact conformation. Consequently, the reduction of the number of compact molecules (Ncom) by the charge-induced unfolding during Ato can be represented by a "decay-law", Ncom(Ato) = N o . e x p { A t o . r - l } .

[1]

Taking into account the quantitative spreading (i.e., known total number of molecules, No), the c o m m o n initial value A ° = 1780 A2, as well as the corresponding final values A e,d obtained by measurement, the following expression for the average molecular area Ac as function of Ato can be derived from Eq. [ 1] : &(&t0)

= Ace °~ -

NITSCH

account and must be investigated in each particular case. Furthermore, it should be emphasized that the kinetics of unfolding may also be influenced by the method of protein isolation and preparation, as has been observed for different supports of catalase (unpublished results); presumably unfavorable conditions during the isolation process (for example low pH-value) may lead to a reduced long term stability of the molecules at the interface and an advanced tendency for unfolding, thereof. On the other side, the confirmed lack of unfolding for certain conditions may also be an interesting aspect in view of protein crystallization at fluid interfaces (cf. (5)). In order to obtain reproducible and quantitative results, this should be relevant not only for pure enzyme layers, but also for the frequently discussed enzyme/lipid monolayers for which a standard waiting period of some minutes just for evaporation of the lipid solvent before compression may not be sufficient. Obviously, such important information can be obtained from conventional film balance experiments described above.

3. r-Relaxations

{Ace "~ - A ° }

× exp{--At0, r - ' }.

AND

[2]

The "decay-time" r has been obtained from a plot of ln(A e"d - Ac) vs. At0. As can be clearly seen from Fig. 2, the calculated values derived from Eq. [2 ] are in good accordance with the experimental data. Thus, for each &to the monolayer may be regarded as consisting of Ncom compact molecules and (No - Ncom) unfolded ones with a uniform degree of unfolding corresponding to the pH-value. Surely, the occurrence of a slow process of unfolding, which is found for catalase at the air/water interface, may not be generalized, as can be seen from similar investigations of a staphylococcus carnousus lipase (8). However, the behavior of catalase demonstrates that a possibly slow molecular unfolding on timescales up to 60 rain has to be taken into Journal of Colloid and Interface Science, Vol. 147, No. 1, November 1991

For ~- ~< 10 m N . m -~, no ,r-relaxation is observed. Thus, it can be concluded that the monolayer is in an equilibrium state. In the range roughly between 10 m N - m - ~ and 20 m N . m -~ the corresponding slow ~r-decrease may refer to a reorientation of the molecules in the interface. As can be clearly seen from Fig. 4, the total relaxation process will be remarkably faster if the compression is stopped at a pressure ~r >~ 20 r a N . m -l (~Trc!). This drastic acceleration clearly points at the significance of 7re in view of the activation of a distinct relaxation. Possibly, this relaxation refers to a mechanism of, for example, the establishment of multilayer assemblies or random aggregates. However, such a mechanism should depend on the molecular degree of unfolding. In contrast, this is not the case as is obvious from the final state

CATALASE MONOLAYERS as well as the relaxation times, which turn out to be independent of the p H value and the degree of unfolding, resp. Furthermore, an irreversible loss of molecules out of the monolayer as reason for the observed relaxation at constant total area can be excluded because the original compression curves can be reproduced in each case by a second compression after a certain waiting period. Thus, for a compression beyond the molecular cross sectional area Ac (i.e., ~r > 7re) it seems reasonable to assume that the monolayer is stabilized by forcing out segments into a subsurface region (9, 19). On the other hand, this mechanism m a y also be responsible for the destabilization of the catalase monolayer under a constant high pressure as is discussed. 4. Desorption Catalase monolayers can be compressed up to relatively high pressures (at least up to 42 m N . m -1 ) without any loss of molecules or any irreversible aggregation (9). From Fig. 6 it is obvious that for molecular desorption at p H 7.2 it is additionally necessary to keep the monolayer at a sufficiently high pressure during a m i n i m u m At ° ~ 14 min ("induction period"). In general, the existence of an induction period like the At ° is characteristic for complex processes (20). Because an induction period is observed even with convection in the bulk phase, At ° cannot be related to transport processes. Therefore, it m a y be concluded that also for ~r-constancy a complex process occurs during which the layer is destabilized as a precondition for molecular desorption. This complex process possibly refers to the reduction of the molecular area (i.e., reduction of n u m b e r of attachment sites by squeezing out of segments) below a certain value which should be critical for desorption (cf. (21 )). Another explanation could be found in a possible intermolecular interaction laterally stabilizing the monolayer against desorption. In order to destabilize the monolayer, it could be necessary for some molecules to be forced out

75

of the interface. Consequently, the remaining molecules at the border of the thus created "holes" will have a reduced n u m b e r of next neighbors after a certain induction period. Thereof, their lateral stabilization is weakened. For periods of 7r-constancy longer than At °, a strong influence on the desorption is exerted by convection in the underlying solution. Thus, it m a y be concluded that desorption is governed by instationary diffusion in this time range. The drawn curve in Fig. 6 has been calculated on the basis of a diffusion limited transport in semi-infinite media, AN~No = 2" c*" a - l "

(D/Tr)l/2

X (At, -- At°) ~/2

[3]

(cf. (22, 23)). In Eq. [3], D = 4 . 1 . 1 0 -7 cmz. s -1 is the diffusion coefficient of catalase (24), At ° = 14 min is the experimentally obtained induction period, c* denotes the subsurface equilibrium concentration for p H 7.2 at ~- = 42 m N . m -1 and is obtained from the linear approximation in Fig. 10 (data from Fig. 6 (11)): c* = 6.4- 1013 molecules/ml, a is the n u m b e r of m o l e c u l e s / c m 2 in the interface just at the onset of desorption (i.e., after 14 min at constant ~- = 42 m N - m - 1 ) : a = 3.3.1013 c m -2. Obviously, there is good accordance with the data obtained with a-constant experiments. Consequently, the con-

A._.~l No i

0.1/

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/

/ /

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zxt° =1411111 I o

I lO

FIG. 10. Evaluation of the equilibrium subsurface concentration c* according to Eq. [3] for desorption at ~= 42 mN. m-I (pH 7.2). Results refer to Fig. 6 (11). Journal of Colloid and Interface Science,

Vol. 147, No. 1, N o v e m b e r 1991

76

MAKSYMIW

AND

NITSCH

centration c* in the liquid just below the sur- an interesting aspect of the investigation of the face must be constant. Thus, it is necessary to properties of such macromolecular layers at assume a kinetic equilibrium between the sur- the air/water interface with advanced techface and the subsurface region as well as in- niques such as X-ray scattering or diffraction stationary diffusion as controlling step. (see, e.g., (6)). Conclusively, the molecular desorption may be regarded as a three-step process: (i) desta5. Adsorption bilization of the monolayer in the course of a The most striking feature of the adsorption complex process, (ii) desorption from the surof catalase is the temperature dependency of face, (iii) diffusive transport into the bulk the induction period r~,0 which occurs in the phase. In view of the first step, the lack of dea(t)-curves. According to the Arrhenius-plot tectable loss of molecules during At ° at ~- = 42 in Fig. 9, the existence of an activation energy m N . m -1 = const, seems reasonable, the corcan be concluded. Such an activation energy responding equilibrium concentration c* in for the adsorption step has also been pointed the subsurface region is built up slowly because out by Ivanova et al. (25) for ovalbumine and of the preceding complex process. As a con~-lactoglobulin. This activation energy may be sequence, the diffusive transport into the bulk related to conformational changes which could phase is negligible in this time range. Thus, in be necessary for the transition of a molecule the course of the induction period the molecfrom the bulk phase into the interfacial region. ular desorption is limited by the complex proFor the catalase adsorption this energy barrier cess. can be estimated from Fig. 9 to be about 180 At pH 5.0, the remarkably increased stakJ. m o l - i. bility against desorption is reasonable with reThe maximal pressure rradmaxwhich can be gard to a high number of molecular sites of obtained by adsorption, exclusively, is about attachment because of almost completely un24 m N . m -l (Fig. 8). Thus, ira%ax is remarkfolded molecules. ably smaller than the maximal a- up to which In contrast to the desorption behavior at a monolayer can be compressed, reversibly. pH 7.2 as well as pH 5.0, there is no detectable The fact that no desorption from a monolayer loss for pH 5.8 during a 60 min 7r-constancy occurs even under r-constancy at a compaat 42 m N - m -1 . This higher stability seems rable ~r (Fig. 6 ) points at a stabilization of the intelligible also because of the slightly admolecules in the monolayer upon their contact vanced degree of unfolding and the correwith the air/water interface. Such a mechasponding higher number of attachment sites nism may be attributed to what was proposed compared to pH 7.2. However, with regard to to be a "hydrophobic anchoring" of the enthe reduced but nonvanishing desorption tenzyme molecules at the polar/nonpolar interdency at pH 5.0, it is necessary to assume a face, again. further mechanism of monolayer stabilization at pH 5.8. Presumably, the minimal net charge CONCLUSION of the catalase molecules at this pH (roughly the isoelectric point) causes a higher stability 1. In conventional film balance investigavia reduced protein solubility (19). tions of catalase monolayers at the air/water It should be emphasized here that the over- interface, a pH-controlled unfolding of the all remarkably high stability of the catalase enzyme molecules is observed: pH-dependent, monolayers against desorption is the basis for the molecular configuration ranges from a their quantitative transfer onto solid substrates compact state to almost completely unfolded via LB technique (see, e.g., (19)). Further- ones. The confirmed conservation of a commore, the monolayer stability should also be pact configuration at a pH beyond the isoJournal of Colloid and Interface Science, Vol. 147, No. 1, November 1991

CATALASE MONOLAYERS

electric point demonstrates that enzymes do not necessarily unfold on their contact with the water surface. 2. The kinetics o f the charge-induced process o f unfolding are resolved and turn out to resemble slow first-order processes. Obviously, the possibility o f the occurrence o f such slow unfolding processes has to be taken into account in the investigation of enzymatic monolayer systems, in general, 3. Slow r-relaxations are observed when the monolayer compression is stopped at a pressure ~r ~ 20 m N . m -~ . In comparison, a vast relaxation process is activated at surface pressures beyond ,~20 m N . m -1 . This emphasizes again that the critical pressure re, which amounts to about 20 m N . m -1 , represents a distinguished state o f the e n z y m e monolayer. 4. The ~r-relaxations in the pressure range ~r > 7re are shown to be completely independent o f the pH. Thus, the underlying relaxation mechanisms turn out to be not determined by the molecular degree of unfolding. This observation as well as the complex desorption kinetics obtained from ~r-constancy experiments turn out to be starting points for further investigations. ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschatt (SFB 266). We thank E. Gebhart, T. Pietral3, W. Stfckelhuber, and P. Watzlowik for experimental assistance. REFERENCES 1. Ancelin, H., Zhu, D. G., Petty, M. C., and Yarwood, J., Langmuir 6, 1068 (1990). 2. Haas, H., and Mrhwald, H., Thin Solid Films 180, 101 (1989). 3. Sriyudthsak, M., Yamagishi, H., and Moriizumi, T., Thin SolidFilms 160, 463 (1988).

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4. Okahata, Y., Tsuruta, T., Ijiro, K., and Ariga, K., Langmuir 4, 1373 (1988). 5. Yoshimura, H., Matsumoto, M., Endo, S., and Nagayama, K., Ultramicroscopy 32, 265 (1990). 6. Kjaer, K., Als-Nielsen, J., Helm, C. A., TippmannKrayer, P., and Mrhwald, H., J. Phys. Chem. 93, 3200 (1989). 7. Rehm, H.-J., and Reed, G., Eds., "Biotechnology," Vol. 7a. VCH Verlagsgesellschaft,Weinheim/New York, 1987. 8. Nitsch, W., Maksymiw, R., and Erdmann, H., J. Colloid Interface Sci. 141, 322 ( 1991 ). 9. Nitsch, W., and Maksymiw, R., Colloid Polym. Sci. 268, 452 (1990). 10. Trurnit, H. J., J. ColloidSci. 15, 1 (1960). 11. Bradford, M. M., Anal. Biochem. 72, 248 (1976). 12. Adamson, A. W., "Physical Chemistry of Surfaces." Wiley, New York, 1982. 13. Murthy, M. R. N., Reed lII, T. J., Sicignano, A., Tanaka, N., and Rossmann, M. G., Z Mol. Biol. 152, 465 (1981). 14. Reed III, T. J., Murthy, M. R, N., Sicignano, A., Tanaka, N., Musick, W. D. L., and Rossmann, M. G., Proc. Natl. Acad. Sci. U.S.A. 78, 4767 (1981). 15. Bull, H. B., Adv. Protein Chem. 3, 95 (1947). 16. Yamashita, T., and Bull, H. B., J. Colloid Interface Sci. 24, 310 (1967). 17. Schroeder, W. A., Shelton, J. R., Schelton, J. B., Robberson, B., Apell, G., Fang, R. S., and Bonaventura, J., Arch. Biochem. Biophys. 214, 397 (1982). 18. Nicholls, P., and Schonbaum G. R., in "The Enzymes" (P. D. Boyer, H. A. Lardy, and K. Myrb/ick, Eds.), p. 147. Academic Press, New York, 1963. 19. MacRitchie, F., Adv. Colloid Interface Sci. 25, 341 (1986). 20. Moore, J. W., and Pearson, R. G., "Kinetics and Mechanism," Wiley, New York, 1981. 21. MacRitchie, F., Colloids Surf. 41, 25 (1989). 22. Crank, J., "The Mathematics of Diffusion," Clarendon, Oxford, 1975. 23. Nitsch, W., Kremnitz, W., and Schweyer, G., Ber. Bunsenges. Phys. Chem. 91, 218 (1987). 24. van Holde, K. E., "Physical Biochemistry," PrenticeHall, Englewood Cliffs, N J, 1985. 25. Ivanova, M. G., Panaiotov, J., Bois, A. G., Gargouri, Y., and Verger, R., J. Colloid Interface Sci. 136, 363 (1990).

Journalof Colloidand Inte(faceScience.Vol.147,No. i, November1991