Journal of Biochemical and Biophysica! Methods, 25 ( 1992) 45 - 53
45
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-022X/92/$~J5.(10
JBBM 00950
A new micromethod for mechano-chemical research V.N. Morozov, A.V. Gorelov and T.A. Z e n c h e n k o Institute of Theoretical and Experimental Biophysics of the Academy of Sciences of the USSR, Pllshchino, Moscow Region (Russia) (Received 10 December 1991) (Accepted 28 April 1992)
Summary A new micromethod to study activity of enzymes in crystalline and amorphous solid samples subjected to tension is described. Both static (up to 200%) and dynamic (with an amplitude up to 50% and frequency of 10-3-50 Hz) deformation may be applied to the sample during the activity measurement. Strain-induced changes in activity of thin (3-10/~m) needle-like strips (0.2-0.5 p.m long). cut out of the microtome sections of cross-linked crystals of pancreatic carboxypeptidase A is measured in a droplet of substrate solution (with volume of 10-6-10-91)with a pH-microeletrode technique. Key words: Crystalline enzyme; Deformation effect; Mechano-chemistry
Introduction Protein molecules are subjected to mechanical stresses both in living cells (as actin and myosin molecules in contracting muscles [1]) and in biotechnological processes (hydrodynamic stresses and shear deformations in filtration, in flow reactors and other biotechnological procedures [2-4]). Two approaches have been developed to study effects of deformation of enzyme molecules on their catalytic activity. One consisted in applying controlled velocity gradients to enzyme solutions [2,3], another used deformation of protein molecules covalently bound to a surface of deformable polymer fibers [5]. Both approaches suffer from serious drawbacks. Unsteady and uncontrollable deformation of protein molecules in the Correspondence address: V.N. Morozov, Institute of Theoretical and Experimental Biophysics of the Academy of Sciences of the USSR, Pushchino, Moscow Region, 142292, Russia.
46
9
0 11
12
Fig. 1. Schematic for the mechanical part of the device for measurement of enzyme activity in thin crystalline samples subjected to deformation. (1) Supporting base; (2) C-hook; (3) wedge slide; (4) screw-and-nut pusher; (5) clamp; (6) flat spring; (7) sample holder; (8) micromanipulator; (9) humid chamber; (10) clamp; (11) fiat spring; (12) permanent magnet; (13) coil; (14) sample holder.
hydrodynamic method presents a proble~=a in interpreting the experimental results on a molecular level. The method using enzymes immobilized via numerous cross links to the surface of deformable polymers is also not free from limitations: forces applied to the molecules on the surface are randomly distributed by value and randomly directed. One of the ways to produce controllable deformations is to use enzyme monocrystals as an object in these studies. Ordered packing of molecules in crystals provides a uniform distribution of load between molecules, thus permitting the force applied to each individual molecule to be readily calculated. By deforming the crystal along different directions one could also deform the molecules in different directions, thus affecting various degrees of freedom in enzyme molecules. Recently this approach has been advanced in studies of stress-induced denaturation of lysozyme molecules in crystals [6]. Here we present a technique developed for mechano-chemical researches with cross linked enzyme monocrystals as objects. Materials and Methods
General design A mechanical setup of the device is shown in Fig. 1. It consists of 6 simple micromanipulators, 8 controlling positions of pH-microelectrode, reference dec-
47
trode, stirrer, 2 micropipettes, and suction microcapiilary. Each micromanipulator allows adjustment of its holder in 3 dimensions with a precision of a few/zm. The sample attached by its ends to two holders (7 and 14) is placed in a thermostated humid chamber (9). The holders are attached to the double springs (6 and 11), fixed on L-clamps (5 and 10). The left clamp is made to be adjustable in two directions in the plane. The right one can adjust height. Both clamps are fastened on the C=hook (2), which can move in wedge slides (3). This enables uniaxial displacement of the sample in the chamber without disturbing its strain. The screw-and=nut pusher (4) on the left clamp (5) allows the application of static deformation to the sample. One of the cantilevered springs is bent by the pusher touching its end. Since the holder is attached closer to the spring attachment point the holder deflection is about 1/10 of the pusher pass. The arc scale marked on the pusher head facilitates the measurement of sample deformation. The right=hand L-clamp (10) includes a generator of mechanical oscillations. It consists of a permanent rare earth magnet (12) fixed to the end of a long cantilevered spring (11) and a solenoid coil (13). Pulling the magnet in and out of the coil with alternating current applied is transformed into displacements of the sample holder, as described above for static deformation. With 6 holders needed to be introduced into a small volume it is difficult to design a measuring vessel for substrate solution. It was decided, therefore, to perform measurements in a free drop. The method for making the droplets and keeping their volume constant will be considered below.
Sample preparation procedure has been described in our recent papers [6,7] for cross linked lysozyme crystals. Monoclinic crystals of pancreatic carboxypeptidase A (CPA) used here were grown according to the method described in Ref. 8. The crystals were fixed at 25°C for 6 h in 0.1% glutaraldehyde solution prepared on 20 mM veronal buffer with 0.2 N NaCl. Usually, samples measured 300-400/zm x 30~m x 5/zm. Attachment of the sample to the holders. As shown in Fig. 2, the sample (1) is glued by both ends to microdroplets of supercooled ultra=pure sulfur (5) placed at the open end of glass capillaries (4 and 9) each about 100/zm in diameter. The sample is initially glued to one holder in a special device equipped with a micromanipulator, then it is placed in the humid chamber and dipped into a supercooled sulfur droplet on the end of another holder. The droplet is then crystallized by touching it with a tiny cotton fiber previously contacted with sulfur dust. Humid chamber (9) seen in Fig. 1 is made of a piece of brass with 8 grooves cut radially. Through these grooves all microcapillaries and holders penetrate the chamber. The grooves are hermetically sealed with petroleum jelly. The top of the chamber is covered with a microscope cover-glass. To maintain the humidity in the chamber, a droplet of the substrate or buffer solution is placed on its bottom. The sample is illuminated either through the transparent bottom, made of a glass cylinder, or through the cover. All attachment operations and deformation measurements are made under stereo microscope control ( x 100 (10)).
48
8
•
12
Fig. 2. Enlarged top view of the central part of the schematic presented in Fig. 1, showing attachment of the sample and its surroundings when measuring catalytic activity. (1) sample; (2) glass capillary of Sb-microelectrode; (3) Sb tip; (4) glass capillary; (5) sulphur drop; (6) vibrating reed mixer; (7) microcapillary holding droplet of substrate solution; (8) salt bridge of a reference electrode; (9) glass capillary; (10) aspirator; (l 1) microdroplet of substrate solution; (12)buffer-filled capillary.
The chamber is thermostated by a flow of water through a jacket. To prevent dew formation on the cover glass when the chamber temperature is considerably different from that of the room, the chamber was covered with a thermostated cover made of two parallel glasses with a flow of water between them.
Microelectrode fabrication.
The antimony microelectrode design is shown schematically in Fig. 3A. A small amount of Sb placed in a pyrex glass tube is melted in a slight flux of nitrogen through the tube and then is extended in a flame together with the tube to obtain the capillary with exterior diameter of 20-50 ~ m and diameter of Sb core 10-20 ~m. The capillary is then cut into pieces 10-15 mm long. A piece is then inserted by one end into melted Wood's alloy (3) in the glass capillary (1). Copper wire (2) melted in the alloy makes electric contact. The free end of the Sb filled microcapillary was treated before measurements in a drop of 40% hydrofluoric acid for half an hour to make the tip (5) free of covering glass layer (4). The electrode was calibrated by dipping it into standard buffer solutions. The response time of the electrode in the buffer solutions did not e~ceed a few seconds.
49
A
f/////////////~
1
2
3
/
/
/
4
5
B
- ,
/ 1
,
\
/ 2
3
/ / 4
5
Fig. 3. Design of Sb-microelectrode (A) and capillary for making microdroplets of solution (B). A: (1) glass capillary; (2) Cu wire; (3) Wood's alloy; (4) microcapillary filled with Sb; (5) Sb tip. B: (1) glass capillary; (2) solution; (3) thermoplastic polymer; (4) thick-walled capillary; (5) microdroplet.
A few disadvantages of the Sb-microelectrode, namely its low mechanical strength and stability, as well as short operating time due to dissolving of the Sb tip (in a few days), made us design Ir microelectrodes. Ir wire of 0.125 mm in diameter (Aldrich) was mechanically sharpened and covered with an oxide layer in an oven at 800°C after wetting its tip with a concentrated solution of NaOH [9]. After soaking in water for a day, lr microelectrodes demonstrated stable nearly Nemstian response for a few weeks. A standard calomel electrode was used as a reference. It was connected with the substrate droplet via a capillary filled with KCI agar-agar gel.
Micropipettes, as shown in Fig. 3B, were used to form microdroplets of solutions. Substrate solution (2), placed in a wide capillary (1), was then displaced by applying a pressure of about 1 atm to the microcapillary (4). When the meniscus reaches the end of the microcapillary the solution penetrates it and, if the pressure exceeds a critical value (Per = 2or~r, where or is water surface tension, r is the internal radius of the microcapillary; Pcr is approx. 0.4 atm for r = 3 ~m), a microdroplet (5) grows on the end of the microcapillary. When its diameter reaches a required value the pressure is thrown off. Immediately after the meniscus of the liquid (2) comes out of contact with the microcapillary, the changing of the droplet diameter stops, its volume remaining constant provided the humidity in the chamber corresponds to the activity of water in the solution. Special experiments revealed no changes in the droplet volume for at least 10 min when the same solution was placed at the bottom of the chamber to humidify air.
50
To successfully operate both the internal surface of the large capillary (1) and external (not the internal one or the end faces), the surface of the microcapillary (4) should be made hydrophobic with silane treatment. Activity measurements. Before measurement of activity, the sample was placed in buffer solution to check the electrode stability. Then buffer droplet was exchanged for substrate droplet. The sample can penetrate the droplet only partly, as shown in Fig. 2, or the droplet can cover the sample completely, from one sulfur droplet to another. The latter position is advantageous in keeping all of the sample accessible to the solution independently on its deformation. In contrast, the former allows work with much smaller volumes of solution, i.e., to considerably increase the effective concentration of the enzyme. During the activity measurements the solution in the substrate droplet is extensively stirred by the quartz vibrating reed (6). The reed is attachted to a steel needle. The resonant oscillations of the reed are excited through the steel needle with a small electromagnet'. It is possible to change substrate concentration without opening the chamber and refilling the micropipette. For this operation one of the two micropipettes is filled with a concentrated substrate solution, the other with the buffer. To obtain a required concentration, microdroplets of certain sizes of both the substrate solution and of the buffer are blown and mixed. Since the precision of concentration is mainly determined by that of measuring droplet diameters, ti is preferable to blow
pH
Exteneion by 10%
7.4
~
7.2
Releue
~
Extension by 40%
---~ Releue 1rain
~
Fig. 4. An example of using the device for recording enzyme reaction in crystalline CPA subjected to deformation in the [010] direction. Sample (350/zm×85 ?zm×7 gin) in a 0.2-?zl drop of 40 mM hippuryI-DL-phenyllactic acid dissolved in 10 mM veronal buffer (pH 7.0), with 0.2 M NaCl. Hydrolysis by crystalline CPA. Arrows denote apllication and removal of static strain.
51
large droplets, mix them and then diminish the volume of the mixture to the required value using the suction capillary (10). Buffer is also used to wash the sample from substrate and products between measurements. Specific enzyme activity was calculated from measured rates of pH change using the value of the buffer capacity of the substrate solution (measured in a separate experiment), droplet volume (measured under the microscope with a precision of about 5-10%), and amount of enzyme (calculated from substrate-accessible sample volume and crystal lattice parameters, length of the sample and its width being measured under the microscope; for thickness determination Linnik's interferometer was used [7]). Deformation experiments. It is convenient to deform a sample during the recording of the enzyme reaction, thus obtaining internal control. Static deformation of the sample is controlled either directly by measuring length under the microscope, or using the micrometer scale of the pusher. Dynamic deformation can be applied together with static. The upper limit of the available frequencies is determined by the resonant frequency of 60 Hz of the
Activity increase ratio
7 13 6
/
5
/
°
13
13
0
I
I
I
20
40
60
80
Strain, %
Fig. 5. Strain dependence of catalytic activity of CPA in crystalline samples. Other conditions are as described in the legend to Fig. 4. Each point is an average of 3-5 independent measurements. The sample has been subjected to 68% strain in a previous experiment.
52
In V, r a M / r a g r a i n C
o ~
~" o...o D "iF
\% \
-.l 5 I-
\n 2
\
~E3
-25
..... 3
I
I
I
I
31
3.2
33
34
3.5
-1
T
3
36
-I
xlO, K
Fig. 6. Temperature dependence of esterase activity of CPA. See legend to Fig. 4 for experimental conditions.
generator of mechanical oscillations. Maximum amplitude of dynamic deformation reaches 130 ~m (corresponding to about 30% dynamic strain of the sample).
Results and Discussion
The device described here can be used both to study mechano-chemical interactions in enzyme molecules and characteristic properties of enzyme reactions within protein crystals and films. Data presented in Figs. 4-6 represent examples of the device being used in different modes. Fig. 4 presents an example of recording the enzyme kinetics in a crystalline sample subjected to mechanical stress: esterase activity of a cross linked CPA crystal is seen to considerably increase as a result of stretching. Release of the strain restores the initial activity almost completely. As seen from Fig. 5, strain dependence of the effect shows complex behavior, presumably reflecting different mechanisms under different extensions. The temperature dependence of CPA activity in the crystalline state is different from that in solution, where no break in activity vs. the inverse temperature plot was found [10]. A detailed analysis of these and other data concerning the enzyme reaction within crystalline samples is forthcoming. Nevertheless, it is now clear that the method may help in solving a few fundamental problems in enzyme catalysis; namely, to clear up the role of domain and subunit motion in the catalytic act, since deformation may greatly modulate the probability of such motions within crystaili~ae enzymes, and to
53
understand mechanisms underlying crystallization-induced activity changes already well documented for many enzymes [11]. Simplified description of the method and its application The micromethod described here is specially designed for studies of enzyme activity in small cross linked protein crystals subjected to mechanical stress. Undoubtedly, it may also be used in biochemical studies of small muscles and other systems of biological motility, where dependence of enzyme reactions upon deformation is of great importance. The micromethod also has a serious advantage in studies of mechano-chemical coupling in synthetic and natural polymers, such as polypeptides [12], since more rapid equilibration of microspecimens with their surrounding solution is possible and strain-induced pK shifts in much lower fractions of ionizable groups may be followed due to their greater effective concentration in microdroplet. For example, the volume of droplet solution could be only about twice as large as the sample volume: for CPA crystals with a 25-mM protein concentration [13] this would give an effective concentration of 12 mM or 0.4 g/m!! High effective concentrations also give an opportunity to measure trace enzymatic activities in protein samples.
References 1 Bukatina, A.E. (1988) Enzymatic Energy Transducers: General principles. M. Moscow. 2 Tirrel, M. (1975) Shear modification of enzyme kinetics. Biotech. Bioeng. 17, 299-308. 3 Charm, St. and Wong Bing, L. (1981) Action of shear deformation on enzymes. Enz. Microbiol. Technol. 3, 111-118. 4 Ferry, A. and Grazi, E. (1982) Mechano-chemical energy transduction in biological systems. Biochem. J. 205, 281-284. 5 Berezin, I.V., Kiibanov, A.M., Samokhin, G.P. and Martinek, K. (1976) Mechanochemistry of immobilized enzymes: a new approach to studies in fundamental enzymology. In: Mosbach, K. (Ed.), Methods in Enzymology, Academic. Press, pp. 558-571. 6 Gorelov, A.V. and Morozov, V.N. (1987) Mechanical denaturation of globular protein in the solid state. Biophys. Chem. 28, 199-205. 7 Morozov, V.N. and Morozova, T.Ya. (1981) Viscoelastic properties of protein crystals. Triclinic crystals of hen egg white lysozyme in different conditions. Biopolymers 20, 451-467. 8 Quiocho, F.A. and Richards, F.M. (1964) Intermolecular cross-linking of a protein in the crystalline state: carboxypeptidase-A. Proc. Natl. Acad. Sci. USA 52, 833-839. 9 Glab, S., Hulanicki, A., Edwall, G. and Ingman F. (1989) Metal-metal oxide and metal oxide electrodes as pH sensors. Anal. Chem. 21, 29-46. 10 Snoke, .I.E. and Neurath, H. (1949) .I. Biol. Chem. 181, 789-802. 11 Rupley, J. (1969) Comparison of protein structure in the crystal and in solution. In: Timasheff, S.N. and Fasman, G.D. (Eds.), Structure and Stability of Biological Macromolecules, Marcel Dekker, New York, pp. 255-319. 12 Urry, D.W., Peng, S.Q., Hayes, L., Jaggard, J. and Harris, D.R. (1990) A new mechanism of mechanochemical coupling: stretch-induced increase in carboxyl pK a as a diagnostic. Biopolymers 30, 215-218. 13 Lipscomb, W.N. (1973) Enzymatic activity of carboxypeptidase A" in solution and in crystals. Proc. Natl. Acad. Sci. USA 70, 3797-3801.
45
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-022X/92/$~J5.(10
JBBM 00950
A new micromethod for mechano-chemical research V.N. Morozov, A.V. Gorelov and T.A. Z e n c h e n k o Institute of Theoretical and Experimental Biophysics of the Academy of Sciences of the USSR, Pllshchino, Moscow Region (Russia) (Received 10 December 1991) (Accepted 28 April 1992)
Summary A new micromethod to study activity of enzymes in crystalline and amorphous solid samples subjected to tension is described. Both static (up to 200%) and dynamic (with an amplitude up to 50% and frequency of 10-3-50 Hz) deformation may be applied to the sample during the activity measurement. Strain-induced changes in activity of thin (3-10/~m) needle-like strips (0.2-0.5 p.m long). cut out of the microtome sections of cross-linked crystals of pancreatic carboxypeptidase A is measured in a droplet of substrate solution (with volume of 10-6-10-91)with a pH-microeletrode technique. Key words: Crystalline enzyme; Deformation effect; Mechano-chemistry
Introduction Protein molecules are subjected to mechanical stresses both in living cells (as actin and myosin molecules in contracting muscles [1]) and in biotechnological processes (hydrodynamic stresses and shear deformations in filtration, in flow reactors and other biotechnological procedures [2-4]). Two approaches have been developed to study effects of deformation of enzyme molecules on their catalytic activity. One consisted in applying controlled velocity gradients to enzyme solutions [2,3], another used deformation of protein molecules covalently bound to a surface of deformable polymer fibers [5]. Both approaches suffer from serious drawbacks. Unsteady and uncontrollable deformation of protein molecules in the Correspondence address: V.N. Morozov, Institute of Theoretical and Experimental Biophysics of the Academy of Sciences of the USSR, Pushchino, Moscow Region, 142292, Russia.
46
9
0 11
12
Fig. 1. Schematic for the mechanical part of the device for measurement of enzyme activity in thin crystalline samples subjected to deformation. (1) Supporting base; (2) C-hook; (3) wedge slide; (4) screw-and-nut pusher; (5) clamp; (6) flat spring; (7) sample holder; (8) micromanipulator; (9) humid chamber; (10) clamp; (11) fiat spring; (12) permanent magnet; (13) coil; (14) sample holder.
hydrodynamic method presents a proble~=a in interpreting the experimental results on a molecular level. The method using enzymes immobilized via numerous cross links to the surface of deformable polymers is also not free from limitations: forces applied to the molecules on the surface are randomly distributed by value and randomly directed. One of the ways to produce controllable deformations is to use enzyme monocrystals as an object in these studies. Ordered packing of molecules in crystals provides a uniform distribution of load between molecules, thus permitting the force applied to each individual molecule to be readily calculated. By deforming the crystal along different directions one could also deform the molecules in different directions, thus affecting various degrees of freedom in enzyme molecules. Recently this approach has been advanced in studies of stress-induced denaturation of lysozyme molecules in crystals [6]. Here we present a technique developed for mechano-chemical researches with cross linked enzyme monocrystals as objects. Materials and Methods
General design A mechanical setup of the device is shown in Fig. 1. It consists of 6 simple micromanipulators, 8 controlling positions of pH-microelectrode, reference dec-
47
trode, stirrer, 2 micropipettes, and suction microcapiilary. Each micromanipulator allows adjustment of its holder in 3 dimensions with a precision of a few/zm. The sample attached by its ends to two holders (7 and 14) is placed in a thermostated humid chamber (9). The holders are attached to the double springs (6 and 11), fixed on L-clamps (5 and 10). The left clamp is made to be adjustable in two directions in the plane. The right one can adjust height. Both clamps are fastened on the C=hook (2), which can move in wedge slides (3). This enables uniaxial displacement of the sample in the chamber without disturbing its strain. The screw-and=nut pusher (4) on the left clamp (5) allows the application of static deformation to the sample. One of the cantilevered springs is bent by the pusher touching its end. Since the holder is attached closer to the spring attachment point the holder deflection is about 1/10 of the pusher pass. The arc scale marked on the pusher head facilitates the measurement of sample deformation. The right=hand L-clamp (10) includes a generator of mechanical oscillations. It consists of a permanent rare earth magnet (12) fixed to the end of a long cantilevered spring (11) and a solenoid coil (13). Pulling the magnet in and out of the coil with alternating current applied is transformed into displacements of the sample holder, as described above for static deformation. With 6 holders needed to be introduced into a small volume it is difficult to design a measuring vessel for substrate solution. It was decided, therefore, to perform measurements in a free drop. The method for making the droplets and keeping their volume constant will be considered below.
Sample preparation procedure has been described in our recent papers [6,7] for cross linked lysozyme crystals. Monoclinic crystals of pancreatic carboxypeptidase A (CPA) used here were grown according to the method described in Ref. 8. The crystals were fixed at 25°C for 6 h in 0.1% glutaraldehyde solution prepared on 20 mM veronal buffer with 0.2 N NaCl. Usually, samples measured 300-400/zm x 30~m x 5/zm. Attachment of the sample to the holders. As shown in Fig. 2, the sample (1) is glued by both ends to microdroplets of supercooled ultra=pure sulfur (5) placed at the open end of glass capillaries (4 and 9) each about 100/zm in diameter. The sample is initially glued to one holder in a special device equipped with a micromanipulator, then it is placed in the humid chamber and dipped into a supercooled sulfur droplet on the end of another holder. The droplet is then crystallized by touching it with a tiny cotton fiber previously contacted with sulfur dust. Humid chamber (9) seen in Fig. 1 is made of a piece of brass with 8 grooves cut radially. Through these grooves all microcapillaries and holders penetrate the chamber. The grooves are hermetically sealed with petroleum jelly. The top of the chamber is covered with a microscope cover-glass. To maintain the humidity in the chamber, a droplet of the substrate or buffer solution is placed on its bottom. The sample is illuminated either through the transparent bottom, made of a glass cylinder, or through the cover. All attachment operations and deformation measurements are made under stereo microscope control ( x 100 (10)).
48
8
•
12
Fig. 2. Enlarged top view of the central part of the schematic presented in Fig. 1, showing attachment of the sample and its surroundings when measuring catalytic activity. (1) sample; (2) glass capillary of Sb-microelectrode; (3) Sb tip; (4) glass capillary; (5) sulphur drop; (6) vibrating reed mixer; (7) microcapillary holding droplet of substrate solution; (8) salt bridge of a reference electrode; (9) glass capillary; (10) aspirator; (l 1) microdroplet of substrate solution; (12)buffer-filled capillary.
The chamber is thermostated by a flow of water through a jacket. To prevent dew formation on the cover glass when the chamber temperature is considerably different from that of the room, the chamber was covered with a thermostated cover made of two parallel glasses with a flow of water between them.
Microelectrode fabrication.
The antimony microelectrode design is shown schematically in Fig. 3A. A small amount of Sb placed in a pyrex glass tube is melted in a slight flux of nitrogen through the tube and then is extended in a flame together with the tube to obtain the capillary with exterior diameter of 20-50 ~ m and diameter of Sb core 10-20 ~m. The capillary is then cut into pieces 10-15 mm long. A piece is then inserted by one end into melted Wood's alloy (3) in the glass capillary (1). Copper wire (2) melted in the alloy makes electric contact. The free end of the Sb filled microcapillary was treated before measurements in a drop of 40% hydrofluoric acid for half an hour to make the tip (5) free of covering glass layer (4). The electrode was calibrated by dipping it into standard buffer solutions. The response time of the electrode in the buffer solutions did not e~ceed a few seconds.
49
A
f/////////////~
1
2
3
/
/
/
4
5
B
- ,
/ 1
,
\
/ 2
3
/ / 4
5
Fig. 3. Design of Sb-microelectrode (A) and capillary for making microdroplets of solution (B). A: (1) glass capillary; (2) Cu wire; (3) Wood's alloy; (4) microcapillary filled with Sb; (5) Sb tip. B: (1) glass capillary; (2) solution; (3) thermoplastic polymer; (4) thick-walled capillary; (5) microdroplet.
A few disadvantages of the Sb-microelectrode, namely its low mechanical strength and stability, as well as short operating time due to dissolving of the Sb tip (in a few days), made us design Ir microelectrodes. Ir wire of 0.125 mm in diameter (Aldrich) was mechanically sharpened and covered with an oxide layer in an oven at 800°C after wetting its tip with a concentrated solution of NaOH [9]. After soaking in water for a day, lr microelectrodes demonstrated stable nearly Nemstian response for a few weeks. A standard calomel electrode was used as a reference. It was connected with the substrate droplet via a capillary filled with KCI agar-agar gel.
Micropipettes, as shown in Fig. 3B, were used to form microdroplets of solutions. Substrate solution (2), placed in a wide capillary (1), was then displaced by applying a pressure of about 1 atm to the microcapillary (4). When the meniscus reaches the end of the microcapillary the solution penetrates it and, if the pressure exceeds a critical value (Per = 2or~r, where or is water surface tension, r is the internal radius of the microcapillary; Pcr is approx. 0.4 atm for r = 3 ~m), a microdroplet (5) grows on the end of the microcapillary. When its diameter reaches a required value the pressure is thrown off. Immediately after the meniscus of the liquid (2) comes out of contact with the microcapillary, the changing of the droplet diameter stops, its volume remaining constant provided the humidity in the chamber corresponds to the activity of water in the solution. Special experiments revealed no changes in the droplet volume for at least 10 min when the same solution was placed at the bottom of the chamber to humidify air.
50
To successfully operate both the internal surface of the large capillary (1) and external (not the internal one or the end faces), the surface of the microcapillary (4) should be made hydrophobic with silane treatment. Activity measurements. Before measurement of activity, the sample was placed in buffer solution to check the electrode stability. Then buffer droplet was exchanged for substrate droplet. The sample can penetrate the droplet only partly, as shown in Fig. 2, or the droplet can cover the sample completely, from one sulfur droplet to another. The latter position is advantageous in keeping all of the sample accessible to the solution independently on its deformation. In contrast, the former allows work with much smaller volumes of solution, i.e., to considerably increase the effective concentration of the enzyme. During the activity measurements the solution in the substrate droplet is extensively stirred by the quartz vibrating reed (6). The reed is attachted to a steel needle. The resonant oscillations of the reed are excited through the steel needle with a small electromagnet'. It is possible to change substrate concentration without opening the chamber and refilling the micropipette. For this operation one of the two micropipettes is filled with a concentrated substrate solution, the other with the buffer. To obtain a required concentration, microdroplets of certain sizes of both the substrate solution and of the buffer are blown and mixed. Since the precision of concentration is mainly determined by that of measuring droplet diameters, ti is preferable to blow
pH
Exteneion by 10%
7.4
~
7.2
Releue
~
Extension by 40%
---~ Releue 1rain
~
Fig. 4. An example of using the device for recording enzyme reaction in crystalline CPA subjected to deformation in the [010] direction. Sample (350/zm×85 ?zm×7 gin) in a 0.2-?zl drop of 40 mM hippuryI-DL-phenyllactic acid dissolved in 10 mM veronal buffer (pH 7.0), with 0.2 M NaCl. Hydrolysis by crystalline CPA. Arrows denote apllication and removal of static strain.
51
large droplets, mix them and then diminish the volume of the mixture to the required value using the suction capillary (10). Buffer is also used to wash the sample from substrate and products between measurements. Specific enzyme activity was calculated from measured rates of pH change using the value of the buffer capacity of the substrate solution (measured in a separate experiment), droplet volume (measured under the microscope with a precision of about 5-10%), and amount of enzyme (calculated from substrate-accessible sample volume and crystal lattice parameters, length of the sample and its width being measured under the microscope; for thickness determination Linnik's interferometer was used [7]). Deformation experiments. It is convenient to deform a sample during the recording of the enzyme reaction, thus obtaining internal control. Static deformation of the sample is controlled either directly by measuring length under the microscope, or using the micrometer scale of the pusher. Dynamic deformation can be applied together with static. The upper limit of the available frequencies is determined by the resonant frequency of 60 Hz of the
Activity increase ratio
7 13 6
/
5
/
°
13
13
0
I
I
I
20
40
60
80
Strain, %
Fig. 5. Strain dependence of catalytic activity of CPA in crystalline samples. Other conditions are as described in the legend to Fig. 4. Each point is an average of 3-5 independent measurements. The sample has been subjected to 68% strain in a previous experiment.
52
In V, r a M / r a g r a i n C
o ~
~" o...o D "iF
\% \
-.l 5 I-
\n 2
\
~E3
-25
..... 3
I
I
I
I
31
3.2
33
34
3.5
-1
T
3
36
-I
xlO, K
Fig. 6. Temperature dependence of esterase activity of CPA. See legend to Fig. 4 for experimental conditions.
generator of mechanical oscillations. Maximum amplitude of dynamic deformation reaches 130 ~m (corresponding to about 30% dynamic strain of the sample).
Results and Discussion
The device described here can be used both to study mechano-chemical interactions in enzyme molecules and characteristic properties of enzyme reactions within protein crystals and films. Data presented in Figs. 4-6 represent examples of the device being used in different modes. Fig. 4 presents an example of recording the enzyme kinetics in a crystalline sample subjected to mechanical stress: esterase activity of a cross linked CPA crystal is seen to considerably increase as a result of stretching. Release of the strain restores the initial activity almost completely. As seen from Fig. 5, strain dependence of the effect shows complex behavior, presumably reflecting different mechanisms under different extensions. The temperature dependence of CPA activity in the crystalline state is different from that in solution, where no break in activity vs. the inverse temperature plot was found [10]. A detailed analysis of these and other data concerning the enzyme reaction within crystalline samples is forthcoming. Nevertheless, it is now clear that the method may help in solving a few fundamental problems in enzyme catalysis; namely, to clear up the role of domain and subunit motion in the catalytic act, since deformation may greatly modulate the probability of such motions within crystaili~ae enzymes, and to
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understand mechanisms underlying crystallization-induced activity changes already well documented for many enzymes [11]. Simplified description of the method and its application The micromethod described here is specially designed for studies of enzyme activity in small cross linked protein crystals subjected to mechanical stress. Undoubtedly, it may also be used in biochemical studies of small muscles and other systems of biological motility, where dependence of enzyme reactions upon deformation is of great importance. The micromethod also has a serious advantage in studies of mechano-chemical coupling in synthetic and natural polymers, such as polypeptides [12], since more rapid equilibration of microspecimens with their surrounding solution is possible and strain-induced pK shifts in much lower fractions of ionizable groups may be followed due to their greater effective concentration in microdroplet. For example, the volume of droplet solution could be only about twice as large as the sample volume: for CPA crystals with a 25-mM protein concentration [13] this would give an effective concentration of 12 mM or 0.4 g/m!! High effective concentrations also give an opportunity to measure trace enzymatic activities in protein samples.
References 1 Bukatina, A.E. (1988) Enzymatic Energy Transducers: General principles. M. Moscow. 2 Tirrel, M. (1975) Shear modification of enzyme kinetics. Biotech. Bioeng. 17, 299-308. 3 Charm, St. and Wong Bing, L. (1981) Action of shear deformation on enzymes. Enz. Microbiol. Technol. 3, 111-118. 4 Ferry, A. and Grazi, E. (1982) Mechano-chemical energy transduction in biological systems. Biochem. J. 205, 281-284. 5 Berezin, I.V., Kiibanov, A.M., Samokhin, G.P. and Martinek, K. (1976) Mechanochemistry of immobilized enzymes: a new approach to studies in fundamental enzymology. In: Mosbach, K. (Ed.), Methods in Enzymology, Academic. Press, pp. 558-571. 6 Gorelov, A.V. and Morozov, V.N. (1987) Mechanical denaturation of globular protein in the solid state. Biophys. Chem. 28, 199-205. 7 Morozov, V.N. and Morozova, T.Ya. (1981) Viscoelastic properties of protein crystals. Triclinic crystals of hen egg white lysozyme in different conditions. Biopolymers 20, 451-467. 8 Quiocho, F.A. and Richards, F.M. (1964) Intermolecular cross-linking of a protein in the crystalline state: carboxypeptidase-A. Proc. Natl. Acad. Sci. USA 52, 833-839. 9 Glab, S., Hulanicki, A., Edwall, G. and Ingman F. (1989) Metal-metal oxide and metal oxide electrodes as pH sensors. Anal. Chem. 21, 29-46. 10 Snoke, .I.E. and Neurath, H. (1949) .I. Biol. Chem. 181, 789-802. 11 Rupley, J. (1969) Comparison of protein structure in the crystal and in solution. In: Timasheff, S.N. and Fasman, G.D. (Eds.), Structure and Stability of Biological Macromolecules, Marcel Dekker, New York, pp. 255-319. 12 Urry, D.W., Peng, S.Q., Hayes, L., Jaggard, J. and Harris, D.R. (1990) A new mechanism of mechanochemical coupling: stretch-induced increase in carboxyl pK a as a diagnostic. Biopolymers 30, 215-218. 13 Lipscomb, W.N. (1973) Enzymatic activity of carboxypeptidase A" in solution and in crystals. Proc. Natl. Acad. Sci. USA 70, 3797-3801.
