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A simple rapid-mixing apparatus for reactions of 10−3 to 1 sec duration
ANALYTICAL BIOCHEMISTRY 71, 544-549 (1976)
A Simple Rapid-Mixing Apparatus for Reactions of 10 -3 to 1 sec Duration ()RJAN ZETTERQVIST, SVEN M.g, RDH AND N I L S SANDSTROM
Institute of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsala, Box 575, S-751 23 Uppsala (Sweden) Received September 18, 1975; accepted October 29, 1975 A four-syringe, rapid-mixing apparatus of the stopped-flow, rapid-quenching type has been developed to make possible two or three consecutive mixings fop the study of fast, partial enzymatic reactions. The range of incubation time is 10-a to 1 sec and is varied by changing the length of the outlet tubing or the flow rate. The volume of the syringes is adjustable from 0.5 to 2.5 ml. Two, three or four syringes may be used simultaneously and thus allow mixing of two solutions or allow consecutive mixings with a short, predetermined time interval. The quenching time is estimated to be about 1 msec.
In a previous report from this laboratory (1) a rapid-mixing apparatus of the stopped-flow, rapid-quenching type, with two syringes, was described. This apparatus, which adopted some features of the stoppedflow apparatus presented by Sturtevant (2), was used for studies on nucleoside diphosphokinase (1), a-chymotrypsin (3), Streptomyces griseus protease (4) and (Na*,K*)-stimulated ATP phosphohydrolase (5). During the further investigation of the ATP phosphohydrolase it became necessary to perform rapid-mixing experiments with addition of potassium ions a short time after phosphorylation of the enzyme in the presence of sodium ions and (a2p)ATP. Therefore, the present apparatus with four syringes was developed to make possible the serial coupling of two or three mixing chambers. Since the flow resistance in the new apparatus was higher, the steel spring used to move the plungers of the syringes of the previous apparatus was replaced by a piston, driven by compressed air. This gave constant rate of flow over a wide range of reaction times. The new apparatus is described in the present communication. It has so far been used in experiments with three enzymes, viz (Na ÷,K+)-stimulated ATP phosphohydrolase (6-8), nucleoside diphosphokinase (9) and phosphoglucomutase (10). APPARATUS
General Design of the Rapid-Mixing Apparatus Some features of the rapid-mixing apparatus were described in previous reports (6,7). A detailed drawing is shown in Fig. 1. The different parts 544 Copyright © 1976by AcademicPress, Inc.
545
RAPID-MIXING APPARATUS
A° 10cm
®
@ ®
@ ®
®
@
FIG. 1. Construction of the rapid-mixing apparatus. (A) Front view and view from the right side. (B) Rear view and view from the left side. For details A through Z see under Apparatus.
of the apparatus were attached to a vertical steel support (A), mounted onto a base plate. Four-jet mixers (B), Y-connections (C), and blocks with syringes (D) were made from plexiglass. Holders (E) of the mixers and Y-connections were made from stainless steel. To allow easy removal of these the vertical support had 1 x 1 cm holes into which hooks (F1)
546
ZETTERQVIST, MARDH AND SANDSTR~)M
were inserted. The hooks were attached to the support by a screw (F2). Each block with syringes was attached to the suport by a knob at the rear (G). The syringes were vertical to avoid the trapping of air bubbles. The plungers (I) of the syringes were made from stainless steel with a head of Teflon (H). A three-way stopcock (K1), made from Teflon, was turned to direct the flow either from the reservoir to the syringe or from the syringe to the mixer. It was operated by a key, inserted into a hexagonal, steel-lined hole in the centre of the stopcock-head. A shallow groove was made along the full length of the stopcock and a fully inserted position of the stopcock was secured by a flat-headed screw (K2). The lower end of the plunger was inserted into a T-shaped slide (L) of a movable block of stainless steel (M) and secured by one knob (N) for each pair of syringes. By the aid of this knob an iron rod (O) was pressed against the lower end of each plunger. The block was attached to the piston of a driving device (P) (1300 DV pneumatic cylinder, diameter 50 mm, stroke length 25 mm, Mecman, Gothenburg, Sweden) which was operated with compressed air at 0.18-0.35 MPa (i.e., about 26-50 psi). The upward and downward movement was initiated by turning the handle (R) of a two-way vent (S) (Mecman 464/60). This vent was connected to the compressed air via hole (b). At position (a) and (c) sound-dampers (Mecman 338/125) were inserted. A micrometer screw (T), attached to the moving block, and a stationary vertical steel rod (U) on the driving device controlled the volume of the syringes which was adjustable from 0.5 to 2.5 ml. The rate of push was monitored by a storage oscilloscope (Tectronix Model 564) and a straight linear potentiometer (V1) (type RLP-75, Swema, Stockholm, Sweden) connected to a 10 V dc source. The rod (V2) of the potentiometer slide was anchored to the moving block and the slide was connected to the oscilloscope. The sweep was triggered when two metal pins (V3), in endto-end contact, were separated as the plungers began to move. The position of the upper pin was secured by a locking screw (V4). Fine adjustment of the distance between the pins was achieved by screwing the lower pin (screw head V~), which was secured by another locking screw (V6). In order to obtain slow filling, the reversed air stream was passed through a reduction valve (X) (Norgren Regulator R06, 50 psi, Stockholm, Sweden) before reaching the cylinder. In addition, the air from the space below the piston of the pneumatic cylinder was passed through a constriction vent (Y) (Mecman 344/125), adjustable with a screwdriver. The function of the vent (Y) was a coarse adjustment, and that of the reduction valve (X) was a fine adjustment of the rate of filling the syringes. The reduction valve also reduced the load on the micrometer screw (T). The design of the vent and the reduction valve allowed an unconstricted air pathway when the syringes were emptied.
RAPID-MIXING APPARATUS
547
Temperature Control It was possible to run experiments at a temperature _+5°C that of the room temperature by connecting a circulating water system (Z1) of the plexiglass blocks with a thermostated water bath, placed on the rear shelf (Zz) of the vertical support. The temperature of the plexiglass blocks, mixers and Y-connections was monitored by a thermometer inserted into a bore (Za). The solutions fed into the syringes were thermostated in the water bath. When the temperature of the water bath was kept 5°C above or below the room temperature, the difference in temperature between the bath and the plexiglass block was less than I°C. A greater difference between ambient temperature and the temperature of reaction solution will require special isolation of syringe blocks, tubing, mixing chambers and Y-connections.
Mixers, Y-Connections and Syringes The details of the four-jet mixers were given in a previous report (1). As can be inferred from Fig. 1 of Ref. (1) the bore of the four channels was 0.5 mm and that of the mixing space 1.5 mm. Details of the plexiglass Y-connection are shown in Fig. 2A. The bore of the channels was 0.5 mm. The screws for connection of the polyethene tubing were the same as for the four-jet mixer (1). Figure 2B shows details of a syringe-plunger. The flanges of the plunger head which was made from Teflon, minimized friction and provided elastic and efficient tightning over a wide temperature range, i.e., 0-95°C. Inner diameter of the syringe bore in the plexiglass block was 11.000
b
A
B
5cm
FIG. 2. (A) Section through a Y-connection of plexiglass at the height of the channels (diam, 0.5 mm). Broken lines indicate screw thread of the bores, into which hollow screws (M 4.5 x 0.75) with tubing were inserted [c.f. Fig. 1 of Ref. (1)]. (B) Longitudinal section through a plunger. (a) plunger head of Teflon, screwed onto a stainless steel rod, the lower end of which was cut to fit into the T-shaped slide of the movable block of the apparatus (see details L and M of Fig. 1). For further data, see text.
548
ZETTERQVIST, MARDH AND SANDSTROM
+ 0.005 mm and outer diameter of the Teflon plunger head was 11.025 + 0.005 mm. The diameter of the steel rod of the plunger was 10.00 mm. The head was screwed onto the rod, the end of which was made a screw with the length of 7 mm and the screw thread being M 5 x 0.8.
Tubing and Connections The different parts of the apparatus were connected by low density polyethene tubing (delivered by Noax, Bandhagen, Sweden). All tubings used for connection had a 0.6 mm wall-thickness, but the inner diameter could be varied in order to increase or decrease the resistance of flow and to regulate the flow rate. The tubing most often used, had an inner diameter of 1 mm which allowed incubation times in the range 1-1000 msec. The length of the tubing which connected syringes with mixers was about 15 cm, while the length of the outlet tubing and the tubing connecting the Y-connection with the mixers was varied with the reaction time desired. The ends of the polyethylene tubing were softened in a flame and then immediately pressed in between the end of the hollow screw and the 45 ° shaped tip of a stainless flanging tool, till the end of the tubing was cooled. These flanges functioned as gaskets between the screws and the flat-bottomed bores in the plexiglass parts.
Experimental Setup In most experiments with two consecutive mixings the arrangement was as described in Fig. 1 of Ref. (6). However, where symmetrical flow was important, an arrangement with three mixers and two Y-connections was chosen [see Fig. 1 of Ref. (7)]. Once the flow rate had been chosen by setting the proper air pressure, the reaction time was varied by changing the length of the outlet tubing.
Operation of the Apparatus After turning the 3-way stopcock (K1) to the appropriate position, the syringes were filled by pushing the handle (R) downwards, thus reversing the flow of the compressed air. The 3-way stopcock was turned again and the tubing which connected each syringe with the four-jet mixer was filled with solution from the syringe. The mixing chambers, the tubing which connected mixing chambers with Y-connections, and the outlet tubing were all filled with distilled water in order to achieve a constant resistance of flow from the beginning of the experiment. The metal pins (V3) were adjusted to gently touch each other. In order to quench the reaction, the outlet tubing was dipped into a beaker with base or acid (6-10). With 5 ml cold 10% (w/w) HCIO4 the estimated quenching time was about 1 msec (6). The reaction was started by moving the handle (R) upwards. After
RAPID-MIXING APPARATUS
549
quenching, the mixture was removed and assayed for the reaction products. The amount of the assayed reaction product was corrected for losses of the material left in the tubing, mixers and Y-connections.
Rinsing and Cleaning After each experiment, tubing, mixers and Y-connections were rinsed with distilled water. Before the next experiment these spaces were filled with water as mentioned above. After each series of experiments, tubing, mixers, Y-connections and syringes were rinsed with a mild detergent and with water. A more thorough cleaning was performed after 2 weeks of usage. The blocks with the syringes were then taken apart and soaked in water, then wiped clean with Kleenex tissue. The fine channels of the plexiglass parts were rinsed by use of compressed air and a thin thread of stainless steel. Since the construction of the apparatus allowed simple disconnection and remounting of these parts without special tools, the cleaning procedure was completed within 1 hr.
CONCLUSION The apparatus described in the present report permits a considerable flexibility in the design of experiments on partial reactions of enzymes (6-10). Other designs are also possible, e.g., the use of three consecutive mixings of reactants. By omitting the immediate quenching, the apparatus may be used as an efficient mixer to initiate reactions of several seconds duration, e.g., reactions to be followed spectrophotometrically or to be quenched at a later stage by manual addition of a quenching solution.
ACKNOWLEDGMENTS This work was supported by the Swedish Medical Research Council, Project 13X-50.
REFERENCES 1. W~ilinder, O., Zetterqvist, 0., and Engstrrm, L. (1969)J. Biol. Chem. 244, 10601064. 2. Sturtevant, J, M. (1964) in Rapid Mixing and Sampling Techniques in Biochemistry (Chance, B., Eisenhardt, R. H., Gibson, Q. H., and Lonberg-Holm, K. K., eds.), pp. 89-102, Academic Press, New York. 3. W~ihlby,S. (1970)Acta Chem. Scand. 24, 2429-2434. 4. W/ihlby, S. (1970)Acta Chem. Scand. 24, 703-704. 5. M~trdh, S., and Zetterqvist, O. (1972) Biochim. Biophys. Acta 255, 231-238. 6. M~trdh, S., and Zetterqvist, O. (1974) Biochim. Biophys. Acta 350, 473-483. 7. M~rdh, S. (1975) Biochim. Biophys. Acta 391,448-463. 8. M~rdh, S. (1975) Biochim. Biophys. Acta 391, 464-473. 9. Edlund, B., and W~tlinder, 0. (1974) FEBS Lett. 38, 225-228. 10. W~ilinder, O., and Joshi, J. G. (1974)J. Biol. Chem. 249, 3166-3169.
A Simple Rapid-Mixing Apparatus for Reactions of 10 -3 to 1 sec Duration ()RJAN ZETTERQVIST, SVEN M.g, RDH AND N I L S SANDSTROM
Institute of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsala, Box 575, S-751 23 Uppsala (Sweden) Received September 18, 1975; accepted October 29, 1975 A four-syringe, rapid-mixing apparatus of the stopped-flow, rapid-quenching type has been developed to make possible two or three consecutive mixings fop the study of fast, partial enzymatic reactions. The range of incubation time is 10-a to 1 sec and is varied by changing the length of the outlet tubing or the flow rate. The volume of the syringes is adjustable from 0.5 to 2.5 ml. Two, three or four syringes may be used simultaneously and thus allow mixing of two solutions or allow consecutive mixings with a short, predetermined time interval. The quenching time is estimated to be about 1 msec.
In a previous report from this laboratory (1) a rapid-mixing apparatus of the stopped-flow, rapid-quenching type, with two syringes, was described. This apparatus, which adopted some features of the stoppedflow apparatus presented by Sturtevant (2), was used for studies on nucleoside diphosphokinase (1), a-chymotrypsin (3), Streptomyces griseus protease (4) and (Na*,K*)-stimulated ATP phosphohydrolase (5). During the further investigation of the ATP phosphohydrolase it became necessary to perform rapid-mixing experiments with addition of potassium ions a short time after phosphorylation of the enzyme in the presence of sodium ions and (a2p)ATP. Therefore, the present apparatus with four syringes was developed to make possible the serial coupling of two or three mixing chambers. Since the flow resistance in the new apparatus was higher, the steel spring used to move the plungers of the syringes of the previous apparatus was replaced by a piston, driven by compressed air. This gave constant rate of flow over a wide range of reaction times. The new apparatus is described in the present communication. It has so far been used in experiments with three enzymes, viz (Na ÷,K+)-stimulated ATP phosphohydrolase (6-8), nucleoside diphosphokinase (9) and phosphoglucomutase (10). APPARATUS
General Design of the Rapid-Mixing Apparatus Some features of the rapid-mixing apparatus were described in previous reports (6,7). A detailed drawing is shown in Fig. 1. The different parts 544 Copyright © 1976by AcademicPress, Inc.
545
RAPID-MIXING APPARATUS
A° 10cm
®
@ ®
@ ®
®
@
FIG. 1. Construction of the rapid-mixing apparatus. (A) Front view and view from the right side. (B) Rear view and view from the left side. For details A through Z see under Apparatus.
of the apparatus were attached to a vertical steel support (A), mounted onto a base plate. Four-jet mixers (B), Y-connections (C), and blocks with syringes (D) were made from plexiglass. Holders (E) of the mixers and Y-connections were made from stainless steel. To allow easy removal of these the vertical support had 1 x 1 cm holes into which hooks (F1)
546
ZETTERQVIST, MARDH AND SANDSTR~)M
were inserted. The hooks were attached to the support by a screw (F2). Each block with syringes was attached to the suport by a knob at the rear (G). The syringes were vertical to avoid the trapping of air bubbles. The plungers (I) of the syringes were made from stainless steel with a head of Teflon (H). A three-way stopcock (K1), made from Teflon, was turned to direct the flow either from the reservoir to the syringe or from the syringe to the mixer. It was operated by a key, inserted into a hexagonal, steel-lined hole in the centre of the stopcock-head. A shallow groove was made along the full length of the stopcock and a fully inserted position of the stopcock was secured by a flat-headed screw (K2). The lower end of the plunger was inserted into a T-shaped slide (L) of a movable block of stainless steel (M) and secured by one knob (N) for each pair of syringes. By the aid of this knob an iron rod (O) was pressed against the lower end of each plunger. The block was attached to the piston of a driving device (P) (1300 DV pneumatic cylinder, diameter 50 mm, stroke length 25 mm, Mecman, Gothenburg, Sweden) which was operated with compressed air at 0.18-0.35 MPa (i.e., about 26-50 psi). The upward and downward movement was initiated by turning the handle (R) of a two-way vent (S) (Mecman 464/60). This vent was connected to the compressed air via hole (b). At position (a) and (c) sound-dampers (Mecman 338/125) were inserted. A micrometer screw (T), attached to the moving block, and a stationary vertical steel rod (U) on the driving device controlled the volume of the syringes which was adjustable from 0.5 to 2.5 ml. The rate of push was monitored by a storage oscilloscope (Tectronix Model 564) and a straight linear potentiometer (V1) (type RLP-75, Swema, Stockholm, Sweden) connected to a 10 V dc source. The rod (V2) of the potentiometer slide was anchored to the moving block and the slide was connected to the oscilloscope. The sweep was triggered when two metal pins (V3), in endto-end contact, were separated as the plungers began to move. The position of the upper pin was secured by a locking screw (V4). Fine adjustment of the distance between the pins was achieved by screwing the lower pin (screw head V~), which was secured by another locking screw (V6). In order to obtain slow filling, the reversed air stream was passed through a reduction valve (X) (Norgren Regulator R06, 50 psi, Stockholm, Sweden) before reaching the cylinder. In addition, the air from the space below the piston of the pneumatic cylinder was passed through a constriction vent (Y) (Mecman 344/125), adjustable with a screwdriver. The function of the vent (Y) was a coarse adjustment, and that of the reduction valve (X) was a fine adjustment of the rate of filling the syringes. The reduction valve also reduced the load on the micrometer screw (T). The design of the vent and the reduction valve allowed an unconstricted air pathway when the syringes were emptied.
RAPID-MIXING APPARATUS
547
Temperature Control It was possible to run experiments at a temperature _+5°C that of the room temperature by connecting a circulating water system (Z1) of the plexiglass blocks with a thermostated water bath, placed on the rear shelf (Zz) of the vertical support. The temperature of the plexiglass blocks, mixers and Y-connections was monitored by a thermometer inserted into a bore (Za). The solutions fed into the syringes were thermostated in the water bath. When the temperature of the water bath was kept 5°C above or below the room temperature, the difference in temperature between the bath and the plexiglass block was less than I°C. A greater difference between ambient temperature and the temperature of reaction solution will require special isolation of syringe blocks, tubing, mixing chambers and Y-connections.
Mixers, Y-Connections and Syringes The details of the four-jet mixers were given in a previous report (1). As can be inferred from Fig. 1 of Ref. (1) the bore of the four channels was 0.5 mm and that of the mixing space 1.5 mm. Details of the plexiglass Y-connection are shown in Fig. 2A. The bore of the channels was 0.5 mm. The screws for connection of the polyethene tubing were the same as for the four-jet mixer (1). Figure 2B shows details of a syringe-plunger. The flanges of the plunger head which was made from Teflon, minimized friction and provided elastic and efficient tightning over a wide temperature range, i.e., 0-95°C. Inner diameter of the syringe bore in the plexiglass block was 11.000
b
A
B
5cm
FIG. 2. (A) Section through a Y-connection of plexiglass at the height of the channels (diam, 0.5 mm). Broken lines indicate screw thread of the bores, into which hollow screws (M 4.5 x 0.75) with tubing were inserted [c.f. Fig. 1 of Ref. (1)]. (B) Longitudinal section through a plunger. (a) plunger head of Teflon, screwed onto a stainless steel rod, the lower end of which was cut to fit into the T-shaped slide of the movable block of the apparatus (see details L and M of Fig. 1). For further data, see text.
548
ZETTERQVIST, MARDH AND SANDSTROM
+ 0.005 mm and outer diameter of the Teflon plunger head was 11.025 + 0.005 mm. The diameter of the steel rod of the plunger was 10.00 mm. The head was screwed onto the rod, the end of which was made a screw with the length of 7 mm and the screw thread being M 5 x 0.8.
Tubing and Connections The different parts of the apparatus were connected by low density polyethene tubing (delivered by Noax, Bandhagen, Sweden). All tubings used for connection had a 0.6 mm wall-thickness, but the inner diameter could be varied in order to increase or decrease the resistance of flow and to regulate the flow rate. The tubing most often used, had an inner diameter of 1 mm which allowed incubation times in the range 1-1000 msec. The length of the tubing which connected syringes with mixers was about 15 cm, while the length of the outlet tubing and the tubing connecting the Y-connection with the mixers was varied with the reaction time desired. The ends of the polyethylene tubing were softened in a flame and then immediately pressed in between the end of the hollow screw and the 45 ° shaped tip of a stainless flanging tool, till the end of the tubing was cooled. These flanges functioned as gaskets between the screws and the flat-bottomed bores in the plexiglass parts.
Experimental Setup In most experiments with two consecutive mixings the arrangement was as described in Fig. 1 of Ref. (6). However, where symmetrical flow was important, an arrangement with three mixers and two Y-connections was chosen [see Fig. 1 of Ref. (7)]. Once the flow rate had been chosen by setting the proper air pressure, the reaction time was varied by changing the length of the outlet tubing.
Operation of the Apparatus After turning the 3-way stopcock (K1) to the appropriate position, the syringes were filled by pushing the handle (R) downwards, thus reversing the flow of the compressed air. The 3-way stopcock was turned again and the tubing which connected each syringe with the four-jet mixer was filled with solution from the syringe. The mixing chambers, the tubing which connected mixing chambers with Y-connections, and the outlet tubing were all filled with distilled water in order to achieve a constant resistance of flow from the beginning of the experiment. The metal pins (V3) were adjusted to gently touch each other. In order to quench the reaction, the outlet tubing was dipped into a beaker with base or acid (6-10). With 5 ml cold 10% (w/w) HCIO4 the estimated quenching time was about 1 msec (6). The reaction was started by moving the handle (R) upwards. After
RAPID-MIXING APPARATUS
549
quenching, the mixture was removed and assayed for the reaction products. The amount of the assayed reaction product was corrected for losses of the material left in the tubing, mixers and Y-connections.
Rinsing and Cleaning After each experiment, tubing, mixers and Y-connections were rinsed with distilled water. Before the next experiment these spaces were filled with water as mentioned above. After each series of experiments, tubing, mixers, Y-connections and syringes were rinsed with a mild detergent and with water. A more thorough cleaning was performed after 2 weeks of usage. The blocks with the syringes were then taken apart and soaked in water, then wiped clean with Kleenex tissue. The fine channels of the plexiglass parts were rinsed by use of compressed air and a thin thread of stainless steel. Since the construction of the apparatus allowed simple disconnection and remounting of these parts without special tools, the cleaning procedure was completed within 1 hr.
CONCLUSION The apparatus described in the present report permits a considerable flexibility in the design of experiments on partial reactions of enzymes (6-10). Other designs are also possible, e.g., the use of three consecutive mixings of reactants. By omitting the immediate quenching, the apparatus may be used as an efficient mixer to initiate reactions of several seconds duration, e.g., reactions to be followed spectrophotometrically or to be quenched at a later stage by manual addition of a quenching solution.
ACKNOWLEDGMENTS This work was supported by the Swedish Medical Research Council, Project 13X-50.
REFERENCES 1. W~ilinder, O., Zetterqvist, 0., and Engstrrm, L. (1969)J. Biol. Chem. 244, 10601064. 2. Sturtevant, J, M. (1964) in Rapid Mixing and Sampling Techniques in Biochemistry (Chance, B., Eisenhardt, R. H., Gibson, Q. H., and Lonberg-Holm, K. K., eds.), pp. 89-102, Academic Press, New York. 3. W~ihlby,S. (1970)Acta Chem. Scand. 24, 2429-2434. 4. W/ihlby, S. (1970)Acta Chem. Scand. 24, 703-704. 5. M~trdh, S., and Zetterqvist, O. (1972) Biochim. Biophys. Acta 255, 231-238. 6. M~trdh, S., and Zetterqvist, O. (1974) Biochim. Biophys. Acta 350, 473-483. 7. M~rdh, S. (1975) Biochim. Biophys. Acta 391,448-463. 8. M~rdh, S. (1975) Biochim. Biophys. Acta 391, 464-473. 9. Edlund, B., and W~tlinder, 0. (1974) FEBS Lett. 38, 225-228. 10. W~ilinder, O., and Joshi, J. G. (1974)J. Biol. Chem. 249, 3166-3169.