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An apparatus for sequentially combining microvolumes of reagents by infrasonic mixing
ANhYTICAL
BIOCHEMISTRY
138, 329-334 (1984)
An Apparatus for Sequentially Combining Microvolumes Reagents by Infrasonic Mixing’
of
MERRILL N. CAMIEN AND ROBERT C. WARNER Department of Molecular Biology and Biochemistry, University of Calijornia. Iwine, California 92717 Received September 2, 1983 A method employing high-speed infrasonic mixing for obtaining timed samples for following the progress of a moderately rapid chemical reaction is described. Drops of 10 to 50 pl each of two reagents are mixed to initiate the reaction, followed, after a measured time interval, by mixing with a drop of a third reagent to quench the reaction. The method was developed for measuring the rate of denaturation of covalently closed, circular DNA in NaOH at several temperatures. For this purpose the timed samples were analyzed by analytical ultracentrifugation. The apparatus was tested by determination of the rate of hydrolysis of 2,4dinitrophenyl acetate in an alkaline buffer. The important characteristics of the method are (i) it requires very small volumes of sample and reagents; (ii) the components of the reaction mixture are preequilibrated and mixed with no transfer outside the prescribed constant temperature environment; (iii) the mixing is very rapid, and (iv) satisfactorily precise measurements of relatively short time intervals (approximately 2 set minimum) between sequential mixings of the components are readily obtainable. KEY WORDS: mixing; infrasonic mixing; kinetic sampling device; denaturation of DNA, rapid reaction; hydrolysis of 2,4dinitrophenyl acetate.
Studies of the kinetics of renaturation of denatured, covalently closed, circular DNA reported previously from this laboratory (1) employed methods that are generally applicable only to reactions that proceed negligibly at 0°C but with easily measurable rates at elevated temperatures (25 to 50°C). These methods were successful because the reactants could be premixed at 0°C rapidly brought to and held at the desired reaction temperature for a predetermined time, and then rapidly returned to 0°C before neutralization. Ongoing extensions of these studies have revealed that alkaline denaturation reactions generally proceed with measurable rates only at relatively low temperatures, for which the previously described methods do not apply. In addition, it was desired to extend the rate measurements to faster reactions. Attempts to ’ This investigation was supported by NIH Research Grant GM28769 from the Institute of General Medical Sciences. 329
circumvent this difficulty culminated in methodology presented in the following section of this report. We describe here a simple apparatus for mixing reagents to initiate a chemical reaction and for quenching it after a measured reaction time. It is designed for reactions having half times of 5 s or greater and for reagent volumes in the range of 2 to 30 ~1. It is easily thermostated and allows preequilibration of the reagents with the desired temperature. MATERIALS
AND METHODS
Apparatus and its me. Essential features of the apparatus for infrasonic mixing are displayed diagramatically in Fii. 1A. A doublewalled Plexiglas water bath with a sidearm for overflow and with outside dimensions of 5.5 in. square by 7.25 in. high is shown at lower right in Fig. 1A. The double-walled construction prevents external fogging at low bath temperatures and preserves optimal visibility 0003-2697184 $3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any fom rcwved.
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CAMIEN I
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I
FIG. 1. (A) Diagrammatic representation of system components for infrasonic mixing apparatus. The components shown from left to right are an infrasonic generator, consisting of a speaker coil and a rubber dropper bulb, an electrical solenoid valve, a three-port three-position manual valve, a l-ml manually operated syringe; and a reaction vessel, consisting of a 5-cm length of 4-mm-diameter Teflon tubing, and its support assembly immersed in a double-walled Plexiglas water bath with a sidearm for overflow. (B) An expanded, cutoff view of the bottom of the support assembly and the reaction vessel, which contains the DNA sample solution forming a seal (center), the NaOH solution in a free droplet (right of center), and the Tris-HCl solution in a free droplet (left of center) configured for temperature equilibration prior to mixing.
of the reaction vessel and its contents under all experimental conditions. Low temperatures (in range from 3 to 20°C with ambient temperatures near 24°C) are maintained constant in the bath with a tolerance of less than 0.003”C by additions of ice water to the bath through a thermoregulated solenoid valve (from a reservoir of ice and water located above the bath), while the contents of the bath are stirred with a magnetic stir bar. Higher constant temperatures are obtained in the same manner but with the addition of an immersion heater powered to maintain an unregulated temperature barely above the desired regulated temperature. A mixture of ice and water is employed in the bath for work at 0°C. The reaction vessel, consisting of a 5-cm length of thin-walled Teflon tubing (AWG 7 LW NAT tubing, supplied by Albert H. Surprenant, Inc.-4.3-mm outside diameter, 0.2mm wall thickness) is shown suspended inside the water bath in Fig. IA and in expanded view in Fig. 1B. During equilibration and incubation of the reactants within the reaction
WARNER
vessel before and after mixing, the reaction vessel is held in place by a support assembly, consisting of two 7.5-in. lengths of glass tubing with an outside diameter selected to fit snugly into the ends of the reaction vessel. The lengths of glass tubing are bent at one end as shown in Fig. IA to form a U when fitted into the two ends of the reaction vessel. The top of the U is held in place by sandwiching it between two Plexiglas bars (held together by rubber bands), which also serve to support the assembly from the top edges of the water bath. A 5.5-in. length of rubber tubing, left permanently connected to the left arm of the support assembly, is fitted with a connector at its distal end to facilitate connecting the support assembly to the train of system components shown at the upper center and left of Fig. 1A. Before the reaction vessel is put in place in the support assembly, the solutions to be combined sequentially are inserted by micropipet into the reaction vessel in the configuration shown in Fig. 1B. A micropipet made of Teflon tubing with an inside diameter of 0.6 mm and marked at 15 ~1 (approximately 54 mm from the end) by notching with a razor blade is operated with a Hamilton syringe while viewing it with five power magnifying lenses to obtain a precision of 0.03 ~1. First, two pipet loads (30 ~1) of DNA solution in 2 mM EDTA, pH 8, with a total DNA content approximating 1.2 pg, are combined to form a large droplet in the center of the reaction vessel, which is then tapped gently to cause the droplet to form a seal across the center of the vessel (see Fig. 1B). Then a droplet of from 5 to 10 ~1 (high precision not required) of Tris-HCl solution is placed in the vessel approximately 1 cm to the left of the DNA solution. Finally, using the same pipet as for the DNA solution a precise 15-~1 pipet load of NaOH solution containing NaCl and EDTA is positioned approximately 5 mm to the right of the DNA solution, and the vessel is then immediately put in place in the support assembly and immersed in the constant-temperature bath. The amounts of NaCl and
APPARATUS
FOR
INFRASONIC
EDTA in the NaOH solution are adjusted to yield final concentrations in the reaction mixture of 1.28 M NaCl and 4.667 mM EDTA. The concentration of the Tris-HCl solution is adjusted to provide, in the volume employed, roughly two equivalents of Tris-HCl per equivalent of NaOH in the final mixture. The two system components immediately to the left of the water bath in Fig. IA are a syringe and a three-port, three-position manual valve connected together by a T tube, which connects to the reaction vessel support assembly. In each of the three valve positions, one of the three valve ports is closed while continuity is maintained between the other two. Before the syringe and valve assembly are connected to the reaction vessel support assembly, the valve is set for continuity between the T tube and the outside air. The valve port connecting to the T tube is then closed before the opposite port is connected to the electrical solenoid valve (normally closed) shown immediately to the left of the manual valve in Fig. IA. After the latter connection is made, the manual valve is set for continuity between the T tube and the closed solenoid valve. These manipulations of the manual valve before making the described connections are required to avoid pressure changes in the air column to the left of the seal formed by the DNA solution in the reaction vessel since such changes would produce movements of the DNA solution resulting in premature mixing of the latter with either the NaOH or the Tris-HCl solution. The solenoid valve (Fig. IA) is mounted under a Plexiglas slab next to a 5-in., 8-ohm speaker from which the speaker cone is removed. A rubber dropper bulb connected to the solenoid valve is positioned between the speaker coil and a brass disk supported by the Plexiglas slab as shown in Fig. 1A. The speaker coil is continuously energized with a 25-Hz sinusoidal current from a signal generator equipped with a 5-W amplifier, and this electrical energy is transduced to infrasonic energy in the dropper bulb. Amplification of the sinusoidal signal is adjusted with the solenoid
MIXING
331
valve opened and with a dummy solution seal in the reaction vessel to produce the maximum agitation of the solution achievable without danger of disrupting the seal. Complete assembly of the system as described is generally accomplished within less than a minute following immersion of the reaction vessel in the constant-temperature bath. The syringe is then employed to move the DNA solution seal to within I mm of the NaOH solution, and the separate solutions in the reaction vessel are given additional time for temperature equilibration to a total of 3 min. The solenoid valve, operated electrically, is then opened to feed infrasonic energy to the DNA solution, which is thereby caused to mix rapidly with the NaOH solution, while the Tris-HCl solution, being safely remote from the DNA solution and not forming a closed seal, is left unaffected. A Rockwell AIM-65 microcomputer, programmed for the purpose, opens the solenoid valve and simultaneously begins timing the reaction when the operator presses the keyboard space bar. The solenoid valve is closed automatically after 30 s, and the operator again uses the syringe to move the solution seal (now comprising the reaction mixture of DNA and alkali) to within 1 mm of the droplet of TrisHCl solution. Pressing the button of a handheld momentary switch (after approximately the desired time interval) then reopens the solenoid valve to cause instantaneous mixing of the Tris-HCl solution with the reaction mixture (thereby stopping the reaction) and simultaneously produces a printout of lapsed time to the nearest hundredth second on the computer display. When reaction times of less than 30 s are to be employed, the syringe is used to move the reaction mixture while it remains in a state of sonic agitation into the region where it will capture and mix with the droplet of Tris-HCl. This operation is observed with five power lenses, and the button of the hand-held momentary switch is pressed at the moment that capture of the Tris-HCl droplet is seen. Lapsed time is printed out in the same manner as before and, in either case,
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sonic mixing continues without interruption for an additional 30 s. The operator then disassembles the system, following the reverse procedure from that employed during assembly. The reaction vessel is removed from its support assembly and placed in a tightly capped vial for temporary storage in a freezer prior to assay. Immediately before the stopped reaction mixture is assayed, its volume is reduced to approximately 10 ~1 by evaporation under a stream of dry nitrogen as illustrated in Fig. 2. First, one end of the reaction vessel is trimmed off with a razor blade to a distance of ap proximately 5 mm from the seal formed by the reaction mixture. This end of the reaction vessel is then fitted over the short vertical arm of a J formed from a Pyrex rod of appropriate diameter. The other end of the reaction vessel is then likewise trimmed, and positioned under a nitrogen jet as shown (Fig. 2). The low-pressure gas is carefully turned on and regulated with a needle valve to produce a slight concavity of the upper surface of the reaction
mixture. A beaker of room-temperature water supported under the J rod by a lab-jack is then raised to put the water level barely above the level of reaction mixture in the trimmed reaction vessel. The water serves as a roomtemperature heat source to replace heat removed by evaporation. The seal formed by the reaction mixture remains intact during evaporation; however, its thickness is progressively reduced and can readily be estimated by inspection. A thickness of approximately 1 mm, which indicates the desired final volume, is reached within a few minutes. In practice, three evaporation assemblies are employed simultaneously, and the resulting samples are assayed together in a three-sample analytical ultracentrifuge run. The analytical ultracentrifuge procedures employed for these assays, including computerized data acquisition and data processing, have been described elsewhere ( 1). Hydrolysis of DNPA.2 Alkaline hydrolysis of DNPA was employed as a separate reaction to test the performance of the mixing apparatus. A stock solution of 12.5 ItIM DNPA in isopropanol was diluted immediately before use with 9 vol of ice-cold 5.56 mM HCl to produce 1.25 mM DNPA in 10% isopropanol and 5 IIIM HCl (kept in ice). Phosphate buffers N2 GAS MANIFOLD with pH values of 12.48 and 12.06, measured a NEEDLE VALVE at 0- 1“C after dilution with 2 vol of 10% isopropanol in 5 mM HCl, were prepared with 0.1 M &PO4 and K2HP04 in 4: 1 and 1: 1 ratios. The procedures described in the preSAMPLE IN ceding paragraphs were followed to react 30 REACTION VESSEL ~1 of the 1.25 IIIM DNPA with 15 ~1 of 0.1 M phosphate buffer at 0°C and to stop the tSUPPORT ROD reaction by adding 15 ~1 of 1 N HCI to the reaction mixture. The stopped reaction mixFIG. 2. Diagrammatic representation of apparatus used ture was immediately diluted with 140 ~1 of to reduce volume of reaction mixture by evaporation in water and transferred to a microcuvette, and stream of N2 gas prior to assay. The reaction vessel, absorbance of the resulting mixture at 360 nm trimmed as described in the text and containing the stopped was measured to determine the amount of reaction mixture, is supported under the N2 gas jet by a 2,4-dinitrophenol produced in the reaction. J-shaped Pyrex rod. The lower part of the J, including A maximal absorbance value was obtained by the reaction vessel up to the level of the upper surface of allowing the reaction of the DNPA and phosthe reaction mixture, is immersed in a beaker of room-
J
temperature water (not shown), which serves as a heat source during the evaporation.
* Abbreviation used: DNPA, 2,4dinitrophenyl
acetate.
APPARATUS
FOR INFRASONIC
phate buffer to proceed at room temperature for 20 min before stopping the reaction and reading the absorbance. A reagent blank was obtained by mixing 30 ~1 of 1.25 mM DNPA with 30 ~1 of a mixture of equal volumes 0.1 M phosphate buffer and 1 N HCl, diluting the resulting mixture with 140 ~1 of water, and reading absorbance of the final solution at 360 nm. The fraction of DNPA with respect to DNPA plus product in each stopped reaction mixture was estimated as ((M - B) - (R - B))/ (M - B), where M is the maximal absorbance value, B is the reagent blank, and R is the absorbance of the diluted reaction mixture. RESULTS
Data, produced in denaturation experiments with G4 replicative form DNA, typical of those readily obtainable with the apparatus and procedures, are shown in Fig. 3. The data (Fig. 3) define regular, smooth curves approximated by those drawn through the sequential points and can be judged, on this basis, to exhibit a high level of internal consistency. In this regard, it is noteworthy that except for use of the same DNA and reagent solutions throughout, each point shown in Fig. 3 represents an essentially independent experiment. Interpolation of the Fig. 3 data yields 50% denaturation times of 11.7, 4.4, and 1.8 s under the described conditions at 0, 5, and 10°C respectively. The apparatus and procedures are thus applicable for studies of relatively fast reactions. To test the apparatus with a reaction other than the denaturation of DNA we have made measurements on the alkaline hydrolysis of DNPA at O’C. The data and the linear regression lines are given in Fig. 4. From the intercepts at zero times, net mixing delays of 0.3 and 0.15 s were calculated for the upper and lower curves, respectively. These delays are within the standard deviation of the time measurements and are probably maximum values. The first-order rate constants were 0.0 136 and 0.037 s-‘, log,, respectively. These values yield second-order constants of 6.6 and
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MIXING
II 0
20
40
60
SECONDS
FIG. 3. Denaturation curves for G4 replicative form DNA in 40 mM NaOH at the indicated temperatures. Supercoiled DNA, form I, is denatured in the reaction to yield a more rapidly sedimenting species, form I.+ Relative amounts of forms I and & in the reaction mixtures are determined in an analytical ultracentrifuge. The natural logarithm of the fraction remaining as form I is plotted versus the reaction time in seconds.
7.2 s-i M-’ when combined with (OH-) calculated from the pH and pK, at O’C. We have not found a recorded value for this constant at 0°C but it appears consistent with the value of 49 s-’ M-’ at 25°C obtained by Barman and Gutfreund (2). DISCUSSION
The infrasonic mixing apparatus described here fills a gap in the available devices for rapid sampling when small volumes of reagents must be used and the reaction is too fast to use automated sampling. Flow methods employing two mixing chambers for achieving sampling require large volumes. For example, the device of Thayer and Hinkle (3) was designed to reduce the volume, but 0.5 ml is stated to be the minimum required. It is also difficult in this type of apparatus to maintain a constant temperature. The design of the mixing apparatus was based on empirical observations and on specifications dictated by the nature of our experiments. However, it is flexible and could be improved in various ways. For applications in which smaller sample volumes are desired, the diameter of the reaction vessel tubing could be reduced. However, the total volume of re-
334
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AND WARNER
SECONDS
FIG. 4. First-order plots of rates of hydrolysis of DNPA at 0°C in mixtures of 30 ~1 of 1.25 mM DNPA (in 5 mM HCI) and 15 @I of 0.1 M phosphate buffer with K$‘Q,/ K2HPQ, ratios of 4: 1 (lower data set) and 1: I (upper data set), respectively. The fraction DNPA (ordinate) values are calculated as described in the text. Least-squares linear regression lines are drawn through the data. The slopes are -0.0136 (upper set) and -0.0370 (lower set), and the standard deviations from the regression lines are 0.0080 and 0.0072 log. units for the upper and lower sets, respectively. The zero time intercepts are -0.0040 (upper set) and -0.0055 (lower set).
action mixture of 45 ~1, employed in our procedure, is probably near the upper limit imposed by the requirement that the reaction mixture be contained as a stable seal in the tubing. Improvements could also be made in the precision of the timing to adapt the apparatus for use with faster reactions. We have found that a working frequency of 25 Hz for infrasonic mixing provided stronger mixing with less propensity to disrupt the reaction mixture seal than higher or lower frequencies. Effects of variations in characteristics and dimensions of the system components (speaker coil, rubber dropper bulb, glass and rubber tubing, etc.) on the signal frequency and amplitude required for optimal performance were not investigated. An electrical solenoid valve was employed to start and stop the mixing because it was observed that either turning on the signal generator or closing a switch between the speaker coil and the amplifier produces a strong transient signal which generally results in disruption of the reaction mixture seal. Alternatively,
the transient signal might be attenuated by modifying the switching circuitry. Transparent Teflon tubing was used for fabricating the reaction vessel and micropipet because the marked hydrophobicity of this material facilitates quantitative transfers of aqueous sample and reagent solutions and was essential for the manipulations of the solutions within the reaction vessel. An inside diameter of approximately 4 mm was used for the reaction vessel because a droplet of sample solution will not readily form a stable seal inside a larger diameter, and the sample volume required for a seal of acceptable thickness in tubing of smaller diameter is smaller than we desired. System components, except for the specially constructed water bath and signal generator/ amplifier, were selected from parts which were on hand in the laboratory. The solenoid valve (ASCO No. 8262B208) was from Automatic Switch Company, Florham Park, New Jersey; the three-position manual valve was a Luer 3-way nylon stopcock (Bio-Rad No. 7329009); and the speaker (FoMoCo No. C6VA18932-A646) was from a discarded automobile audio tape player. Requirements for the signal generator/amplifier were determined in preliminary experiments employing a research model signal generator with a standard audio power amplifier. Subsequent work employed a relatively inexpensive and compact signal generator/amplifier designed and built to provide an adjustable sinusoidal output of 0 to 5 V peak-to-peak at 1O-60 Hz with a standard S-ohm speaker load. REFERENCES 1. Strider, W., &mien, M. N., and Warner, R. C. (1981) J. Biol. Chem. 256,7820-7829. 2. Barman, T. E., and Gutfreund, H. (1964) in Rapid Mixing and Sampling Techniques in Biochemistry (Chance, B., Eisenhardt, R. H., Gibson, Q. H., and Lo&erg-Holm, K. K., eds.), pp. 339-344, Academic Press, New York. 3. Thayer, W. S., and Hinkle, P. C. (1979) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 56, pp. 492-496, Academic Press, New York.
BIOCHEMISTRY
138, 329-334 (1984)
An Apparatus for Sequentially Combining Microvolumes Reagents by Infrasonic Mixing’
of
MERRILL N. CAMIEN AND ROBERT C. WARNER Department of Molecular Biology and Biochemistry, University of Calijornia. Iwine, California 92717 Received September 2, 1983 A method employing high-speed infrasonic mixing for obtaining timed samples for following the progress of a moderately rapid chemical reaction is described. Drops of 10 to 50 pl each of two reagents are mixed to initiate the reaction, followed, after a measured time interval, by mixing with a drop of a third reagent to quench the reaction. The method was developed for measuring the rate of denaturation of covalently closed, circular DNA in NaOH at several temperatures. For this purpose the timed samples were analyzed by analytical ultracentrifugation. The apparatus was tested by determination of the rate of hydrolysis of 2,4dinitrophenyl acetate in an alkaline buffer. The important characteristics of the method are (i) it requires very small volumes of sample and reagents; (ii) the components of the reaction mixture are preequilibrated and mixed with no transfer outside the prescribed constant temperature environment; (iii) the mixing is very rapid, and (iv) satisfactorily precise measurements of relatively short time intervals (approximately 2 set minimum) between sequential mixings of the components are readily obtainable. KEY WORDS: mixing; infrasonic mixing; kinetic sampling device; denaturation of DNA, rapid reaction; hydrolysis of 2,4dinitrophenyl acetate.
Studies of the kinetics of renaturation of denatured, covalently closed, circular DNA reported previously from this laboratory (1) employed methods that are generally applicable only to reactions that proceed negligibly at 0°C but with easily measurable rates at elevated temperatures (25 to 50°C). These methods were successful because the reactants could be premixed at 0°C rapidly brought to and held at the desired reaction temperature for a predetermined time, and then rapidly returned to 0°C before neutralization. Ongoing extensions of these studies have revealed that alkaline denaturation reactions generally proceed with measurable rates only at relatively low temperatures, for which the previously described methods do not apply. In addition, it was desired to extend the rate measurements to faster reactions. Attempts to ’ This investigation was supported by NIH Research Grant GM28769 from the Institute of General Medical Sciences. 329
circumvent this difficulty culminated in methodology presented in the following section of this report. We describe here a simple apparatus for mixing reagents to initiate a chemical reaction and for quenching it after a measured reaction time. It is designed for reactions having half times of 5 s or greater and for reagent volumes in the range of 2 to 30 ~1. It is easily thermostated and allows preequilibration of the reagents with the desired temperature. MATERIALS
AND METHODS
Apparatus and its me. Essential features of the apparatus for infrasonic mixing are displayed diagramatically in Fii. 1A. A doublewalled Plexiglas water bath with a sidearm for overflow and with outside dimensions of 5.5 in. square by 7.25 in. high is shown at lower right in Fig. 1A. The double-walled construction prevents external fogging at low bath temperatures and preserves optimal visibility 0003-2697184 $3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any fom rcwved.
330
CAMIEN I
AND
I
FIG. 1. (A) Diagrammatic representation of system components for infrasonic mixing apparatus. The components shown from left to right are an infrasonic generator, consisting of a speaker coil and a rubber dropper bulb, an electrical solenoid valve, a three-port three-position manual valve, a l-ml manually operated syringe; and a reaction vessel, consisting of a 5-cm length of 4-mm-diameter Teflon tubing, and its support assembly immersed in a double-walled Plexiglas water bath with a sidearm for overflow. (B) An expanded, cutoff view of the bottom of the support assembly and the reaction vessel, which contains the DNA sample solution forming a seal (center), the NaOH solution in a free droplet (right of center), and the Tris-HCl solution in a free droplet (left of center) configured for temperature equilibration prior to mixing.
of the reaction vessel and its contents under all experimental conditions. Low temperatures (in range from 3 to 20°C with ambient temperatures near 24°C) are maintained constant in the bath with a tolerance of less than 0.003”C by additions of ice water to the bath through a thermoregulated solenoid valve (from a reservoir of ice and water located above the bath), while the contents of the bath are stirred with a magnetic stir bar. Higher constant temperatures are obtained in the same manner but with the addition of an immersion heater powered to maintain an unregulated temperature barely above the desired regulated temperature. A mixture of ice and water is employed in the bath for work at 0°C. The reaction vessel, consisting of a 5-cm length of thin-walled Teflon tubing (AWG 7 LW NAT tubing, supplied by Albert H. Surprenant, Inc.-4.3-mm outside diameter, 0.2mm wall thickness) is shown suspended inside the water bath in Fig. IA and in expanded view in Fig. 1B. During equilibration and incubation of the reactants within the reaction
WARNER
vessel before and after mixing, the reaction vessel is held in place by a support assembly, consisting of two 7.5-in. lengths of glass tubing with an outside diameter selected to fit snugly into the ends of the reaction vessel. The lengths of glass tubing are bent at one end as shown in Fig. IA to form a U when fitted into the two ends of the reaction vessel. The top of the U is held in place by sandwiching it between two Plexiglas bars (held together by rubber bands), which also serve to support the assembly from the top edges of the water bath. A 5.5-in. length of rubber tubing, left permanently connected to the left arm of the support assembly, is fitted with a connector at its distal end to facilitate connecting the support assembly to the train of system components shown at the upper center and left of Fig. 1A. Before the reaction vessel is put in place in the support assembly, the solutions to be combined sequentially are inserted by micropipet into the reaction vessel in the configuration shown in Fig. 1B. A micropipet made of Teflon tubing with an inside diameter of 0.6 mm and marked at 15 ~1 (approximately 54 mm from the end) by notching with a razor blade is operated with a Hamilton syringe while viewing it with five power magnifying lenses to obtain a precision of 0.03 ~1. First, two pipet loads (30 ~1) of DNA solution in 2 mM EDTA, pH 8, with a total DNA content approximating 1.2 pg, are combined to form a large droplet in the center of the reaction vessel, which is then tapped gently to cause the droplet to form a seal across the center of the vessel (see Fig. 1B). Then a droplet of from 5 to 10 ~1 (high precision not required) of Tris-HCl solution is placed in the vessel approximately 1 cm to the left of the DNA solution. Finally, using the same pipet as for the DNA solution a precise 15-~1 pipet load of NaOH solution containing NaCl and EDTA is positioned approximately 5 mm to the right of the DNA solution, and the vessel is then immediately put in place in the support assembly and immersed in the constant-temperature bath. The amounts of NaCl and
APPARATUS
FOR
INFRASONIC
EDTA in the NaOH solution are adjusted to yield final concentrations in the reaction mixture of 1.28 M NaCl and 4.667 mM EDTA. The concentration of the Tris-HCl solution is adjusted to provide, in the volume employed, roughly two equivalents of Tris-HCl per equivalent of NaOH in the final mixture. The two system components immediately to the left of the water bath in Fig. IA are a syringe and a three-port, three-position manual valve connected together by a T tube, which connects to the reaction vessel support assembly. In each of the three valve positions, one of the three valve ports is closed while continuity is maintained between the other two. Before the syringe and valve assembly are connected to the reaction vessel support assembly, the valve is set for continuity between the T tube and the outside air. The valve port connecting to the T tube is then closed before the opposite port is connected to the electrical solenoid valve (normally closed) shown immediately to the left of the manual valve in Fig. IA. After the latter connection is made, the manual valve is set for continuity between the T tube and the closed solenoid valve. These manipulations of the manual valve before making the described connections are required to avoid pressure changes in the air column to the left of the seal formed by the DNA solution in the reaction vessel since such changes would produce movements of the DNA solution resulting in premature mixing of the latter with either the NaOH or the Tris-HCl solution. The solenoid valve (Fig. IA) is mounted under a Plexiglas slab next to a 5-in., 8-ohm speaker from which the speaker cone is removed. A rubber dropper bulb connected to the solenoid valve is positioned between the speaker coil and a brass disk supported by the Plexiglas slab as shown in Fig. 1A. The speaker coil is continuously energized with a 25-Hz sinusoidal current from a signal generator equipped with a 5-W amplifier, and this electrical energy is transduced to infrasonic energy in the dropper bulb. Amplification of the sinusoidal signal is adjusted with the solenoid
MIXING
331
valve opened and with a dummy solution seal in the reaction vessel to produce the maximum agitation of the solution achievable without danger of disrupting the seal. Complete assembly of the system as described is generally accomplished within less than a minute following immersion of the reaction vessel in the constant-temperature bath. The syringe is then employed to move the DNA solution seal to within I mm of the NaOH solution, and the separate solutions in the reaction vessel are given additional time for temperature equilibration to a total of 3 min. The solenoid valve, operated electrically, is then opened to feed infrasonic energy to the DNA solution, which is thereby caused to mix rapidly with the NaOH solution, while the Tris-HCl solution, being safely remote from the DNA solution and not forming a closed seal, is left unaffected. A Rockwell AIM-65 microcomputer, programmed for the purpose, opens the solenoid valve and simultaneously begins timing the reaction when the operator presses the keyboard space bar. The solenoid valve is closed automatically after 30 s, and the operator again uses the syringe to move the solution seal (now comprising the reaction mixture of DNA and alkali) to within 1 mm of the droplet of TrisHCl solution. Pressing the button of a handheld momentary switch (after approximately the desired time interval) then reopens the solenoid valve to cause instantaneous mixing of the Tris-HCl solution with the reaction mixture (thereby stopping the reaction) and simultaneously produces a printout of lapsed time to the nearest hundredth second on the computer display. When reaction times of less than 30 s are to be employed, the syringe is used to move the reaction mixture while it remains in a state of sonic agitation into the region where it will capture and mix with the droplet of Tris-HCl. This operation is observed with five power lenses, and the button of the hand-held momentary switch is pressed at the moment that capture of the Tris-HCl droplet is seen. Lapsed time is printed out in the same manner as before and, in either case,
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AND WARNER
sonic mixing continues without interruption for an additional 30 s. The operator then disassembles the system, following the reverse procedure from that employed during assembly. The reaction vessel is removed from its support assembly and placed in a tightly capped vial for temporary storage in a freezer prior to assay. Immediately before the stopped reaction mixture is assayed, its volume is reduced to approximately 10 ~1 by evaporation under a stream of dry nitrogen as illustrated in Fig. 2. First, one end of the reaction vessel is trimmed off with a razor blade to a distance of ap proximately 5 mm from the seal formed by the reaction mixture. This end of the reaction vessel is then fitted over the short vertical arm of a J formed from a Pyrex rod of appropriate diameter. The other end of the reaction vessel is then likewise trimmed, and positioned under a nitrogen jet as shown (Fig. 2). The low-pressure gas is carefully turned on and regulated with a needle valve to produce a slight concavity of the upper surface of the reaction
mixture. A beaker of room-temperature water supported under the J rod by a lab-jack is then raised to put the water level barely above the level of reaction mixture in the trimmed reaction vessel. The water serves as a roomtemperature heat source to replace heat removed by evaporation. The seal formed by the reaction mixture remains intact during evaporation; however, its thickness is progressively reduced and can readily be estimated by inspection. A thickness of approximately 1 mm, which indicates the desired final volume, is reached within a few minutes. In practice, three evaporation assemblies are employed simultaneously, and the resulting samples are assayed together in a three-sample analytical ultracentrifuge run. The analytical ultracentrifuge procedures employed for these assays, including computerized data acquisition and data processing, have been described elsewhere ( 1). Hydrolysis of DNPA.2 Alkaline hydrolysis of DNPA was employed as a separate reaction to test the performance of the mixing apparatus. A stock solution of 12.5 ItIM DNPA in isopropanol was diluted immediately before use with 9 vol of ice-cold 5.56 mM HCl to produce 1.25 mM DNPA in 10% isopropanol and 5 IIIM HCl (kept in ice). Phosphate buffers N2 GAS MANIFOLD with pH values of 12.48 and 12.06, measured a NEEDLE VALVE at 0- 1“C after dilution with 2 vol of 10% isopropanol in 5 mM HCl, were prepared with 0.1 M &PO4 and K2HP04 in 4: 1 and 1: 1 ratios. The procedures described in the preSAMPLE IN ceding paragraphs were followed to react 30 REACTION VESSEL ~1 of the 1.25 IIIM DNPA with 15 ~1 of 0.1 M phosphate buffer at 0°C and to stop the tSUPPORT ROD reaction by adding 15 ~1 of 1 N HCI to the reaction mixture. The stopped reaction mixFIG. 2. Diagrammatic representation of apparatus used ture was immediately diluted with 140 ~1 of to reduce volume of reaction mixture by evaporation in water and transferred to a microcuvette, and stream of N2 gas prior to assay. The reaction vessel, absorbance of the resulting mixture at 360 nm trimmed as described in the text and containing the stopped was measured to determine the amount of reaction mixture, is supported under the N2 gas jet by a 2,4-dinitrophenol produced in the reaction. J-shaped Pyrex rod. The lower part of the J, including A maximal absorbance value was obtained by the reaction vessel up to the level of the upper surface of allowing the reaction of the DNPA and phosthe reaction mixture, is immersed in a beaker of room-
J
temperature water (not shown), which serves as a heat source during the evaporation.
* Abbreviation used: DNPA, 2,4dinitrophenyl
acetate.
APPARATUS
FOR INFRASONIC
phate buffer to proceed at room temperature for 20 min before stopping the reaction and reading the absorbance. A reagent blank was obtained by mixing 30 ~1 of 1.25 mM DNPA with 30 ~1 of a mixture of equal volumes 0.1 M phosphate buffer and 1 N HCl, diluting the resulting mixture with 140 ~1 of water, and reading absorbance of the final solution at 360 nm. The fraction of DNPA with respect to DNPA plus product in each stopped reaction mixture was estimated as ((M - B) - (R - B))/ (M - B), where M is the maximal absorbance value, B is the reagent blank, and R is the absorbance of the diluted reaction mixture. RESULTS
Data, produced in denaturation experiments with G4 replicative form DNA, typical of those readily obtainable with the apparatus and procedures, are shown in Fig. 3. The data (Fig. 3) define regular, smooth curves approximated by those drawn through the sequential points and can be judged, on this basis, to exhibit a high level of internal consistency. In this regard, it is noteworthy that except for use of the same DNA and reagent solutions throughout, each point shown in Fig. 3 represents an essentially independent experiment. Interpolation of the Fig. 3 data yields 50% denaturation times of 11.7, 4.4, and 1.8 s under the described conditions at 0, 5, and 10°C respectively. The apparatus and procedures are thus applicable for studies of relatively fast reactions. To test the apparatus with a reaction other than the denaturation of DNA we have made measurements on the alkaline hydrolysis of DNPA at O’C. The data and the linear regression lines are given in Fig. 4. From the intercepts at zero times, net mixing delays of 0.3 and 0.15 s were calculated for the upper and lower curves, respectively. These delays are within the standard deviation of the time measurements and are probably maximum values. The first-order rate constants were 0.0 136 and 0.037 s-‘, log,, respectively. These values yield second-order constants of 6.6 and
333
MIXING
II 0
20
40
60
SECONDS
FIG. 3. Denaturation curves for G4 replicative form DNA in 40 mM NaOH at the indicated temperatures. Supercoiled DNA, form I, is denatured in the reaction to yield a more rapidly sedimenting species, form I.+ Relative amounts of forms I and & in the reaction mixtures are determined in an analytical ultracentrifuge. The natural logarithm of the fraction remaining as form I is plotted versus the reaction time in seconds.
7.2 s-i M-’ when combined with (OH-) calculated from the pH and pK, at O’C. We have not found a recorded value for this constant at 0°C but it appears consistent with the value of 49 s-’ M-’ at 25°C obtained by Barman and Gutfreund (2). DISCUSSION
The infrasonic mixing apparatus described here fills a gap in the available devices for rapid sampling when small volumes of reagents must be used and the reaction is too fast to use automated sampling. Flow methods employing two mixing chambers for achieving sampling require large volumes. For example, the device of Thayer and Hinkle (3) was designed to reduce the volume, but 0.5 ml is stated to be the minimum required. It is also difficult in this type of apparatus to maintain a constant temperature. The design of the mixing apparatus was based on empirical observations and on specifications dictated by the nature of our experiments. However, it is flexible and could be improved in various ways. For applications in which smaller sample volumes are desired, the diameter of the reaction vessel tubing could be reduced. However, the total volume of re-
334
CAMIEN
AND WARNER
SECONDS
FIG. 4. First-order plots of rates of hydrolysis of DNPA at 0°C in mixtures of 30 ~1 of 1.25 mM DNPA (in 5 mM HCI) and 15 @I of 0.1 M phosphate buffer with K$‘Q,/ K2HPQ, ratios of 4: 1 (lower data set) and 1: I (upper data set), respectively. The fraction DNPA (ordinate) values are calculated as described in the text. Least-squares linear regression lines are drawn through the data. The slopes are -0.0136 (upper set) and -0.0370 (lower set), and the standard deviations from the regression lines are 0.0080 and 0.0072 log. units for the upper and lower sets, respectively. The zero time intercepts are -0.0040 (upper set) and -0.0055 (lower set).
action mixture of 45 ~1, employed in our procedure, is probably near the upper limit imposed by the requirement that the reaction mixture be contained as a stable seal in the tubing. Improvements could also be made in the precision of the timing to adapt the apparatus for use with faster reactions. We have found that a working frequency of 25 Hz for infrasonic mixing provided stronger mixing with less propensity to disrupt the reaction mixture seal than higher or lower frequencies. Effects of variations in characteristics and dimensions of the system components (speaker coil, rubber dropper bulb, glass and rubber tubing, etc.) on the signal frequency and amplitude required for optimal performance were not investigated. An electrical solenoid valve was employed to start and stop the mixing because it was observed that either turning on the signal generator or closing a switch between the speaker coil and the amplifier produces a strong transient signal which generally results in disruption of the reaction mixture seal. Alternatively,
the transient signal might be attenuated by modifying the switching circuitry. Transparent Teflon tubing was used for fabricating the reaction vessel and micropipet because the marked hydrophobicity of this material facilitates quantitative transfers of aqueous sample and reagent solutions and was essential for the manipulations of the solutions within the reaction vessel. An inside diameter of approximately 4 mm was used for the reaction vessel because a droplet of sample solution will not readily form a stable seal inside a larger diameter, and the sample volume required for a seal of acceptable thickness in tubing of smaller diameter is smaller than we desired. System components, except for the specially constructed water bath and signal generator/ amplifier, were selected from parts which were on hand in the laboratory. The solenoid valve (ASCO No. 8262B208) was from Automatic Switch Company, Florham Park, New Jersey; the three-position manual valve was a Luer 3-way nylon stopcock (Bio-Rad No. 7329009); and the speaker (FoMoCo No. C6VA18932-A646) was from a discarded automobile audio tape player. Requirements for the signal generator/amplifier were determined in preliminary experiments employing a research model signal generator with a standard audio power amplifier. Subsequent work employed a relatively inexpensive and compact signal generator/amplifier designed and built to provide an adjustable sinusoidal output of 0 to 5 V peak-to-peak at 1O-60 Hz with a standard S-ohm speaker load. REFERENCES 1. Strider, W., &mien, M. N., and Warner, R. C. (1981) J. Biol. Chem. 256,7820-7829. 2. Barman, T. E., and Gutfreund, H. (1964) in Rapid Mixing and Sampling Techniques in Biochemistry (Chance, B., Eisenhardt, R. H., Gibson, Q. H., and Lo&erg-Holm, K. K., eds.), pp. 339-344, Academic Press, New York. 3. Thayer, W. S., and Hinkle, P. C. (1979) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 56, pp. 492-496, Academic Press, New York.