A simple stopped-flow temperature-jump apparatus

ANALYTICAL

BIOCHEMISTRY

117, 164- 169 ( 198 1)

A Simple Stopped-Flow A. S. VERKMAN, Biophysical

Laboratory,

Temperature-Jump

JAMES A. DIX, Harvard

Medical

Apparatus

AND A. A. PANDKCIO

School,

Boston,

Massachusetts

021 I.5

Received April 3, 1981 A stopped-flow temperature-jump apparatus that has been used to extend the capabilities of a temperature-jump apparatus described previously (Verkman et al., Anal. Biochem. 102, 189-195, 1980) is described. The observation cell is a single cylinder made from ultraviolettransmitting Lucite. Stainless steel electrodes are fitted axially into each end of the cylinder, and solutions enter into and exit from the cylinder by passing through holes drilled perpendicular to the cylinder axis at the level of the electrodes. A triggering circuit provides a variable delay between solution mixing and the temperature-jump discharge. The mixing time for this instrument is under 1 ms, the dead time is under 60 ms, and the heating time is under 3 us. Solution reactions are followed by measuring optical absorption, fluorescence, or scattering with signal-to-noise ratios generally exceeding 5OO:l. The performance of this apparatus is illustrated by several membrane-substrate interactions.

The kinetics of fast reactions have been studied extensively in recent years by relaxation methods (1). The stopped-flow temperature-jump technique, in which solutions are mixed rapidly and then subjected to a temperature jump after a set delay, is a versatile method to study a wide variety of systems (2,3). Microsecond time resolution may be obtained for chemical systems in a nonequilibrium state, allowing detailed characterization of transient reaction intermediates and enzyme complexes (4). In biomembrane systems, the stopped-flow temperature-jump technique is especially suited to the study of membrane transport asymmetry, where rapid binding kinetics may be studied in membranes exposed to substrate at each surface independently. We describe here the electronics, mixing chamber, and observation cell that may be incorporated into and extend the capabilities of a preexisting temperature-jump instrument (5) so that stopped-flow temperaturejump measurements may be performed. The design criteria are high signal-to-noise ratio, visible and uv optical detection, and freedom from solution cavitation and solution eddies, which may cause observation of unheated solution. 0003-2697/81/150164-06$02.00/O Copyright 0 I98 I by Academic Press, Inc. All rights of reproduction in any form reserved.

MATERiALS

Phenol red was obtained from Fisher Scientific Company (Fair Lawn, N. J.), phloretin was purchased from K and K Laboratories (Plainview, N. Y.), and 4,4’-dibenzamido-2,2’-disulfonic stilbene (DBDS)’ was synthesized by the method of Kotaki et al. (6). Single-wall phosphatidylcholine vesicles were prepared by sonication (7) and human red cell ghost membranes were prepared by hypotonic cell lysis (8). DESIGN OF APPARATUS

Figure 1 shows a schematic of the stoppedflow temperature-jump apparatus. The details for activating the temperature-jump discharge have been described in a previous paper (5). A manual trigger starts a waveform recorder and activates the high-voltage discharge. The observed absorption, fluorescence, or scattering signal, indicating the extent of solution reaction, is detected by a photomultiplier and then amplified, digitized, and transferred to a computer for analysis. We have previously described the I Abbreviations used: DBDS, 4,4’-dibenzamido-2,2’disulfonic stilbene; PC, phosphatidylcholine. 164

STOPPED-FLOW

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Piow DRIVE

FIG. 1. Schematic of stopped-flow temperature-jump apparatus. shown) initiates the stopped-flow trigger, which activates a delay After the delay, the pulse sequence generator triggers a waveform the temperature jump. Solution reaction photomulitplier signal is amplified, filtered, for analysis as described previously (5).

is detected by absorption, fluorescence, or scattering. The and then digitized, recorded, and transferred to a computer

battery-driven 150-W mercury arc or tungsten filament lamp. A 600-W mercury-xenon lamp (Conrad Haovia Model 941 BOO, Newark, N. J.) with power supply (Electra Powerpacs Corp., Cambridge, Mass.) has been added to increase the intensity for fluorescence energy transfer and polarization measurements. Figure 2 is a more detailed block diagram of the circuitry included in the block labeled

FIG. 2. Trigger selection. The set/reset flip-flop either by a manual push button or by a microswitch the microswitch mode is selected, a variable delay and

the temperature-jump

discharge.

A microswitch on the plow drive (not timer in the trigger selection circuit. recorder and spark-gap discharge for

Trigger Selection in Fig. 1. When the apparatus is operated in the temperature-jump mode, the trigger signal which initiates the high-voltage discharge is generated manually by depressing a push button. Mechanical switches, including microswitches, are often characterized by a bouncing of the contact surfaces when activated. This produces a series of electrical pulses of indeterminate duration, and it is standard practice

allows the temperature-jump on the plow of the stopped-flow is interposed between the time

discharge to be fired syringe drive. When when the flow ceases

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VERKMAN,

DIX

to electrically condition the output of mechanical switches so as to produce a clean switching action. The electrical conditioning is referred to as “switch debouncing” and is designated in Fig. 2. A short delay is incorporated into this circuit so that an oscilliscope may be triggered a short time before the high-voltage discharge takes place. When the apparatus is operated in a stopped-flow temperature-jump mode, the trigger signal which initiates the discharge is derived from a microswitch activated by the plow on the syringe drive as solution flow stops. The microswitch output is debounced for reasons described above and the conditioned signal is then delayed before being used to initiate the discharge. The delay is incorporated to allow solution reaction to take place for a desired time before the temperature-jump discharge is produced. As a safety precaution, a circuit that will allow the discharge system to be triggered by only one of the two possible sources at a given time has been included. The operator must choose which source is to be enabled by depressing one of two pushbuttons. The choice made is displayed via light-emitting diodes and directs a “gating” circuit to accept trigger inputs only from the source indicated. The logic components used in the circuits are chosen from a family of devices called HiNIL (specifically the 300 series manufactured by Teledyne Semiconductor, Mountain View, Calif.), which are characterized by high noise immunity. Such devices ignore small transients that might otherwise cause false triggering. The mixing chamber and observation cell are shown in Fig. 3. The dual-jet mixing chamber is simple to construct from a single Lucite block and mixes two solutions rapidly and efficiently (9). The observation cell is constructed from uv-transmitting Lucite (Acryline type OPl; American Cyanamid, Wallingford, Conn.), so that absorption, fluorescence, and scattering measurements may be performed in the wavelength range 280750 nm. Solutions enter the Lucite mixing

AND

PANDISCIO

h B \

I

I

LOWER ELECTRODE

L---J

FIG. 3. Schematic of mixing chamber and observation cell. Solutions are mixed in a dual-jet mixing chamber and enter the observation cell at the level of the lower electrode. Flow exits from the observation cell at the level of the upper electrode. Rubber O-ring seals prevent leakage of solution from the interface between the electrodes and Lucite observation cell. The electrode diameter is I cm and the spacing between electrodes is 1.2 cm. The mixing chamber and observation cell are drawn to scale.

chamber at the level of the lower electrode and exit at the level of the upper electrode. The choice of solution entrance and exit paths was found empirically to minimize solution cavitation, ensure complete replacement of old solution with freshly mixed solution, and minimize observation of unheated solution during a stopped-flow temperaturejump measurement. Calibration experiments? performed using 0.1 M Fe(NO,), in 0.1 M HzS04 with 0.1 M KSCN show that the mixing time of a stopped-flow experiment is
Dionex

Instrument

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200 TIME

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FIG. 4. Search for eddy currents following a stopped-flow temperature-jump of phenol red. Phenol red (40 PM) in 100 mM Tris + 100 mM KCI, pH 7.6, 25°C was mixed with identical buffer not containing phenol red. When a 4°C-temperature jump is applied after mixing, there is the expected fast acid-base relaxation of phenol red, which could be followed by any transient artifacts, such as eddy currents, which might arise from unheated solution entering the observation light path.

by means of a Teflon three-way valve (Type 50, Rheodyne, Berkeley, Calif.). Solution outflow is unrestricted; flow is stopped at the syringe drive, where the Lucite block limits forward plow motion (10). For faster reaction times (cl00 ms), an air-driven syringe

drive (11) is under construction to replace the hand-driven syringe drive. The temperature-jump electrodes consist of polished stainless steel cylinders that fit to within one micron into the Lucite cylinder (5). Rubber O-rings prevent solution leak-

0.38 E c

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FIG. 5. Stopped-flow temperature-jump study of phloretin binding phloretin was mixed with an equal volume of 100 PM PC vesicles in and 100 mM Tris at pH 7.3, 23°C. Increasing transmittance at 328 phloretin to vesicles. The inserts show the results of a 4°C-temperature after the phloretin and vesicles are mixed.

to PC vesicles. 0.75 ml of 100 FM solutions containing 100 mM KCI nm indicates increased binding of jump performed at selected times

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DIX,

AND

PANDISCIO

age. Sample temperature is controlled to within 0.5”C over the temperature range 550°C by passing water through the hollow electrodes and by circulating temperaturecontrolled air into a Lucite box enveloping the electrodes, syringe drive, and observation cell. PERFORMANCE

EXPONENTIALS

OF APPARATUS

To investigate whether eddies arising from solution entrance and exit paths cause observation of unheated solution, phenol red was mixed with buffer and then temperature-jumped as shown in Fig. 4. The initial rise results from a rapid acid-base shift in phenol red induced by the temperature jump. The trace should then remain high and level until solution cooling has occurred, which, for a temperature-jump experiment, is approximately 1 s. If convective eddy currents exist, there will be premature cooling observable as a downward deflection of the trace in Fig. 4 over hundreds of milliseconds. The experiment shows no effect of eddy currents over the useful measurement time in a stopped-flow temperature-jump experiment. Figure 5 shows the binding of phloretin to phosphatidylcholine (PC) vesicle membranes. When phloretin is mixed with PC vesicles, there is a rapid, diffusion-limited binding to the outer vesicle surface (30-200 ps) followed by a slower (0. l- 10 s) movement of phloretin from sites at the outer to inner vesicle surfaces. ( 12,13). A temperature jump was performed at three selected times after the solutions were mixed; the resultant traces demonstrate the unbinding of phloretin from outer vesicle sites induced by the temperature jump. The amplitude and time course of each trace yield information about the mechanism and rate constants of the binding process. The signal-tonoise ratio for these experiments was approximately 4O:l for a fractional change in transmittance of 3%. Figure 6 shows a fluorescence stoppedflow temperature-jump application. The stil-

I I

2

3

4

TIME ki 6. Stopped-flow temperature-jump study of DBDS binding to ghost membranes. The DBDS binding to red-cell ghost membranes was measured by Ruorescence. Lower trace: 0.75 ml of I pM DBDS was mixed with an equal volume of ghosts at a membrane band 3 concentration of 40 nM in 28.5 mM sodium citrate, pH 7.4, 25°C. Upper trace: a 3OCtemperature jump was applied 100 ms after the same solutions were mixed. The solid smooth lines are single exponential fits to the data.

bene inhibitor of anion exchange, DBDS, is rapidly mixed with human red cell ghost membranes, and the fluorescence enhancement of DBDS associated with binding is observed ( 14). The lower trace shows a simple stopped-flow experiment. In order to search for rapid, temperature-induced conformational changes in the DBDS-binding protein (band 3), a 3YJ-temperature jump is performed immediately after mixing as shown in the upper trace. Since the observed time course is not altered significantly by the additional temperature jump, there are no important temperature-dependent band 3 conformational changes which might make this system difficult to study by the temperature-jump technique alone. The stopped-flow temperature-jump apparatus described is an easily built and valuable addition to a preexisting temperaturejump system. The system has an excellent signal-to-noise ratio and efficient mixing

STOPPED-FLOW

without significant cavitation or convection, and it allows the rapid accumulation and computer analysis of kinetic data. Several potential biomembrane applications of the stopped-flow temperature-jump technique have been explored. Detailed schematics of the electronics are available upon request. ACKNOWLEDGMENTS We wish to thank B. Corrow, R. Dooley, and W. Kazolias for their excellent technical assistance and Professor A. K. Solomon for advice and encouragement. A.S.V. wishes to acknowledge the Prudential Insurance Company for a Medical Scientist Scholarship. This work was supported by NIH GM 15692.

REFERENCES I.

Hammes, G. G. (1974) in Techniques of Chemistry (Weissberger, A., ed.), Vol. VI, Wiley, New York. 2. Eigen, M., and deMaeyer, L. (1964) in Rapid Mixing and Sampling Techniques in Biochemistry (Chance, B., Eisenhardt, R. H., Gibson, Q. H., and Lonberg-Holm, K. K., eds.), Academic Press, New York.

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3. Erman, J. E., and Hammes, G. G. (1966) Rev. Sci. Instrum. 37, 746-750. 4. Bernasconi, C. F., and Muller, M. C. (1978) J. Amer. Chem. Sot. 100, 5530-5533. 5. Verkman, A. S., Pandiscio, A. A., Jennings, and Solomon, A. K. (1980) Anal. Biochem. 189-195. 6. Kotaki, A., Naoi, Biophys. Acta

M., and Yagi, 249, 547-566.

R. (1971)

M., 102,

Biochim.

7. Huang, C., and Thompson, T. E. (1974) in Methods in Enzymology (F1eisher.S. and Packer, L., eds.), Vol. 32, pp. 485-489, Academic Press, New York. 8. Dodge, J. T., Mitchell, C., and Hanahan, D. T. (1963) Arch. Biochem. Biophys. 100, 119-130. 9. Paganelli, C. V., and Solomon, A. K. (1957) J. Gen. Physiol. 41, 259-277. 10. Sidel, V. W., and Solomon, A. K. (1957) J. Gen. Physiol. 41, 243-257. 11. Levin, S. W., Levitt, R. L., and Solomon, A. K. (1980) J. Biochem. Biophys. Methods 3, 255-

272. 12. Verkman, Physiol. 13. Verkman,

A. S., and Solomon, 75, 673-692. A. S. (1979) Ph.D.

A. K. (1980) thesis,

Harvard

J. Gen. Univ.

14. Dix, James A., Verkman, A. S., Solomon, A. K., and Cantley, L. C. (1979) Nature (London) 282,

520-522.