Construction of up-and-down temperature-jump apparatus

ANALYTICAL

BIOCHEMISTRY

87-90

149,

Construction

(1985)

of Up-and-Down

Temperature-Jump

HIROSHI Department

of Food

Science

and Technology,

Apparatus

NARATANI

Faculty

of Agriculture,

Kyoto

University,

Kyoto

606, Japan

Received February 4, 1985 A fast up-and-down temperature-jump apparatus whose dead time is about 60 ms was constructed. The principle of the method is to let the sample solution flow to the observation cell through a capillary in a heat-exchange chamber. Bubbling and cavitation effects in the observation cell at large up or down temperature jumps were eliminated by application of a nitrogen gas pressure of 2-5 bar. The down-temperature-jump method is especially effective for measuring temperature-induced conformational transitions of biopolymers and their assemblies. Q 1985 Academic Ress, Inc. KEY WORDS: temperature jump; up-and-down T jump; protein folding; ribonuclease A; rapid head exchange; relaxation method.

may contribute to kinetic measurements of temperature-induced conformational transitions of biopolymers.

The usual temperature-jump methods are up type, applying single or multiple energy pulses to the sample solution (1). The energy pulse is derived from electric current, laser, microwave, etc, with a measureable time range from 30 ns to a few seconds. The maximum jump height is about 0.2- 15 “C; a temperature jump by energy-pulse methods tends to cause cavitation in the sample solution. On the other hand, rapid down-type temperature jumps are not easy because homogeneous cooling of the sample solution cannot be achieved rapidly. The easiest way is to cool the sample solution cell holder by exchanging heat from a circulating water bath; the sample solution is cooled slowly by heat exchange through the surrounding wall. F. M. Pohl succeeded in producing large upand-down temperature jumps within a few seconds using the above idea in 1969 (2). Apparatuses which followed, by other authors, are in principle the same (3,4). Attainment of homogeneous heating or cooling by previous heat-exchange methods, needed at least a few seconds. The purpose of the method reported in this paper is to reduce the time resolution one or two orders below a few seconds. The extension of the measurable time range of the down-temperature jump

MATERIALS

AND

METHODS

Reagent-grade nickel nitrate, asparagine, phenol red, boric acid, and 5-sulfosalycilic acid were purchased from Nakarai Chemicals, Ltd. Bovine pancreas ribonuclease A type XII-A was from Sigma. The schematic structure of the apparatus is shown in Fig. 1. The apparatus consists of three parts: reservoir, heat-exchange chamber, and observation cell block. The temperature of each part can be controlled separately with circulating-water baths. The reservoir is a glass cylinder of about 10 ml. The heatexchange chamber is made of copper and has a rectangular capillary to contain the sample solution (Fig. 2). The dimensions of the capillary are 0.5 X 1 mm cross section and 344 mm length. The whole surface of the heat exchange chamber is gold plated. The optical cell is made of ceramic with quartz windows and an optical path length of 10 mm. The cell is enclosed in a metal block whose temperature is controlled by a circulating-water bath. A nitrogen gas pressure 87

0003-2697185

$3.00

Copyright Q 1985 by Academic Press. Inc. All rights of reproduction in any form reserved

HIROSHI

88

NAKATANI

ELECTROMAGNETIC VALVE

DON

CIRC;LATING WATER

CHAMBER

nc. 1. Schematic structure of apparatus. A sample solution flows from reservoir to electromagnetic valve through heat-exchange chamber and observation cell. The solution from the electromagnetic valve can be recovered without damage.

of 2-5 bar was maintained inside the reservoir, heat-exchange chamber, and observation cell. The flow start and stop are controlled by an electric valve at the end of the optical cell block. A Union Giken stopped-flow apparatus (RA- 1300) was used with slight modification for the flow control, signal detection, and recording. Actually, the temperature of the heat-exchange chamber and cell block were set to be the same. About 300 ~1 of the sample solution was consumed for one shot. RESULTS

The nickel-asparagine system is a reversible metal-ligand complex-formation reaction. The relaxation has been observed using the pH indicator phenol red by the Joule-heating

7 lgm50c C130C FTG. 3. Relaxation spectra of Ni(II)-asparagine system using phenol red as an indicator: Ni(II), 0.5 mrq asparagine, 20 mM; phenol red, 10 pM; KNO,, 0.1 M; pH 7.7. Final temperature is 23’C and initial temperature is shown in the figure. The upper signal is from 13 to 23°C without Ni(I1). The vertical coordinate is absorbance at 560 nm.

temperature-jump method (5). Since relaxation time is 100 ms- 1 s, the signal could be observed by the present apparatus. As shown in Fig. 3, the direction of the relaxation signal changes with the direction of the temperature change. The relaxation time in Fig. 3 depends on the final temperature but does not depend on the jump height of the temperature and direction of the temperature change. There was no relaxation signal for the phenol redasparagine system at the same pH.

Reservoir (10rnl) Observation

I

I

Cell

optical

path

200 ms

05 xl m&apillary 344 m m path ( total 117 ~1 )

50 m m

FIG. 2. Structure of heat exchange chamber and its connections with reservoir and observation cell.

Fuz. 4. Relaxation spectra of boric acid-5-sulfosalycilic acid system: boric acid, 50 mM; 5-sulfosalycilic acid, 1.5 mM; pH 5.3 (0.1 M acetate buffer in 0.1 M NaCI). The vertical coordinate is absorbance change at 320 nm. The final temperature is 20°C. The initial temperature is 10°C (upper signal) and 30’C (lower signal), respectively.

UP-AND-DOWN

T-JUMP

A standard reaction system was used to check the reliability of observed relaxation times. The borate-5-sulfosalycilic acid system is suitable for this purpose because it is a reversible reaction and is monitored spectroscopically without additional indicators, and relaxation time ranges around 1 s- 100 ms (6). As shown in Fig. 4, the relaxation signals at 20°C were observed by up-and-down temperature-jumps; the relaxation times are 3 10 and 330 ms, respectively, under the conditions used here. The relaxation time which was observed by mixing boric acid and 5sulfosalycilic acid with a stopped-flow method (Union-Giken RA- 1300 apparatus) is 320 ms under the same final condition. The agreement of the relaxation times with both methods is satisfactory. The transition temperature of ribonuclease A is between 50 and 60°C at neutral pH (7). Applicability of the apparatus was tested making use of the denaturation-renaturation kinetics of ribonuclease A at pH 7.55. Only a single relaxation was observed in the time range of the present apparatus. The relaxation time did not depend on the jump height of the temperature and the direction of the temperature change as shown in Fig. 5. The observed relaxation time is shown in Fig. 6

APPARATUS

t

I 2.9

89

I

I

I

3.0

I

I

3.1

1

3.2 jQQ& T

FIG. 6. Plot of reciprocal relaxation time (I/T, s-‘) vs reciprocal absolute temperature (l/Z’). The filled circle is from up-temperature jump and open circle is from down-temperature jump.

as a function of temperature. The overall features of the plots are comparable to the results at low pH range (2). The fastest relaxation signal observed by the present apparatus is shown in Fig. 7. The relaxation time is 29 ms. By comparison with the static signal change the dead time was estimated using the equation (8) Ao td = r ln -

A

-

L0.19:

FIG. 5. Relaxation spectra of ribonuclease A denaturation-renaturation reaction at pH 7.55. The final temperature is 57°C and initial temperature is shown in figure. Ribonuclease A concentration is 64.3 PM. Buffer of Tris-perchlorate (1 mM) in 0.1 M NaCIOs was used. The vertical coordinate is absorbance at 287 nm.

where td and r are the dead time and observed relaxation time, A0 and A are the static and observed kinetic signal amplitudes, respectively. Although fast phase exists in ribonuclease A denaturation which is not observed by the present apparatus, its amplitude is

20msec

FIG. 7. Unfolding signal of ribonuclease A at high temperature region. Temperature jump from 59.1 to 73.1”C: ribonuclease A concentration, 62.2 pM; other conditions are the same as in Fig. 5.

90

HIROSHI NAKATANI

much smaller than that of slow phase (9). The estimated dead time was 58 ms. It may be a little shorter if the contribution of the fast phase is subtracted from the static signal amplitude in the above equation. DISCUSSION

to cooling or heating was observed in the present apparatus (see, for example, the upper trace of Fig. 3). This suggests that heat exchange is almost complete within 100 ms for a temperature change of at least + 15 “C. Fluorescence detection is possible by changing the observation cell and the position of photomultiplier. Applications of other detection methods are in progress.

The present apparatus reduces the time resolution by one or two orders for downACKNOWLEDGMENT temperature jumps as compared to previous The technical assistance of Mr. T. Nagamura in the methods. Most kinetic studies of protein construction of the apparatus is gratefully acknowledged. denaturation and renaturation were carried out in the acidic pH region in a denaturant REFERENCES medium to reduce transition temperature 1. Crooks, J. E. (1983) J. Phys. E 16, 1142-I 147. and reaction rates. Furthermore, at temper2. Pohl, F. M. (1969) Eur. J. Biochem. 4,373-377. atures above 60°C bubbling and cavitation 3. Renter, W., Mandelkow, E.-M., Mandelkow, E., and effects disturb observation of relaxation sigBordas, J. (1983) Nucl. Instrum. Methods 298, nals. These disturbing effects at the higher 535-540. 4. Ranck, J. L., Letellie, L., Schechter, E., Krop, B., temperature region are eliminated by appliPernot, P., and Tardieu, A. (1984) Biochemistry cation of a nitrogen gas pressure of 2-5 bar 23,4955-496 1. to the sample solution (no leakage was de5. Osugi, J., Nakatani, H., and Fujii, T. (1969) Nippon tected up to 90°C). Kagaku Zassi 90, 529-534. If heating or cooling is insufficient, artifact 6. Qeen, A., Davis, L., and Con, A. (1979) Canad. J. Chem. 57, 920-923. signals may appear due to the temperature 7. Garel, J. R., and Baldwin, R. L. (1975) J. Mol. Biol. change at the observation cell. This effect is 94,621-632. easily checked using pH indicator-buffer sys8. Nakatani, H., and Hiromi, K. (1980) J. Biochem. tems with relaxation times much shorter 87, 1805-1810. than the observable time range of the present 9. Tsong, T. Y., Baldwin, R. L., and Elson, E. L. (\ 197 1,j Proc. Nat/ Acad. Sci. USA 68.27 12-27 15. apparatus. No apparent relaxation signal due