Rapid-Kinetics and Spectroscopy: Stopped-Flow; Fluorescence, Absorbance, Circular Dichroism, Anisotropy Spectrometry

Rapid Mixing Temperature Jump System - mT-Jump

• Hot and cold jumps • T-changes as high as several ten of degrees • Low volume consumption • Millisecond resolution

This new T-jump instrument achieves temperature changes by mixing two solutions of different initial temperatures T1 and T2. The final temperature of the mixture (T3) is calculated from the initial temperatures T1 and T2 and the mixing ratio of the two solutions.

Three thermoelectric Peltier elements are used to control the initial temperatures of the two solutions and that of the observation cell after mixing. The mT-jump accessory is installed on a Bio-Logic stopped-flow basis.

The system is fully controlled from the Bio-Kine32 software: the user defines the mixing ratio for the reaction and the amplitude of the temperature jump.
The temperature of each Peltier element is then automatically adjusted. Automatic accumulation of shots is possible to optimize the signal to noise ratio.

The mT-jump system is equipped with a standard Bio-Logic stopped-flow observation cell. It is compatible with all modes of observations such as circular dichroism, fluorescence, absorbance, and fluorescence anisotropy.

A complementary instrument to classical T-jump

The aim of classical T-jump instruments is to record fast heat induced relaxations of reactions. This is normally achieved by applying fast electric current pulses or more recently infrared laser pulses to the sample. Time resolution of classical T-jump can reach a few µs but the kinetics cannot be recorded for more than a few ms due to temperature dissipation, which could be a limitation in some applications. Most classical temperature jump devices are limited to 5°C and are unable to perform cooling jumps.

Bio-Logic’s mT-jump is based on Stopped-Flow technology and thus offers millisecond resolution. The amplitude of the jump is entirely controlled by the user (up to 40°C jump) and the jump can be in both directions (cooling or heating jump) which offers new investigation domains, for example: refolding studies. Protein folding and refolding can easily be studied without adding denaturant to the protein! Detection with classical T-jump instrumentation is also limited to techniques such as absorbance and fluorescence whereas mT-jump is compatible with all optical methods (including circular dichroism, FT-IR, fluorescence anisotropy…)

Example of application: Cold jump induced refolding of cytochrome-c

In classical protein refolding studies, the protein is stored in denaturants such as urea or guanidine hydrochloride 8M.
The refolding is then initiated by diluting the unfolded protein with buffer.
The mT-jump accessory offers a new approach to refolding studies by observing the thermal refolding process. The unfolded protein is stored at a high temperature (T1), the refolding is initiated by diluting the protein with cold buffer (T2). The amplitude of the temperature jump is hereby defined by the mixing ratio applied in the stopped-flow sequence. In the present example, horse heart cytochrome-c is stored at 85°C (unfolded state as determined by thermal denaturation). Refolding is initiated by a 2-fold dilution with cold buffer (60 μl of protein is mixed with 120 μl of buffer). The refolding is followed by measuring tryptophan fluorescence (excitation: 280 nm, and detection using 320 nm cut-off filter) in a FC-15 cuvette (2 ms dead time using SFM-20).

Cold jumps with amplitudes from 5°C to 25°C are shown in figure 1.
For cold jumps with small amplitudes (85°C to 80°C for example) the protein remains unfolded and no kinetics is observed.

Fig 1: refolding of cytochrome-c, starting temperature 85°C

For larger jumps, a burst phase is clearly observed and kinetics can be fitted using a two exponentials model. The cold jump from 85°C to 60° is shown in figure 2. The quality of the stop at 90ms and the even distribution of residuals of the fit demonstrates the performance of the Stopped-Flow mixing.

A plot of the final levels of fluorescence versus temperature will provide the thermal unfolding curve of cytochrome-c (sigmoidal shape not shown).

Fig 2: refolding of cytochrome-c, after a cold jump from 85°C to 60°C observed by florescence

Specifications

Temperature control specifications
• Full software control through USB connection.
• Temperature range : +5°C / +90°C
• Precision of temperature reading: 0.01°C
• Maximum temperature jump : +/- 40°C
• Temperature stability for S1 and S2 in stationary state: < +/- 0.2°C at 80°C after 15 minutes of equilibrium.
• Temperature stability in the cuvette after the T-jump: variation < 1% per 30s
• Precision of the T-jump: defined by user calibration (down to +/- 0.1°C)
• Temperature probe is included.

Mixing specifications
• Compatible with SFM-20, SFM-300 and SFM-400
• Mixing Ratio from 1/1 to 1/4.
• Sample consumption per shot : 45 µl to 220 µl.
• Dead time: depends on cuvette, stopped-flow model and mixing ratio (1.7ms using SFM-20, FC-08 cuvette and 1/1 mixing ratio)
• compatible with MOS-200; MOS-250, MOS-450/AF-CD, Diode Array detector and external devices
• Storage lines : 150 µl
• optional FT-IR cell

Please see the new section in Selection of scientific articles using the Bio-Logic rapid kinetics systems: Stopped-flow (mT jump)

If you are interested, please contact us