Extending applications of subzero mixing for studying chemical reactions with T-cycle EPR

Extending applications of subzero

mixing for studying chemical

reactions with T-cycle EPR

THESIS

submitted in partial fulfillment of the requirements for the degree of

BACHELOR OF SCIENCE

in PHYSICS

Author : M.D. van der Veen

Student ID : 1482858

Supervisor : Dr. P. Gast

2ndcorrector : Prof. dr. E.J.J. Groenen

Extending applications of subzero

mixing for studying chemical

reactions with T-cycle EPR

M.D. van der Veen

Huygens-Kamerlingh Onnes Laboratory, Leiden University P.O. Box 9500, 2300 RA Leiden, The Netherlands

December 5, 2018

Abstract

To improve existing methods for measuring chemical kinetics a technique has been developed: subzero mixing technique. In this procedure reagents are mixed in a cold viscous solution, preventing the components from reacting with each other during mixing. The chemical kinetics of the samples can be measured using temperature cycle EPR. For this bachelor

thesis it has been studied whether the subzero mixing method can be applied to investigate the reaction between myoglobin and azide. Furthermore, this method has been used to prepare and measure samples containing TEMPOL and various concentrations of sodium dithionite in a

Contents

1 Introduction 7

2 Theory 9

2.1 Energy level splitting 9

2.1.1 Zeeman effect 9

2.1.2 Hyperfine interaction 10

2.2 Electron Paramagnetic Resonance 10

2.3 Subzero mixing 12

3 Methodology 13

3.1 EPR spectrometer 13

3.2 Sample preparation 13

3.2.1 General preparation method 13

3.2.2 TEMPOL and sodium dithionite 15

3.3 Measurements 15

4 Results and discussion 19

4.1 Myoglobin and azide 19

4.2 Glycerol and sucrose solutions 21

4.2.1 Viscosity 21

4.2.2 Temperature scans TEMPOL 22

4.3 TEMPOL and sodium dithionite in a sucrose solution 23 4.4 TEMPOL and sodium dithionite in a glycerol solution 24

5 Conclusion 29

Chapter

1

Introduction

A method that can be used to track chemical reactions is that of the Rapid Freeze Quench (RFQ). The method consists of three steps: mixing, aging and freezing. By varying the length of an aging tube and subsequent rapid freezing of the mixture, the reaction can be stopped at different points in time. After freezing, the samples can for example be measured using Electron Paramagnetic Resonance (EPR). Such a measurement allows for finding short-lived intermediate species included in the reaction. These species are not present at the start or end of the reaction, but do occur somewhere in the process.

Advantages of RFQ are that it is suitable for various reactions and that the method has a high time resolution, down to 5 ms. There are however also some disadvantages to the Rapid Freeze Quench. Firstly, the technique is difficult to use. Furthermore, for each time point a new sample is needed. This means the measurements require a lot of resources and there could exist differences between the various samples.

To improve the measurement technique for tracking chemical reactions with EPR, a procedure has been developed: the subzero mixing technique in combination with a temperature jump measurement. In this technique the different components are mixed at a low temperature, in a solution with a high viscosity, allowing for mixing without the components already reacting. The samples can then be measured over a certain time period us-ing temperature cycle EPR to follow the chemical kinetics.

So far this preparation and measurement method has been performed on a reaction between TEMPOL and sodium dithionite in a 50% glycerol so-lution. TEMPOL is a stable radical, that gives a clearly distinguishable EPR-spectrum. TEMPOL can be reduced using dithionite. The reduced TEMPOL is not paramagnetic and therefore cannot be detected by EPR

8 Introduction

For the subzero mixing technique in combination with temperature cycle EPR to be a suitable replacement for the RFQ, it is however needed that the method can also be applied to other chemical reactions and with a time resolution comparable to that of RFQ. The aim of this research is to find out whether it is possible to extend the use of the subzero mixing technique. The study will focus on two questions:

• Can the subzero mixing technique in combination with EPR be used for measuring the reaction between myoglobin and azide?

• Is it possible to measure faster reactions between TEMPOL and dithion-ite than in the already performed research?

To answer the first question it will be investigated at what temperature a sample of myoglobin and azide remains sufficiently stable to mix with the subzero mixing method. It will also be investigated whether it is possible to prepare the samples at that temperature.

To answer the second question, measurements will first be performed with samples in a 60% sucrose solution. The sucrose solution is assumed to have a viscosity similar to the 50% glycerol solution at -50 ◦C, but at a higher temperature. This indicates the sucrose solution can be used to pre-pare the samples and measure the reaction at a higher temperature. Since reaction speed increases with temperature this would allow for tracking faster reactions.

In addition, measurements will be performed with samples in a 50% glyc-erol solution, but with different concentrations of sodium dithionite. The reaction speed increases with increasing concentration of sodium dithion-ite. If it is possible to prepare and measure samples with a higher concen-tration of sodium dithionite, this would thus allow for measuring faster reactions. On the other hand it is possible that a lower concentration of sodium dithionite would allow for measuring at higher temperatures, which also increases the reaction speed.

Chapter

2

Theory

2.1

Energy level splitting

EPR stands for Electron Paramagnetic Resonance. The term Electron Spin Resonance (ESR) is used as well. It makes use of the splitting of energy levels of the electron in a magnetic field. To understand EPR it is needed to first know what causes these splittings.

2.1.1

Zeeman effect

The magnetic moment of an electron, µe, is equal to:

µe = −geµBS (2.1)

In this equation geis the g-factor of an electron, which is for a free electron

approximately 2.0023, µB is the Bohr magneton of the electron and S is the

spin angular momentum. When a magnetic field is applied, the magnetic moment of the electron will tend to align, either parallel or anti-parallel to this magnetic field. This causes a splitting of the energy levels of the electron, that is known as the Zeeman effect. For a magnetic field H, the energy is given by:

E= − ~H· ~µe = geµBHms (2.2)

Here the axis of the magnetic field is taken as the z-axis, msis the

quantum-number corresponding to the z-component of the spin angular momentum of the electron. For a free electron with spin 1/2, the value of ms can be

10 Theory Nucleus Spin 1_{H 1/2} 12_{C 0} 13_{C 1/2} 14_{N 1} 15_{N 1/2} 16_{O 0} 17_{O 5/2} 55_{Mn 5/2}

Table 2.1:Some abundant atoms and their nuclear spin.

2.1.2

Hyperfine interaction

In molecules, the electrons also interact with the nuclear spin of the nuclei, which can have a magnetic dipole moment as well. This causes a dipole-dipole interaction, which is named the hyperfine interaction and causes a further splitting of the energy levels. For each value of the nuclear spin quantum number mI the energy level is split in 2I+1 levels, with I the

nuclear spin angular momentum. Table 2.1 shows the nuclear spin of some abundant atoms.

The energy levels for s= 1₂ are given by:

E =geµBHms+hA0msmI (2.3)

Here h is the Planck constant and A0is the hyperfine coupling constant in

frequency units.

2.2

Electron Paramagnetic Resonance

In the most simple case the EPR-mechanism can be described by Figure 2.1. A single electron can exist in only two spin states and when a magnetic field is applied, the energy levels split up. When the electron is simultane-ously irradiated with an electromagnetic field, it can be excited from the ms = −1₂ state to the ms = 1₂ state. This happens when the energy

differ-ence between the two spin states is equal to the energy of the photons in the electromagnetic radiation. For an electromagnetic radiation frequency ν, when both the Zeeman effect and the hyperfine interaction are taken into account, this is given by:

2.2 Electron Paramagnetic Resonance 11

Figure 2.1:The basic principle of Electron Paramagnetic Resonance Spectroscopy.

Figure 2.2:A basic setup for EPR-spectroscopy.

In an EPR spectrometer a magnetic field is applied, while simultane-ously irradiating the sample with microwaves. The microwave frequency is kept constant during the measurement, while the magnetic field sweeps over a set range. An absorption spectrum can then be obtained as a func-tion of the magnetic field. The EPR spectrometer modulates the magnetic field to obtain a higher signal to noise ratio. The result that is measured after lock-in detection, is the first derivative of the absorption spectrum. A basic, schematic EPR set-up looks like Figure 2.2. The set-up consists of a sample cavity, an electromagnet, a klystron for emitting the microwaves, modulation coils for modulation of the magnetic field and various detec-tors. The spectrometer is connected to a computer.

EPR spectrometers are categorized based on the electromagnetic radia-tion frequency they use. The spectrometer that is used for the experiments in this report is an X-band spectrometer, indicating it operates at a fre-quency of about 9.5 GHz. Such spectrometers are the most common.

12 Theory

2.3

Subzero mixing

To prepare samples that can be used to track chemical reactions, the sub-zero mixing technique has been developed. In this procedure the two reagents are mixed in a very viscous, but not yet frozen, solution at a tem-perature well below 0 ◦C. Since the reactivity decreases with decreasing temperature, the components can be mixed without (fully) reacting. This leads to homogeneous samples containing the two components. A more detailed description of the sample preparation method will be given in chapter 3. After the samples have been prepared, the chemical kinetics can be investigated with a T-jump measurement. For such a measurement a series of spectra is taken. At set time intervals the sample is irradiated with a 1500 nm laser pulse. This laser pulse causes the temperature of the sample to momentarily rise, which activates the reaction. The reaction is stopped again when the laser is turned off and the temperature decreases. Comparison of the spectra taken before and after the increment of the tem-perature can be used to follow the reaction. The function of the laser pulse in the T-jump measurement is analogous to that of the aging tube in RFQ. Since a complete T-jump measurement can take several hours, it is needed that the sample remains sufficiently stable at the temperature at which measurements are performed.

Chapter

3

Methodology

3.1

EPR spectrometer

The EPR spectrometer that was used for the experiments described in this report is a Bruker EMX spectrometer, which operates at X-band frequen-cies (9.5 GHz). The EMX is equipped with a temperature controller that makes use of liquid nitrogen in combination with a heater. Nitrogen gas is cooled by letting it flow through a Dewar filled with liquid nitrogen; the cooled nitrogen gas flows into a Quartz dewar inside the cavity and thereby cools the sample. Provided that the nitrogen flow is sufficient, this controller can stabilize the temperature of the cavity with an accuracy of 1 K.

3.2

Sample preparation

The sample preparation is an important part of this research. The prepa-ration method will first be described in general using the samples with myoglobin and azide as an example. Later will be explained which ad-justments have to be made for the samples with TEMPOL and sodium dithionite.

3.2.1

General preparation method

First stock solutions of the two components of the reaction have to be pre-pared in a 50 % glycerol solution (V/V). These stock solutions can then be used to prepare the samples. A schematic drawing of the preparation

14 Methodology

Figure 3.1: A schematic drawing of the setup used for sample preparation. The setup consist of a styrofoam box, with a small layer of liquid nitrogen on the bottom. A flow of cold nitrogen gas is used to cool the setup. The temperature is measured using thermocouples at various points, indicated with T1, T2 and T3. The samples are mixed in a small beaker, which is located on top of an aluminium holder.

setup is shown in Figure 3.1. The entire setup is cooled using liquid ni-trogen and cold nini-trogen gas. The flow of the nini-trogen gas can be man-ually adjusted to decrease or increase the temperature of the setup. The myoglobin mixture is first added to one half of a cooled 5 mL beaker. The beaker has to be kept at an angle of around 40◦ to ensure the solution stays on one half of the beaker. The setup has to cool down to around -50◦C. At this point the solution will become almost solid. The beaker can then be turned around to add the azide solution to the other side of the beaker. It is again important to keep the beaker at an angle of around 40◦ so the two solutions will not touch. This is illustrated in Figure 3.2.

After both solutions have cooled down sufficiently, a pre-cooled spat-ula can be used to mix the two components. To obtain a homogeneous mixture, the solutions have to be mixed for 30 to 60 seconds. The sample capillaries can be filled with the solutions by sticking them into the viscous mixture until there is enough sample in the capillary to measure. Capillary forces can not be used to fill the capillaries. It is again important to pre-cool the capillaries, since heating of the solution will cause the reaction to start. For the samples with myoglobin and azide 100 µL Hirschmann ring-caps micropipettes are used. The samples have to remain stored in liquid

3.3 Measurements 15

Figure 3.2:A schematic drawing to show how the two solutions should be added to the mixing beaker.

nitrogen.

3.2.2

TEMPOL and sodium dithionite

For samples with TEMPOL and sodium dithionite, some adjustments have to be made to the aforementioned procedure. The most significant adjust-ment is the de-oxygenation of the stock solutions, before adding sodium dithionite. To prevent dithionite in an aqueous solution from reacting with oxygen, all solutions have to be de-oxygenated using argon gas for at least an hour. During the entire sample preparation procedure it is important that the sodium dithionite remains free of oxygen. If the sodium dithionite does not completely dissolve in the solvent, it might be needed to briefly sonicate the solution. Samples containing TEMPOL and sodium dithion-ite are not only made in a 50% glycerol solution, like the samples with myoglobin and azide, but also in a 60% sucrose (mass per volume) so-lution. The mixing temperature for the glycerol solution is around -50

◦_{C, as was mentioned in the general description. The mixing temperature}

for the sucrose solution is around -20◦C. For the samples with TEMPOL and sodium dithionite 50 µL BLAUBRAND intraMARK micropipettes are used.

Following these procedures, samples can be prepared with concentrations as shown in tables 3.1 and 3.2.

3.3

Measurements

To measure any of the samples, the sample has to be inserted into the cav-ity. The samples in a capillary are first put into a 4 mm quartz tube that

16 Methodology

TEMPOL (mM) sodium dithionite (mM)

1 25

1 50

1 100

1 250

(a)50% glycerol solution

TEMPOL (mM) sodium dithionite (mM)

1 50

1 250

(b)60% sucrose solution

Table 3.1: The samples with different concentrations of TEMPOL and sodium dithionite used for this report.

myoglobin (mM) azide (mM)

1 3

1 10

Table 3.2: The samples with different concentrations of myoglobin and azide used for this report.

is inserted into the cavity. To prevent the sample from heating it is impor-tant that the sample cavity has already cooled down and that inserting the sample is done rapidly. To tune the spectrometer, the auto-tune function of the EMX has been used. It might in some cases be necessary to first adjust the parameters manually.

The spectrum of the myglobin/azide sample can only be recorded at low temperatures. For this report, the measurements have been performed at a temperature of 140 K. To check whether and how fast the samples re-act at various temperatures, the temperature is increased to the desired value and after a set time (waiting time) decreased again to 140 K to ob-tain the spectrum. For these samples, the decay is determined manually by comparing the difference in intensity between the maximum value and the minimum value of the signal after different waiting times. This maxi-mum and minimaxi-mum value can for example be seen in Figure 4.1 (chapter 4) around 110 mT and around 125 mT, respectively.

For the samples with TEMPOL and sodium dithionite it is needed to first record temperature scans of the TEMPOL solution without the sodium dithionite, as a reference. The temperature sweep option of the EMX was

3.3 Measurements 17

used for this. With this option automatic spectra for a discrete set of in-creasing temperatures are obtained. It is best to fine-tune the EMX before each new scan, to ensure the parameters are still correct.

In contrast to the myoglobin/azide samples, the TEMPOL samples do not require measurement at a low temperature. For measuring the decay of the TEMPOL signal, the temperature can thus be set to the desired value. After setting the number of scans and the time between each scan, the EMX can record multiple spectra. A Python script has been written to de-termine the decay rate by finding the peak value of each spectrum. This script can be found in Appendix A.

The measurements can be repeated for increasing temperatures until the EPR spectrum is not detectable anymore, indicating that all TEMPOL in the sample has reacted.

Chapter

4

Results and discussion

4.1

Myoglobin and azide

To be able to properly investigate the myoglobin/azide samples, reference scans have been taken of the background and of a sample containing just myoglobin. These are shown in Figure 4.1. In Figure 4.2 is shown what the myoglobin signal looks like after background correction. When myo-globin and azide react, the signal will change from the high-spin state of the iron atom (around 120 mT) to a low-spin state (around 350 mT). As can be seen in Figure 4.1 the background around 120 mT, where the high-spin signal appears, is insignificant in comparison with the signal itself.

Using the subzero mixing method, samples have been prepared with myoglobin and azide in a 50% glycerol solution. The ratio between myo-globin and azide is either around 1 to 3 or 1 to 10. The decay of the signal has been measured for various temperatures. Only the high-spin signal was recorded, without the need for a background correction. Measure-ments have been stopped when the signal to noise ratio became too low for a proper analysis without a baseline correction. The decay is shown in Figure 4.3a and Figure 4.3b. The myoglobin/azide samples can only be measured at low temperatures. For this study they have been measured at a temperature of 140 K. This means the samples are definitely stable at the measurement temperature. To be able to measure the chemical kinet-ics with EPR and a temperature jump, the temperature however first has to be increased to a set-off temperature, from which the samples can be heated using a laser pulse. To ensure the measured decay is only a con-sequence of the increment by the laser pulse, the samples have to remain stable at the set-off temperature. As can be concluded from Figure 4.3 a suitable set-off temperature for the 1:3 ratio is -55◦C and for the 1:10 ratio

20 Results and discussion

Figure 4.1:Reference spectra of a myoglobin sample and its background signal.

Figure 4.2: A myoglobin spectrum for various temperatures, corrected for the background signal. A high-spin signal appears around 120 mT.

this temperature is -60◦C.

The samples have been prepared at a temperature between -50 ◦C and -60 ◦C. This indicates some reaction may have already occurred during the mixing, but based on Figure 4.3 we can conclude that this reaction is probably insignificant. Figure 4.3 however shows a large difference for the various temperatures (between -55◦C and -50◦C for the 1:3 ratio and be-tween -55◦C and -45◦C for the 1:10 ratio). This implies the temperature during mixing has to be accurately stabilized.

4.2 Glycerol and sucrose solutions 21

(a)myoglobin:azide = 1:3 (b)myoglobin:azide = 1:10

Figure 4.3:Decay of the high-spin signal of a myoglobin/azide spectrum for dif-ferent concentrations and temperatures shown on a semi-logarithmic scale.

4.2

Glycerol and sucrose solutions

4.2.1

Viscosity

Based on previous experiments by E.G. Panarelli it is known that the 50% glycerol solution can be mixed at a temperature of around -50◦C. To get an idea of the mixing temperature for the 60% sucrose solution, the viscosities of the 50% glycerol solution and the 60% sucrose solution have been calcu-lated for various subzero temperatures (Figure 4.4).[3][4] Literature on the viscosity of glycerol solutions and sucrose solutions for subzero temper-atures is very limited. Therefore the viscosities have been calculated us-ing formulas that have only been confirmed for temperatures above 0◦C. Moreover, freezing temperatures are not taken into account. Comparing the viscosities of the 50% glycerol solution and the 60% sucrose solution indicates that the 60% sucrose solution can be mixed at a higher temper-ature than the 50% glycerol solution. During the sample preparation it turned out that the 60% sucrose solution freezes around -25◦C. Therefore a slightly higher mixing temperature (between -20◦C and -10◦C) has been chosen for the sucrose solution. This implies the 60% sucrose solution can indeed be used to mix and measure at higher temperatures and thus mea-sure faster reactions. It is however yet to be shown that this will work in practice.

22 Results and discussion

Figure 4.4: Viscosity of a 50% glycerol solution and a 60% sucrose solution at subzero temperatures.[3][4]

4.2.2

Temperature scans TEMPOL

Since the EPR spectrum of TEMPOL changes with viscosity and thus with temperature, it is needed to first obtain spectra of TEMPOL at different temperatures for both a glycerol solution and a sucrose solution.

(a)50% glycerol solution (b)60% sucrose solution

Figure 4.5: EPR spectra of TEMPOL in a 50% glycerol solution and in a 60% sucrose solution for different temperatures.

tem-4.3 TEMPOL and sodium dithionite in a sucrose solution 23

perature of a recorded spectrum if needed. Because experiments have al-ready been performed with a 50% glycerol solution, these spectra can also be used to give an indication of the mixing and measurement temperature for the 60% sucrose solution.

As was mentioned earlier in this paragraph, it is known based on previous experiments that samples containing 50 mM TEMPOL and 1 mM dithion-ite in a 50% glycerol solution can be mixed at -50◦C and that the mixtures remain sufficiently stable at -30 ◦C. Comparison between the spectra of TEMPOL in a 50% glycerol solution (Figure 4.5a) with the spectra in a 60% sucrose solution (Figure 4.5b) indicates that the samples in the sucrose so-lution can be mixed at a temperature of approximately -20◦C. This means the sucrose solution can be used for a measurement at higher temperatures and thus with faster reactions compared to the glycerol solution.

4.3

TEMPOL and sodium dithionite in a sucrose

solution

Samples with TEMPOL and sodium dithionite in a 60% sucrose solution have been prepared using the subzero mixing method.

(a)Time scan of a TEMPOL signal (50 mM sodium dithionite).

(b)Spectra showing a TEMPOL signal at -50◦C, that has vanished after 5 min. at 10◦C (250 mM sodium dithionite).

Figure 4.6: Decay of a TEMPOL signal with two different concentrations of sodium dithionite in a 60% sucrose solution.

Since the current preparation setup does not have an automatic tempera-ture controller, the nitrogen gas flow has to be adjusted manually to obtain

24 Results and discussion

the correct temperature. For the sucrose solution to have a temperature suitable for subzero mixing, the temperature has to be in the range of -20

◦_{C and -10}◦_{C. It is difficult to keep the setup stable within this range.}

Fig-ure 4.6a shows a TEMPOL signal that is stable at temperatFig-ures below -30

◦_{c, but decays at -20} ◦_{C. This indicates it is possible to prepare a sample}

in which both TEMPOL (1mM) and sodium dithionite (50mM) occur but have not yet completely reacted. It however also shows that the reaction between TEMPOL and dithionite for these concentrations in a 60% su-crose solution at -20◦C is already significant on a time scale of 5 minutes. This indicates that a sample that is mixed at a temperature between -20

◦_{C and -10}◦_{C has already (partly) reacted. Figure 4.6b shows a TEMPOL}

spectrum of a sample containing TEMPOL (1mM) and sodium dithionite (250 mM). After 5 minutes at a temperature of 10◦C, the TEMPOL signal has disappeared, indicating the TEMPOL is reduced by sodium dithion-ite. This shows it is possible to prepare a sample containing both TEMPOL (1mM) and sodium dithionite (250mM) in which the components have not yet completely reacted. Measurements of other samples show large differ-ences between the various samples, as some samples do not show any decay. This leads to the conclusion that the 60% sucrose solution is not suitable for subzero mixing with the current sample preparation setup.

4.4

TEMPOL and sodium dithionite in a glycerol

solution

Samples have been prepared in a 50% glycerol solution with 1 mM TEM-POL and various concentrations of sodium dithionite. To allow for a com-parison between the different concentrations of sodium dithionite, the de-cay after one minute at -30◦C has been determined for a number of sam-ples, which is shown in Figure 4.7. The decay after one minute increases with increasing concentrations of dithionite, which is in accordance with expectations. In Figure 4.7 it can be seen that there exist large differences between the various samples, especially for high concentrations of sodium dithionite. An explanation for this is that minor differences between the samples become more significant for samples with a high concentration of sodium dithionite, since these samples have a higher reaction speed. Figure 4.7 however also shows a large difference between samples with a concentration of only 25 mM dithionite. This might be due to improper mixing of the two components in the samples, causing variations in con-centration. One of the samples with 25 mM sodium dithionite seems to

4.4 TEMPOL and sodium dithionite in a glycerol solution 25

Figure 4.7:Decay of the TEMPOL signal (in percentage) in a 50% glycerol solution after 1 minute at -30◦C for samples with different concentrations of dithionite.

have a decay after 1 minute of less than 0 %. This apparent increase of the TEMPOL signal can probably be attributed to small adjustments of the parameters by fine-tuning the EMX before each new scan. If the TEMPOL signal and the decay of the signal are sufficiently large, these adjustments should not give a significant difference. Apparently this is not the case for the sample that shows a decay below 0 %.

For samples with different concentrations of sodium dithionite the de-cay of the TEMPOL signal has been measured at different temperatures. The results are shown in Figure 4.8. The TEMPOL signal is expected to decay exponentially in time, appearing as a linear plot on the semi-logarithmic scale. For the lower temperatures this indeed seems to be the case for the different concentrations. For the higher temperatures it seems less clear that the TEMPOL signal decays exponentially. A possible expla-nation for this is the time the EMX needs for taking a single scan. Since the reaction speed increases with increasing temperature, the decay during a single scan might become significant for higher temperatures, which cre-ates larger errors in the measurement. Another explanation is that the temperature controller of the EMX is less accurate for higher

tempera-26 Results and discussion

tures and needs more time to stabilize, which also causes larger errors in the measurement. Other factors that could create errors occur during the preparation of either the stock solutions or the samples. During the sample preparation both temperature controlling and mixing are done manually. Sample preperation is therefore assumed to account for the largest errors, compared to which errors in the preparation of the stock solutions and er-rors in the measurements are minimal. These erer-rors are hard to estimate and therefore not shown in the results.

For measuring the chemical kinetics using EPR in combination with a tem-perature jump it is necessary that the samples remain stable for several hours. From a comparison between Figure 4.8a and Figure 4.8b can be concluded that the measurement temperature for a sample with a 25mM concentration of sodium dithionite is not higher than the measurement temperature for a sample with a 50 mM concentration of sodium dithion-ite. Decreasing the concentration of sodium dithionite will therefore not allow for a measurement at higher temperatures and is thus not a suitable option for measuring reactions with increased reaction speed.

Furthermore, Figure 4.8c and Figure 4.8d show that also for higher con-centrations of sodium dithionite, it is possible to prepare samples using the subzero mixing technique. The figures show that the samples remain stable at -50◦C on a timescale of minutes. Since the mixing time for obtain-ing a homogeneous solution is approximately 30 to 60 seconds, it is rea-sonable to assume that no significant reaction has already occurred during the mixing. A decay of the samples with 100 mM sodium dithionite or 250 mM sodium dithionite is however already noticable after one minute at -30 ◦C. The samples need to remain stable at the measurement tempera-ture for several hours. For measuring samples with higher concentrations of sodium dithionite, it is therefore needed to decrease the temperature at which the samples are measured. This means using a higher concen-tration of dithionite does not automatically lead to the possibility of mea-suring faster reactions. Further measurements would be needed to check whether these reactions (with a higher concentration of dithionite, but at a lower temperature) are faster than the ones previously investigated.

4.4 TEMPOL and sodium dithionite in a glycerol solution 27

(a) 25 mM sodium dithionite (b)50 mM sodium dithionite

(c)100 mM sodium dithionite (d)250 mM sodium dithionite

Figure 4.8: Time scans of samples of 1 mM TEMPOL and different concentra-tions of sodium dithionite in a 50% glycerol solution. The decay for the different concentrations is plotted on a semi-logarithmic scale.

Chapter

5

Conclusion

Samples containing myoglobin and azide can be prepared using the sub-zero mixing method. The kinetics of a myoglobin/azide reaction can thus be investigated using the subzero mixing technique in combination with temperature cycle EPR. The samples are sufficiently stable on a time scale of at least an hour at temperatures of -55 ◦C or lower depending on the concentration.The samples are prepared at a temperature of less than -50

◦_{C, which indicates that though some reaction could already occur during}

the mixing, the reaction rate is probably insignificant. The measurement temperature for the myoglobin/azide samples that was used for the ex-periments in this report is 140 K. The samples are thus stable at the mea-suring temperature. To allow for a measurement using temperature cycle EPR, the samples need to remain stable at the set-off temperature, from which the temperature is increased using a laser pulse. This means the set-off temperature has to be -55◦C or lower depending on the azide con-centration of the samples.

The decay of a TEMPOL signal due to reduction by dithionite has been measured for various concentrations of sodium dithionite. Measurements have been performed on samples in a 50% glycerol solution, as already has been done, and on samples in a 60% sucrose solutions. These measure-ments show that the 60% sucrose solution does not function as a proper replacement for the 50% glycerol solution. Due to the limited temperature range in which the sucrose solution seems suitable for use in the subzero mixing technique, it is too difficult to get a homogeneous mixture of TEM-POL and dithionite. This results in differences between the various sam-ples. A better temperature controller for the mixing might help to improve the reliability of the samples. With the current set-up, it is not possible to use a 60% sucrose solution for measuring faster reactions between

TEM-30 Conclusion

POL and dithionite.

The measurements of samples with increasing concentrations of sodium dithionite, show that the reaction speed increases with concentration, as was expected. Decreasing the concentration of sodium dithionite does not allow for a measurement at higher temperatures and is therefore not a suitable option for obtaining measurements with a higher time resolution. It is possible to prepare samples with higher concentrations of dithionite in a 50% glycerol solution using the subzero mixing method. The tem-perature at which they remain stable enough for measuring is however lower than for the original samples. This means the measurements per-formed for this report are not sufficient to determine whether increasing the concentration of sodium dithionite would allow for measuring a faster reaction between TEMPOL and dithionite in samples prepared using the subzero mixing technique.

In general can be concluded that it is possible to extend the application of the subzero mixing technique in combination with temperature cycle EPR. Further measurements are however needed to show whether this technique can compete with Rapid Freeze Quench in temporal resolution.

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Appendix

A

Python script

The following Python script was used to read in the TEMPOL spectra and find its peaks.

import numpy as np

import m a t p l o t l i b . pyplot as p l t import p i c k l e

### V a r i a b l e s

n p o i n t s = number #number o f p o i n t s per measurement n measurements = number #number o f measurements

f i l e n a m e = ” n a m e o f f i l e . t x t ” #name o f t h e d a t a f i l e w r i t e t o f i l e = ” n a m e o f f i l e n e w . t x t ” #name o f f i l e t o w r i t e t o timedelay = number # time between measurements

### Make a r r a y t o put data i n

measurements = np . t i l e ( np . nan , ( n p o i n t s , n measurements + 1 ) )

### Read i n microwave f r e q u e n c i e s and w r i t e t o a r r a y f r e q = np . genfromtxt ( filename , s k i p h e a d e r =4 ,

s k i p f o o t e r = ( ( n measurements−1)∗( n p o i n t s )+ ( n measurements−1 ) ∗ 2 ) , u s e c o l s = ( 0 , ) )

34 Python script

### Read i n i n t e n s i t i e s f o r a l l measurements and w r i t e t o a r r a y

f o r i i n range ( n measurements ) : data = np . genfromtxt ( filename ,

s k i p h e a d e r = ( 4 ∗ ( i +1)+ n p o i n t s ∗ ( i ) ) , s k i p f o o t e r = ( ( n measurements−i−1)∗( n p o i n t s )+ ( n measurements−i −1 ) ∗ 2 ) , u s e c o l s = ( 2 , ) ) measurements [ : , i +1]= data ### Write a r r a y t o t e x t f i l e np . s a v e t x t ( w r i t e t o f i l e , measurements , d e l i m i t e r = ’ , ’ ) p r i n t ( measurements ) ### Find peaks

peaks = np . amax ( measurements , a x i s =0) p r i n t ( peaks )

### P l o t decay

i n t e n s i t y p e a k s = np . t i l e ( np . nan , n measurements ) f o r i i n range ( n measurements ) :

i n t e n s i t y p e a k s [ i ] = peaks [ i +1]

time = np . arange ( 0 , n measurements ∗ timedelay , timedelay ) p l t . p l o t ( time , i n t e n s i t y p e a k s )