Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The overall goal of this experiment is to follow enzymatic processes in real time by dynamic nuclear polarization enhanced nuclear magnetic resonance. Magnetic resonance in the liquid state enhanced by the dynamic nuclear polarization, DNP, can help answer key questions:how to determine, for instance, the rates of enzymatic transformation of a pair of molecules in cell or in vivo without having to alter this pair of molecules in any way. Installation of our dissolution DNP equipment in our laboratory and the arrival here of Dr.Riccardo Balzan, a PhD in Arnaud Comment Laboratory in Lausanne, is a big step forward for our research.

DNP significantly increases the sensitivity of experiments we can perform on our 500 megahertz NMR spectrometers. This DNP equipment was the first one to be installed in France and I would like to mention that, in rendering this equipment functional, all the members of our NMR team were involved and I would like to mention in particular Gildas Bertho, our research engineer, and Fatiha Kateb, our maitre de conferences. The main advantage of this technique is that it allows direct, real-time quantification of the substrate and its endogenous derivates with a time resolution of seconds.

For the polarizing solution, prepare two milliliters of a 1.12 molar, carbon 13 labeled sodium pyruvate solution doped with 33 millimolar of TEMPO radical in a two-to-one volume of deuterated water to deuterated ethanol for carbon 13 observations. Lower the dynamic nuclear polarization, or DNP, cryostat temperature through the cooldown procedure by clicking the cooldown button on the cryostat software interface. Collect liquid helium in the DNP cryostat through the filling procedure.

Click the filling button on the cryostat software interface. Then place 300 microliters of the optimized polarizing solution in the sample container provided with the DNP polarizer. Freeze the sample container with the sample inside by gently immersing it in a liquid nitrogen bath.

Eliminate the liquid nitrogen that may be present in the sample container after the freezing procedure by extracting the sample container from the liquid nitrogen bath and rotating it upside down. Next, insert the sample into the cryostat by first inserting the sample container in the sample holder. Then place the sample holder in the main cryostat insert before inserting the microwave's wave guide into the sample holder.

Lower the cryostat temperature by starting the one Kelvin cooling procedure. Click the one Kelvin cooling button on the cryostat software interface. Turn the microwave source on and irradiate the sample by clicking the microwaves on button on the cryostat software interface.

Disconnect the coaxial cable of the NMR console detection channel from the spectrometer coil by rotating the connector anti-clockwise by 90 degrees and pulling it. Plug in the coaxial cable of the detection channel to the coaxial connector of the cryostat NMR coil. Then insert the male connector of the NMR console cable in the female connector of the cryostat NMR coil, paying attention to the orientation pin.

Push firmly and rotate the connector clockwise by 90 degrees. Measure the NMR thermal signal in the solid state at the cryostat site using a simple pulse-acquire sequence with uncalibrated, low flip angle pulses repeated at an interval of t equals five minutes. After setting up the measurement, write zg in the command line of the software and press enter on the keyboard.

After initiating the polarization procedure, measure the DNP enhanced NMR signal in the solid state with the same procedure and conditions used for the thermal signal measurement. Disconnect the NMR console detection channel coaxial cable from the cryostat NMR coil by rotating the connector anti-clockwise by 90 degrees and pulling it. Then plug the coaxial cable of the detection channel to the coaxial connector of the spectrometer coil.

Insert the male connector of the NMR console cable in the female connector of the spectrometer coil, paying attention to the orientation pin. Push firmly and rotate the connector clockwise by 90 degrees. Freshly prepare a rabbit muscle LDH solution at one unit per milliliter in reaction buffer with 20 micrograms per milliliter bovine serum albumin.

Next mix 478 microliters of reaction buffer, 2 microliters of LDH solution, and 20 microliters of deuterated water to allow for spectrometer field stabilization, or locking. Then place the 500 microliter solution volume into a 5 millimeter NMR tube. Connect the transfer tube to an automated, custom-made injection device that separates the hyperpolarized solution from the helium gas used for transfer.

The device automatically delivers 500 microliters of hyperpolarized solution into the sample tube. Before dissolution, place the five millimeter NMR tube containing 500 microliters of sample in the isocenter of the 11.74 tesla NMR spectrometer. Insert 5 milliliters of deuterated water in the dissolution insert boiler.

Pressurize and heat the deuterated water by means of the resistive wire until a temperature of approximately 180 to 200 degrees Celsius is reached by clicking the prepare heater button on the cryostat software interface. Click the SS Operations button on the cryostat software interface. Then remove the microwave guide from the dissolution insert.

Slide the dissolution insert down into the sample holder. The dissolution insert has to reach the bottom of the sample holder and must be pushed firmly to make a leak-tight connection with the sample container to avoid deuterated water leaking into the cryostat. Press the hardware trigger to begin the dissolution sequence, a predetermined timed sequence of pneumatic valves operations.

Just after the dissolution, the injection device mixes 500 microliters of hyperpolarlized solution with the sample placed in the NMR spectrometer. After the dissolution, measure the NMR hyperpolarized signal from the sample in the spectrometer by a series of 10 degree, flip angle pulse-acquire sequences spaced by 1.5 seconds to follow the progression of magnetization of the substrate and the product. After setting up the measurement, write zg in the command line of the software and press enter.

Once the magnetization is fully relaxed, measure the NMR thermal signal by a series of 90 degree flip angle pulse-acquire sequences spaced by about three minutes, corresponding to about 3T1. After setting up the measurement, write zg in the command line of the software the press the enter key. Here the time course of spectra obtained from repeated 10 degree flip angle pulses show the sensitivity and time resolution of the technique.

Following peak identification of the pyruvate substrate and lactate product, quantification is performed through signal integration of the metabolites. Enzymatic activity is determined from the product signal time course by fitting with a linear model. To validate the measurement, the same quantity is evaluated by the initial reaction rate, from the ratio of signals of product and substrate.

Once mastered, a complete measurement of enzymatic activity can be performed in about two hours if it is performed properly. After watching this video, you should have an understanding of how to produce a highly polarized substrate for NMR, inject it into a solution containing an enzyme, and follow enzymatic activity using DNP enhanced NMR. When attempting this procedure, it is important to carefully prepare the enzymatic solution with fresh enzyme and cosubstrates, and to avoid any air bubble in the sample tube.

With the development of DNP enhanced magnetic resonance in the liquid state, researchers can now explore substrate uptake and its enzymatic transformation inside various types of cells by measuring the rate of product formation. Don't forget that working with cryogenic equipment and high magnetic fields can be extremely hazardous. Precautions, such as wearing personal protections against cryogens and verifying the absence of magnetic objects should always be taken while performing these measurements.

Unlike classical NMR, dissolution DNP based NMR is limited to an experimental time window of minutes at most, depending on the substrate and the current state of life. This stands in the way of going beyond one D spectroscopy. Dissolution DNP NMR needs development to bring it to the versatility level of classical multi-dimensional NMR.