CRYOFREE

Application note

Low temperature photolysis studies of vitamin B12 using atop-loading OpstistatDry cryostat
Lara Grangel, Samantha J. O. Hardman and Derren J. Heyes
Manchester Institute of Biotechnology, University of Manchester, UK

Introduction

OptistatDry TLEX model
for sample-in-exchange-gas
applications
This application note discusses

This application note reports a new experimental approach to measure
low temperature absorbance spectra using a top-loading sample inexchange gas Cryofree cryostat system, OptistatDry from Oxford
Instruments (Figure 1) and a conventional UV-Vis spectrophotometer. The
set-up has been used for the photolytic studies of vitamin B12, which is
the largest of all vitamins and is a tetrapyrrole-based diamagnetic, sixcoordinate CoIII complex (Figure 2) that is an essential biomolecule for life.

the customer demonstration ofexperiments in our new OptistatDry
TLEX model with top-loading, samplein-exchange-gas, experimental space.
As shown here, the ability to havethe sample-in-exchange-gas rather
than vacuum offers a wide range ofadditional experimental possibilities,
including those on biological samples.

Figure 1. OptistatDry top-loading Cryofree

BackgroundFigure 2. Molecular structure of vitamin B12.

Biologically active forms of vitamin B12 are 5’-deoxy-adenosyl-cobalamin (R= 5’-deoxyadenosyl) and methyl-cobalamin (R= CH3), which act as essential cofactors tonumerous enzymes throughout all kingdoms of life. The key to the reactivity of thesevitamin B12-derivatives is the breakage of the cobalt-carbon bond upon binding ofa substrate to the enzyme. This same process can also be triggered by light as thecobalt-carbon bond is light-sensitive and undergoes photolysis upon illumination
Reference [1]. The photolysis reaction can be monitored by optical spectroscopy and bystudying this process at low temperatures it should be possible to detect novel photointermediates that are important in biology.

CRYOFREE C
Application note

Experimental set-up
A Cary 60 UV-Vis spectrophotometer, equipped with a fibre optic coupler accessory (Agilent Technolgies), was used
for measuring absorbance spectra of samples in the OptistatDry cryostat. Two optical fibres were attached to thespectrophotometer for coupling the Xenon light source through the sample chamber of the cryostat and back to
the detector (Figure 3). The position of the optical fibres could be fine-tuned using a precision translation mount on
the input and output sides of the cryostat to optimise light-throughput and increase the signal-to-noise-ratio.
A transparent acrylic cuvette containing a 1 ml solution of methyl-cobalamin in 1,2-propanediol was placed in
the sample holder and transferred to the top-loading OptistatDry cryostat, which contains four quartz windows
for optical measurements. Absorbance spectra were measured at room temperature and 7 K, before and afterillumination for 1 min, which was provided by an LED at 530 nm (Thorlabs) through a third window.

Figure 3. OptistatDry cryostat coupled to a Cary 60 UV-Vis spectrophometer (Agilent Technologies) by optical fibres.

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Application note

Experimental results
The UV-Vis absorption spectra of methyl-cobalamin obtained at room temperature and 7 K are shown in Figure 4.
Samples that were cooled to 7 K show a sharpening of the absorbance features (Figure 4).
Upon photo-irradiation at room temperature thespectral features show a decrease in absorbance at~530 nm and an increase at ~475 nm (Figure 5). This
is indicative of the formation of a cob(II)alamin radical[2], confirming that photolysis of the Co-C bond has
occurred.

Figure 4. UV-Vis absorption spectrum of methylcobalamin in 1,2-propanediol at 7 K and at room
temperature prior to illumination.

This process can be followed kinetically by following
the decrease in absorbance at 530 nm over time
(Figure 5). However, no spectral changes were observed
upon illumination at 7 K (Figures 5 and 6), which indicatethat photolysis cannot occur at these temperatures.

Figure 6. Absorbance change at 530 nm upon
illumination of methyl-cobalamin in 1,2-propanediol withan LED at 532 nm at 7 K and at room temperature.

Figure 5. UV-Vis absorption spectrum of methylcobalamin in 1,2-propanediol at 7 K and at room
temperature after illumination with an LED at 532 nm.

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Application note

Conclusion and outlook
Absorption spectra of vitamin B12 were successfully recorded at 7 K androom temperature using the OptistatDry cryostat. Direct illumination waspossible via a 3rd optical window, allowing the measurement of light-activatedreactions. The signal-to-noise and stability over long time periods show thatthe compressor has no adverse effect on these measurements. This paves theway for further low temperature photolysis measurements of vitamin B12 at a
range of cryogenic temperatures.
References[1] A.R. Jones et al., Relating localized protein motions to the reaction coordinate in coenzyme
B12-dependent enzymes. FEBS J., 2013, 280, 2997-3008.
[2] A.G. Cole et al., Time-resolved spectroscopic studies of B12 coenzymes: a comparison of
the primary photolysis mechanism in methyl-, ethyl-, n-propyl-, and 5’-deoxyadenosylcobalamin.
Journal of the American Chemical Society. 2002, 124.3, 443-441.

About the OptistatDry Cryofree® cryostat
The OptistatDry provides a temperature controlled sample measurement
environment within a Cryofree cryostat. It offers a range of top and bottom
loading compact cryostats with optical access cooled by a closed cycle
refrigerator. The system is capable of cooling samples to helium temperatures
without the need for liquid cryogens. This provides significant benefits in termsof ease of use and running costs,. The system enables optical measurements
to be carried out on your samples, as shown in this application note.

OptistatDry
Cryofree cryostat

About the Manchester Institute of Biotechnology,
University of Manchester, UK
The Manchester Interdisciplinary Biocentre was completed in 2006 andrepresented the first university-based, purpose-built interdisciplinary research
institute of its kind in the UK.
Focusing on advanced quantitative approaches to specific biotechnology
challenges at the interface between medicine and biology and the physicalsciences, engineering, mathematics and computation the MIB enjoys aunique pluralistic and open research culture that is supported by world-classinfrastructure. The establishment of multi-skilled interdisciplinary teams withcritical mass generates unique capabilities that cannot be realised throughvirtual associations between PIs or research units to develop regional, nationaland international partnerships in biotechnology research.

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July 2016, Issue 3