This article demonstrates a new experimental method to determine low temperature absorbance spectra of samples using Oxford Instruments’ top-loading, exchange gas Cryofree® cryostat system OptistatDry (Figure 1) coupled with a traditional UV-Vis spectrophotometer.
Figure 1. OptistatDry top-loading Cryofree
The experimental set-up has been employed for the photolytic analysis of vitamin B12, a tetrapyrrole-based diamagnetic, six-coordinate CoIII complex (Figure 2), which is a key biomolecule for life. It is also known to be the largest of all vitamins.
Figure 2. Molecular structure of vitamin B12.
Methyl-cobalamin (R= CH3) and 5’-deoxy-adenosyl-cobalamin (R= 5’-deoxyadenosyl) are biologically active forms of vitamin B12. They act as crucial cofactors to various enzymes across all spheres of life.
The breakage of the cobalt-carbon bond, following binding of a substrate to the enzyme, is essential to facilitate the reactivity of the vitamin B12-derivatives. As the cobalt-carbon bond is sensitive to light and subject to photolysis upon illumination, the same process can be activated by light.
Optical spectroscopy can be used to track the photolysis reaction, and examination of this process at low temperatures can help to detect new photo-intermediates that are important in biology.
In this analysis, the absorbance spectra of samples in the top-loading OptistatDry cryostat was measured using a Cary 60 UV-Vis spectrophotometer fitted with a fibre optic coupler accessory (Agilent Technologies). The spectrophotometer was fixed with two optical fibres to join the Xenon light source via the cryostat sample chamber and back to the detector (Figure 3).
In order to increase the signal-to-noise-ratio and optimize light-throughput, the position of the optical fibres can be adjusted using a precision translation mount on the output and input sides of the cryostat.
Figure 3. OptistatDry cryostat coupled to a Cary 60 UV-Vis spectrophometer (Agilent Technologies) by optical fibers.
A transparent acrylic cuvette comprising of 1 ml of methyl-cobalamin solution in 1,2-propanediol was loaded into the sample holder and shifted to the OptistatDry cryostat, which is equipped with four quartz windows for optical measurements.
At 7 K and room temperature, absorbance spectra were determined before and after illumination for one minute. An LED at 530 nm (Thorlabs) provided this illumination through a third window.
Figure 4 shows the UV-Vis absorption spectra of methyl-cobalamin acquired at 7 K and room temperature. Samples that were cooled to 7 K display sharpening of the absorbance features.
The spectral features display an increased and decreased absorbance at ~ 475 nm and ~ 530 nm, (Figure 5) following photo-irradiation at room temperature. This suggests the formation of a cob(II)alamin radical and confirms that photolysis of the Co-C bond has taken place.
This process can be kinetically followed by tracking the decreased absorbance at 530 nm over time (Figure 5), but spectral changes were not seen upon illumination at 7 K (Figures 5 and 6), suggesting that photolysis cannot take place at these temperatures.
Figure 4. UV-Vis absorption spectrum of methylcobalamin in 1,2-propanediol at 7K and at room temperature prior to illumination.
Figure 5. UV-Vis absorption spectrum of methylcobalamin in 1,2-propanediol at 7K and at room temperature after illumination with an LED at 532nm.
Figure 6. Absorbance change at 530nm upon illumination of methyl-cobalamin in 1,2-propanediol with an LED at 532nm at 7K and at room temperature.
At room temperature and 7 K, absorption spectra of vitamin B12 were effectively recorded using the top-loading OptistatDry cryostat. A third optical window allowed direct illumination, enabling the measurement of light-activated reactions. These measurements are not affected by the compressor, as shown by the stability and signal-to-noise over long time. This insight provides a new avenue to make additional low temperature photolysis measurements of vitamin B12 at a cryogenic temperature range.
This information has been sourced, reviewed and adapted from materials provided by Oxford Instruments Nanoscience.
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