Aspectos básicos tenidos en cuenta durante la confección de la Guía 34

Circular Dichroism (CD) is a form of spectroscopy that uses circularly polarised light to probe the structure of molecules. Solution phase circular dichroism requires analyte molecules that have absorption bands in the wavelength range of interest and exhibit a degree of chirality in order to generate a CD signal 1. In terms of automation and sample requirements, CD spectroscopy is not as developed as other structural methods such as NMR 2 and X-ray crystallography 3, both of which are potent methods of obtaining detailed structural information on the secondary, tertiary and in some cases quaternary level structure of proteins and nucleic acids. Synchrotron radiation circular dichroism (SRCD) 4 allows the use of the deep UV (DUV) region, probing the structure of the analyte molecules using light of far higher energy than that available from most bench-top instruments. The light created at a synchrotron light source covers a far greater range of wavelengths than a bench-top instrument at a continuous high photon flux and produces a far greater amount of

photons This expansion of the examined spectral range helps generate far more information about the molecule, potentially allowing the probing of tertiary structural fold information for protein molecules, which dramatically increases the scope of the technique 5. However, the sample requirements for CD are a barrier to its greater uptake and use, both in academic research and industry.

Uses of CD include the analysis of DNA 6,7 _{and use in the analysis of pharmaceutical}

solutions of proteins. Reduction of sample volume would allow less sample waste and reduced risk of sample contamination in reclaimed sample. An additional benefit of reducing sample volumes would be the ability to analyse expensive or difficult to produce samples such as supra-molecular assemblies8, cyclical DNA9, RNA and synthetic assemblies of amino acids 10–– the association of these molecules with both

themselves and other molecules can be easily observed using CD spectroscopy but sample requirements have put barriers in place to the utilization of CD for analysis of these types of sample.

2.1.1

Cuvette circular dichroism

In the past, circular dichroism data has been collected using flat-faced, circular or rectangular quartz cuvettes as the sample holder, or in certain instances CaF2

cuvettes11. The design of the cuvette has many aspects in its favour. Firstly, the optical qualities of a cuvette are very simple and easy to control. There are only four flat interfaces for the light to pass through (air – solid (quartz) – liquid (sample) – solid (quartz) – air). Optics of this simplicity minimise refraction of the light beam, leading to a well-defined optical path for the light beam. Secondly, the size of the aperture for most cuvettes allows easy sample recovery, taking advantage of the fact that CD is a non-destructive technique. Finally, the standard quartz cuvette can be made to a large variety of specifications, including dramatically increasing or reducing the path-length of the cuvette. This variability of path-length allows CD spectroscopy to be used to collect spectra of samples with protein concentrations ranging from 0.001 mg / ml protein solution right up to 10 mg / ml protein solution. In addition to this, the face of the cuvette can be manufactured to be larger than the light beam from the spectrometer, optimising the amount of light passing through the sample.

However, there are a number of issues with currently used cuvettes for CD which sometimes makes them non-ideal for use by the experimentalist:

(i) Sample size—A typical rectangular cell of path length 1 mm requires (with care) ~150 µl of 0.1 mg/ml protein (i.e. 15 µg) in order to generate a good quality CD

spectrum. This issue with sample size reduces the efficacy of CD for biological use, where often the amount of protein generated will not be sufficient to make such a solution.

(ii) Baseline accumulation—In order to generate a CD spectrum it is necessary to generate a spectrum of not only the analyte molecule, but also of the carrier solvent in the same cuvette, be it buffered, saline or organic. This is so any CD generated by the solvent and cuvette can be compensated for in the final recorded spectrum, where only the analyte molecule’s absorbance is plotted. Currently, the same cuvette must be cleaned and dried between baseline and analyte samples. This is a very time- consuming process which places strict limitations on how many samples can be

analysed in a given time frame, and precludes any option of high throughput data collection since perfectly matched CD cuvettes are hard to source.

(iii) Sample contamination— whilst correct experimental procedure can reduce this possibility, bio-macromolecular samples can adhere to quartz, enhancing the risk that a previous sample remains in the cuvette even after cleaning.

These issues, but predominantly the large sample requirements, pose serious limitations on the application of CD spectroscopy in the field of biological research.

2.1.2

Capillary circular dichroism

We therefore proposed quartz capillaries as an alternative method of holding the sample in the path of the light beam. We have found quartz capillaries to be well- suited to such an application, exhibiting remarkably little intrinsic birefringence 11. The capillaries are formed from high-purity fused silica, which is heated until molten and then extruded. The resultant capillary has a very good uniformity (deviation of the outer diameter is a maximum of 50 µm) as well as excellent optical qualities, being generally free from imperfections. Capillary CD (caCD) offers the potential to use smaller sample volumes whilst obtaining good quality data. If we replace a 1 mm rectangular cuvette with a 1 mm internal diameter capillary, the required sample volume has the potential to be reduced. However, to be of any use, this requires the light beam to be tightly focused so as to pass directly through the sample. Assuming this can be achieved, the volume required for a 1 mm path length CD spectrum can be reduced from 200 µl to 3 µl of sample.

The work outlined in this chapter describes how a Jasco J-815 spectrometer was adapted to address these issues through the incorporation of a caCD base-plate, and how the resulting set-up can be used to collect high quality CD data.