Spectroscopic characterization of biological molecules is important for obtaining complete structural information. Several spectroscopic techniques that were earlier used only by chemists are currently available to biologists for characterization of proteins and nucleic acids. One such technique that is being widely used is circular dichroism (CD). We use the Jasco J715 spectropolarimeter for quick measurements of protein structural variations.
The Jasco J715 can be used for recording circular dichroism spectra of several types of biomolecules, including nucleic acids, proteins and peptides. Others also use this machine to obtain CD information of small molecules. A key requirement for this instrument is a nitrogen source; the nitrogen helps to maintain an inert and dust-free atmosphere in the sample chamber and in the regions containing the light source and reflective prisms. A nitrogen cylinder can be used but it is advisable to install a dedicated nitrogen generator on the instrument. In the J715 I use, the nitrogen generator automatically supplies >98% nitrogen throughout the duration of the experiment.
The instrument takes up a good amount of space – 3 feet x 8 feet – but can be mounted on a table. The instrument is relatively noiseless. The light source is a Xenon arc lamp, which has a good lifetime and stabilizes within minutes of switching on. The sample holder is positioned on one end of the instrument. Quartz cuvettes can be purchased as per requirement. The instrument can accommodate cuvettes of different path lengths, with blocks being provided for holding cuvettes of different sizes and thickness. We use a 1 mm path length cuvette for measurement of far-UV CD and 10 mm path length cuvette for measurement of near-UV CD spectra.
The instrument can also measure temperature-dependent CD spectra with the aid of a Peltier cooling and heating unit (provided) covering a range of 4 to 96ºC. The Peltier unit is useful for direct measurements of thermal melts of proteins. It is detachable and can be used only when required. Care should be taken while fixing it so as to avoid water spillage or wiring blocking the light path. The Peltier unit is cooled by an external water-bath. The software can easily regulate the temperature. When the Peltier unit is used, the maximum cuvette size that can be held is a 10 mm path length cell.
The software provided with the instrument is easy and convenient to use. Data files can be automatically numbered. Additionally, the software prompts before files are re-written. The scan speed, scan number, slit width and data points can be directly selected using the software. The software also monitors high tension of the sample while recording so that excessive noise is not entered in the data file. Data recorded as mdeg (millidegree) can be readily converted to molar ellipticity using a formula integrated within the software. Smoothing of data can also be readily carried out using one of the several algorithms provided. The software permits overlay of spectra from different files, which can be colored according to choice. The graphs can be saved as ASCII files for re-plotting using other software or can be directly exported to Microsoft® PowerPoint. The software, however, does not allow for estimation of the secondary structure content.