Circular dichroism under high pressure

Circular dichroism (CD) cells, equipped with quartz window, are used for measurements of CD in the ultraviolet (UV) region under high pressure. We describe here an improvement in these cells, which are typically distorted by high pressure, thus diminishing the CD signal. A slit with a small 3 mm diameter hole was placed in the light source-side in contact with the window, thus adjusting the center of the light path. This method gave reasonable CD spectra of camphorsulfonic acid, poly-glutamic acid, and RNase A at 150 MPa or lower. For measurements at higher pressure, synthetic diamond was used as windows of the CD cell and reliable CD spectra were obtained in the near-UV and visible light region at pressure of 400 MPa or lower, since the diamond strongly absorbed UV light at 230 nm and shorter wavelengths. By monitoring the molar ellipticity at 235 nm, it was possible to estimate pressure-induced changes in the secondary structure of poly-amino acids, myoglobin, and RNase A. The CD spectra of carboxypeptidase Y, lysozyme, and RNase A were also measured at 245–320 nm under high pressure to estimate structural changes which occur at these conditions. CD spectra of RNase A at 0.1–400 MPa were changed by pressurization at 100 to 250 MPa, showing a reversible two-state transition. However, this negligible discrepancy may be corrected when diamond windows are used.