Sample Volume Reduction Using the Schwarzschild Objective for a Circular Dichroism Spectrophotometer and an Application to the Structural Analysis of Lysine-36 Trimethylated Histone H3 Protein

Abstract

With the increasing interest in scarce proteins, reducing the sample volume for circular dichroism (CD) spectroscopy has become desirable. Demagnification of the incident beam size is required to reduce the sample volume for CD spectroscopy detecting transmitted light passed through the sample. In this study, the beam size was demagnified using a focal mirror, and small-capacity sample cells were developed in an attempt to reduce the sample volume. The original beam size was 6 × 6 mm2; we successfully converged it to a size of 25 × 25 μm2 using the Schwarzschild objective (SO). The new sample cell and SO allowed the required sample volume to be reduced to 1/10 (15 → 1.5 μL), when using a 15 μm path length cell. By adopting a smaller sample cell, further sample reduction could be achieved. By using the SO system, the secondary structural contents of the lysine-36 trimethylated histone H3 protein were analyzed. The trimethylation induced the increment of helix structures and decrement of unordered structures. These structural alterations may play a role in regulating cellular function(s), such as DNA damage repair processes.