Development of Online UV-Visible Microspectrophotometer for Protein Crystallography at BL38B1 of SPring-8

Research and Utilization Division, Structural Biology Group, Japan Synchrotron Radiation Research Institute* PHOTON Design Co.** EPL Inc.*** SPring-8/JASRI**** SPring-8/JASRI, RIKEN SPring-8 Center*****
○Nobutaka Shimizu* Kouichi Tomita** Masao Ogaki*** Kazuya Hasegawa**** Masaki Yamamoto*****

The high intensity X-ray of synchrotron radiation gives the radiation damage to a protein crystal. It is difficult to evaluate the damage through structural analysis, and is necessary to obtain the complementary information of the damage against the X-ray diffraction experiment. UV-visible spectroscopy is one of good techniques for obtaining the information of amino acids in the protein. Especially, Trp and Tyr have absorption around 280 nm unlike other amino acids. Moreover, since proteins which bind with metal atoms and organic small molecules have absorption in the visible region, the change of specific position in the protein might be clarified. We developed online UV-Vis. microspectrophotometer at BL38B1 of SPring-8 to analyze the state of protein in the crystal to which X-ray was irradiated.
This spectrophotometer employed a difference dispersive double monochrometer mounted by Czerny-Turner type in order to acquire high brightness in UV region and reject stray light (PDPT0320, PHOTON design). Two types of diffraction gratings (53006BK01-150R and 53006BK01-280R, Newport) were installed into the monochrometer. Mercury-Xeon lamp (L2423, Hamamatsu Photonics) and the photomultiplier (R374, Hamamatsu Photonics) are selected as a light source and a detector, respectively. In the preliminary test using solution samples and ND filters, the absorption spectra were able to be measured up to ~ 3.0 OD from 250 to 700 nm. We will introduce the feature of this spectrophotometer and the measurement results of crystals on the poster.