title: Development of an online UV-visible microspectrophotometer for a macromolecular crystallography beamline.
abstract: Measurement of the UV-visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV-visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury-xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography.
authors.affiliation: Structural Biology Group, SPring-8/JASRI, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Shimizu
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Shimizu
authors.forename: Seiki
authors.initials: S
authors.lastname: Baba
authors.forename: Kazuya
authors.initials: K
authors.lastname: Hasegawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Kumasaka
keywords: SPring-8; UV–visible spectroscopy; microspectroscopy; protein crystallography; radiation damage