Study on Molecular Mechanism of Sea Firefly Bioluminescence : Elucidation of Acitive Site in the Luciferase

1)针对蛋白质跨理性修饰后的结构特征，采用基质辅助激光解吸电离-飞行时间(MALDI)-飞行时间(TOF)联用技术对糖苷异构体低聚糖进行了区分，并确定了其磷酸化位点。根据特定前体离子的源后衰变(PSD)谱，发现糖苷化位异构体相互区分。我们还发现MALDI-TOF/In SOURCE Decay(ISD)质谱学是确定磷肽磷酸化位点的有效方法。2)我们建立了一种利用悬浮在液体石蜡等粘性分散剂中的碘颗粒基质来测定有机小分子的MALDI/TOF质量法。3)合成了一种具有叠氮基团的光反应性腔肠静类似物，其化学和生物发光性质与天然的腔肠静类似。在二乙胺存在下，将合成的腔肠嗪类似物进行光解，主要产物为氮杂环丙烷类化合物。4)用体积排阻层析法从海水中释放的荧光素和荧光素酶溶液中分离纯化了小型海洋介形类甲壳动物Vargula helgendorfi荧光素酶。该酶是一种糖蛋白，其相对分子质量为61.7 kDa，等电点为3.9-5.0，是一种热稳定性蛋白质，在37℃条件下孵育10小时后，其活力仍保持在80%以上。在0.1M的氯化钠、氯化钾和氯化镁存在下，该酶的活力保持不变。

1) In connection with the structure characterization of post-transrationaly modified proteins by means of mass spectrometry, matrix asisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry was applied to differentiation between a pair of glycosidation site isomeric oligosaccharides, and to determine the phosphorylation site of phosphpeptides. The glycosidation site isomers were found to be distinguished from each other on the basis of post source decay (PSD) spectra from spesific precoursor ions. We found also MALDI-TOF/in source decay (ISD) mass spectrometry to be powerful method for determination of the phosphorylation site of phosphopeptides.2) We developed a MALDI/TOF mass method for small organic molecules using an ioorganic particle matrix suspended in a viscous dispersant such as liquid paraffin.3) A photoreactive coelenterazine analogue having an azide group was synthesized, and was found to show similar chemi- and bioluminescence properties to those of natural coelenterazine. Photolysis of the synthesized coelenterazine analogue in the presence of diethylamine gave a azepine derivative derived from the generated nitrene species, as the major product. photoreactive coelenterazine analophotoreactive coelenterazine analo.4) The small marine ostracod crustacean Vargula hilgendorfi luciferase was purified from the gland-ejected luciferin and luciferase solution in seawater by using size-exclusion chromatography. The luferase was found to be a glycoprotein, which has a molecular mass of 61.7 kDa and pI values of 3.9-5.0, and is a sort of heat stable protein, which maintained 80% activity when incubated for 10 hr at 37 ℃. The activities of the enzyme were maintained in the presence of 0.1 M NaCl, KCl, and MgCl_2.