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

1)结合质谱技术对蛋白质的结构表征，采用基质辅助激光解吸电离（MALDI）-飞行时间（TOF）质谱技术对一对糖苷化位点异构的寡糖进行了鉴别，并确定了磷酸化肽的磷酸化位点。糖苷化位点异构体被发现是相互区分的基础上的源后衰变（PSD）光谱从specific precoursor离子。我们还发现MALDI-TOF/源内衰变（ISD）质谱法是用于确定磷酸肽的磷酸化位点的有力方法。2）我们开发了用于小有机分子的MALDI/TOF质谱法，其使用悬浮在粘性分散剂如液体石蜡中的无机颗粒基质。3）合成了具有叠氮基的光反应性腔肠素类似物，并发现其显示出与天然腔肠素相似的化学和生物发光性质。光解的合成腔肠素类似物在二乙胺的存在下，得到了氮杂卓衍生物衍生物所产生的氮烯物种，作为主要产品。光反应性腔肠荧光素类似物光反应性腔肠荧光素类似物4）通过使用尺寸排阻色谱法从海水中的腺体喷射的荧光素和荧光素酶溶液中纯化小型海洋介形类甲壳动物Vargulahilgendorfi荧光素酶。该酶是一种糖蛋白，分子量为61.7kDa，等电点为3.9-5.0，是一种热稳定蛋白，37 ℃孵育10小时仍有80%的酶活性。在0.1 M NaCl、KCl和MgCl_2存在下酶的活性得以维持。

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.