Saito H
Department of Insect Genetics and Breeding, National Institute of Sericultural and Entomological Science, Ibaraki, Japan. saito
Biochim Biophys Acta. 1998 Mar 12;1380(1):141-50. doi: 10.1016/s0304-4165(97)00149-9.
Two different biliverdin-binding proteins, designated BBP-I and BBP-II, were purified from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. These proteins were readily isolated from the hemolymph of fifth instar larvae using two chromatographic steps, hydrophobic interaction chromatography and ion exchange chromatography. Both BBPs were easily separated by Q-Sepharose HP column chromatography. BBP-I has an apparent molecular weight of 24 kDa, as determined by gel-filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native BBP-II had a molecular weight of 48 kDa estimated by gel-filtration. SDS-PAGE revealed a single band with a molecular weight of 26 kDa. Moreover, the molecular weights of BBP-I and BBP-II were determined to be 20,468 and 22,708 by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight/mass spectrometry), respectively. On this basis, BBP-I and BBP-II molecules are assumed to be a monomer and a dimer, respectively. The blue color of BBPs collected from the hemolymph is attributed to the presence of biliverdin IX gamma, which is non-covalently and stoichiometrically bound to the apoprotein, based on absorbance maxima at 359 and 695 nm in methanol:HCl (95:5, v/v). One molecule of BBP-I contains one molecule of biliverdin IX gamma, whereas BBP-II contains two molecules of biliverdin IX gamma. The amino acid compositions of BBP-I and BBP-II are different, although the N-terminal sequences of both BBPs have a 48% identity. These BBPs were found in the hemolymph of fourth and fifth instar larvae. The newly molted fifth instar larvae had the highest concentration of BBP-I in the hemolymph. This gradually decreased during larval development. In contrast to BBP-I, the level of BBP-II was low, and increased slightly at the same developmental stage in S. cynthia ricini larvae.
从蓖麻蚕（Samia cynthia ricini）幼虫的血淋巴中纯化出了两种不同的胆红素结合蛋白，分别命名为BBP-I和BBP-II。使用疏水相互作用色谱和离子交换色谱这两个色谱步骤，很容易从五龄幼虫的血淋巴中分离出这些蛋白。两种BBP通过Q-Sepharose HP柱色谱很容易分离。通过凝胶过滤和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，BBP-I的表观分子量为24 kDa。通过凝胶过滤估计天然BBP-II的分子量为48 kDa。SDS-PAGE显示出一条分子量为26 kDa的单带。此外，通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）测定，BBP-I和BBP-II的分子量分别为20,468和22,708。在此基础上，推测BBP-I和BBP-II分子分别为单体和二聚体。从血淋巴中收集的BBP呈现蓝色，这归因于胆红素IXγ的存在，基于在甲醇：盐酸（95:5，v/v）中359和695 nm处的最大吸光度，胆红素IXγ与脱辅基蛋白非共价且化学计量结合。一个BBP-I分子包含一个胆红素IXγ分子，而BBP-II包含两个胆红素IXγ分子。尽管两种BBP的N端序列有48%的同一性，但BBP-I和BBP-II的氨基酸组成不同。在四龄和五龄幼虫的血淋巴中发现了这些BBP。刚蜕皮的五龄幼虫血淋巴中BBP-I的浓度最高。在幼虫发育过程中，其浓度逐渐降低。与BBP-I相反，BBP-II的水平较低，在蓖麻蚕幼虫的相同发育阶段略有增加。
