Study On Selenium-rich Amino Acids And A Novel Selenoprotein From Silkworm Pupas

Selenium (Se) plays an important role in cancer-prevention. Silkworm pupas have been used as a Chinese traditional medicine since ancient time. In order to find out effective carcinostatic agents, Se-rich amino acids were extracted from Ziyang silkworm pupas. The Se content of Ziyang pupas was measured to be 215 times higher than that of normal ones, and the majority of Se was stored in proteins. Composition analysis showed that Se-rich amino acids from Ziyang pupas had higher amounts of selenomethionine, methionine, cystine, and tyrosine than normal amino acids from normal pupas,which were rich in amino acids containing alkyl side chains. When cultured with human hepatoma cells SMMC-7721, Se-rich amino acids at concentrations of 0.5, 1.5, and 2.5 μmol/L Se significantly and dose-dependently inhibited cell viability, induced changes in cell morphology and cycle, and caused cell apoptosis. On the contrary, normal amino acids did not show any inhibitory effect on SMMC-7721 cells. Sodium selenite or selenomethionine at the same Se concentrations only slightly inhibited the hepatoma cells. Mechanism study showed that selenium-rich amino acids could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. Those results suggested that Se-rich amino acids were effective carcinostatic agents compared with sodium selenite and selenomethionine. The mechanism for their hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation. To investigate their effective component, the high Se-containing protein was prepared from Ziyang silkworm pupas. Using acetone precipitation, DEAE-Sepharose (Fast Flow) anion exchange chromatography and Sephadex G-75 gel filtration chromatography, Se-containing protein was isolated stepwise from silkworm pupa. The obtained proteins were checked for the purity through SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that selenium-containing proteins from silkworm pupa could be separated through DEAE-Sepharose (Fast Flow) anion exchange chromatography and Sephadex G-75 gel filtration chromatography. SDS-PAGE showed that there are two main proteins present in the product after two-column separation. Characterized by peptide mass fingerprinting using MALDI-TOF-MS and searched by Mascot in NCBI databases, a novel protein with pI value of 5.1 was discovered. Amino acid sequences of its six major peptides digested by trypsin were determined by CapLC-ESI-MS/MS, and the result also showed that it is a new protein. Speciation analysis by HPLC-ICP-MS revealed that selenocystine was the only form of Se in the novel protein, which means that the new protein is a selenoprotein. Its biological function remains to be studied.