Study On Preparation, Purification And Opioid Activity Detection Methods Of Opioid Peptide

Opioid peptides are a kind of neuronimmune peptides with hormone and neurotransmitter functions, which confer opioid peptides an important mediator to regulate the central nervous system and the peripheral organs. They can be divided into endogenous opioid peptides and exogenous opioid peptides. Quite a few opioid peptides are from casein. Hidden or inactive in the amino acid sequence of casein, they can be released or activated via specific, enzyme-mediated proteolysis. So in vitro, certain quantity of opioid peptides can be obtained by adjusting appropriate pH, temperature and the time of hydrolysis, using congruent enzyme or combination.In this paper, casein was hydrolyzed via different enzymes and the optimized conditions of producing maximal opioid activities were established. After sedimentation of ethanol and gel filtration chromatography of casein hydrolysate, a sort of opioid peptide was isolated. By analysis of its amino acid composition and compared with the sequence of casein, it was found to be a novel opioid peptide. Then, the spatial structure was speculated. Meanwhile, opioid potencies were measured in GPI (guinea pig ileum) bioassay system and antifatigue effects were identified by the weight-loaded swimming test. The results of the study showed that opioid activities of peptic casein hydrolysate were higher than tryptic and papain casein hydrolysate. The optimum enzyme hydrolysis conditions were: substrate 3% (w/v), pepsin: substrate ratio [E/S] 30000u/g, hydrolysis for 150 minutes at 42℃, pH=2.0.Then, 80% ethanol was used to deposit other miscellaneous proteins, with substances of opioid potency preserved in the supernatant. The third peak of column chromatography on Sephadex G-50 with opioid potency was separated by Sephadex G-15 again. The forth peak with opioid activity was collected. And it was proved to be a pure peptide by Tricine-SDS-PAGE and the high performance capillary electrophoresis. Via analysis of amino acid composition and goodness of fit test, it was corresponding to 144-158 sequence of Alpha-S1-casein: YFYPELFRQFYQLDA, with a molecular weight 2252.48. After query in the international authoritative protein and bioactive peptide database, it can be found that it is a novel opioid peptide. The properties and spatial structures of the peptide were speculated by protein sequence analysis software ANTHEPROT 5.0 and The Swiss-Pdb Viewer. The results indicated that its pI was 4.035.There were 40%helixes, 27%coils, 20%turns, 3% sheets in its second structure, with a sheet in the forth amino residue PRO and four charge areas in the spatial structure. Finally, from GPI, the constriction of guinea pig ileum in vitro was inhabited by the peptide and its IC50 value was 3.177mg/ml.Moreover, it also had obvious antifatigue effects by prolonging the swimming time of mice.