Purification, Structural Characterization And Biological Activity Of Peptides From The Skin Of Rana Nigromaculata

Amphibians is the most primitive terrestrial vertebrates adapt to both terrestrial and aquatic life. Amphibians skin is uncovered and without hair, feathers and shells. Amphibians skin play an important role in the complex and diverse environment to maintain its own living. Amphibians skin contains biological vivid substances with various comploicated biofunctions, and these substances play an important role in resisting the invasion of harmful environmental factors. Meanwhile, the skin of different Amphibians species consists of different bioactive substances. Amphibian's skin is rich in natural resources and used in new bioactive peptides'discovery, refiner and function research. Rana nigromaculata of China is one of animal resource, which has medical value in northeast regionof china. Researches have shown that Rana nigromaculata skin contains various active peptides, such as bradykinin-like peptide, bombesin peptide, growth factor, morphine and moisturizing factors.In this paper, Rana nigromaculata growth in Jinzhou Dengshahe of Dalian of Liaoning was choosed as the experimental material. The isolation, purification and structural characterization of bioactive peptides from the skin of Rana nigromaculata were carried out by gel chromatography and RP-HPLC. The optimized conditions of purification were screened, and seven of skin bioactive peptides were obtained. MS and Q-TOF MS/MS were used to determinate their molecular weight and amino acid sequences. Rana nigromaculata skin secretion was obtained by electrical stimulation. The secretion was purified using VydacTP510 RP-HPLC into 6 single peptides.The results of analysis of amino acid sequences showed that three of these peptides were bradykinin or bradykinin-related peptides, and the other three were antimicrobial peptides. Their molecular weights were from 1060.2 to 3285.96 Dalton, and composed of 9 to 32 amino acids. Their primary structure was RPPGFSPFR,,RPPGFSDSSPLAPGT,LKNVGKEVGFDVENELRLAGHARPLGK,KSPGV GALAGKKPHGLLLSGLKNVGGAEAGFD,LGLGKVLGVGQRALAHSPPK and LVPLV SYHLPVL. After BLAST searched in NCBI Protein Databases, we found that peptides RPPGFSPFR was typical Bradykinin composed of 9 amino acids and RPPGFSDSSPLAPGT had the motif of Bradykinin (RPPGFS-) and belonged to bradykinin-related peptides,named it Ranakinin-RN1.Although the peptide LVPLVSYHLPVL does not contain the conserved domain of bradykinin, we still named it as Ranakinin-RN2 due to its higher homology bradykinin activity. Meanwhile, the rest of peptides shared a lower homoloy with some known amphibian skin peptides. Therefore, they are the new peptides, named them as nigromin-1,nigromin-2, nigromin-3, respectively.The bradykinin activity of bradykinin and bradykinin-related peptides was tested by assaying the contractile activity on isolated guinea pig ileum. The results showed Bradykinin-RN and Ranakinin-RN1 contracted guinea pig ileum in a positive linear relationship, bradykinin activity of Bradykinin-RN was more potent than Ranakinin-RN1. bradykinin activity of Ranakinin-RN2 was inhibited after reaching its maximum, showing that the peptide might be the bradykinin antagonist peptide.