Purification And Isolation Of Buthus Martensi Karsch And Its Bioactivity Upon Analgesia Based On Vanilloid Receptor 1

Part I: The purification and isolation of Buthus martensi Karsch (BmK) ncurotoxin 2-2-5-5-1 and 2-2-5-6-1BmK neurotoxins 2-2-5-5-1 and 2-2-5-6-1 were purified from scorpion crude venom by classical chromatographic method. Sephadex G-25, Sephadex G-50 size exclusion column and CM Sephadex C-25 ion-exchange column were used to primarily purify the crude venom and then RP-HPLC was used for further purifying the venom. Four chromatographic peak were obtained with Sephadex G-25 column and 3 with Sephadex G-50 from the previously fraction 2. Peak 2-2 from the previously peak 2 were applied for CM Sephadex C-25 column. Five fractions were observed and peak 2-2-5 was applied to further purification with RP-HPLC. Within the 9 fractions from the former peak 2-2-5, fraction 2-2-5-5 and 2-2-5-6 displayed relative purification by HPLC chromatography. RP-HPLC procedure was employed to further purify these peaks. Eventually we secured purified fractions 2-2-5-5-1 and 2-2-5-6-1.Part II : The identification of peptides 2-2-5-5-1 and 2-2-5-6-1Study upon primary characterization and identification of these peptides referred to four parts as follows: (1) The result of ESI-MS showed that both the fractions 2-2-5-5 and 2-2-5-6 contain two components. The MW of the two components of 2-2-5-5 are 7214.36(i.e. 2-2-5-5-1) and 7253.11 respectively and the ratio of the relative abundance is 100:20.77. The MW of the two components of 2-2-5-6 are 7036.85(i.e. 2-2-5-6-1) and 3180.69 respectively and the ratio of the relative abundance is 100:30.03. (2) Both of 2-2-5-5-1 and 2-2-5-6-1 reveal end absorption by UV scan. The absorbency is the highest at 206nm or so. (3) Acid amino analysis was used to determinate the relative ratio of the acid amino residues: Asp: Thr: Ser: Glu: Gly: Ala: Val: Met: lie: Leu: Tyr: Phe: Lys: His: Arg 4: 1: 8: 4: 10: 4: 4: 2: 2: 2: 2: 2:2:1: 2. The sum of the number of the ratio is in agreement with the length of the peptide. The peptides of BmK that have a closely ratio to 2-2-5-5-1 were not found. (4) No signal was detected by Edman degradation. It is probably that these two peptides are N-terminal blocked.Part HI: The study upon analgesic activity of BmK purified fractions based on vanilloid receptor 1(VR1)The analgesic activity screen test based on VR1 showed that 2-2-5 has the positive activity to VR1, which indicated 2-2-5 probably has exciting activity on plasma calcium via VR1. Whole-cell patch clamp recording showed the fractions 2-2-5-5 and 2-2-5-6 have obviously effect on potassium and sodium channel respectively. All of the above demonstrated the potential of the peptides for analgesia and for tool drug upon ion channels.