Studies On The Variation Of Venom Roteome And Enzymatic Activities Between Adult And Neonate Venomous Snakes

Snake venom is comprised of complex proteins and peptides. because of the variations of prey and digest in evolution, snake venom always possesses significant intraspecific variation in composition. In this study, I determined the protein profiles and enzymatic activities of newborn and adult snake venoms from short-tailed pit-viper (Gloydius brevicaudus), five-paced pit-viper (Deinagkistrodon acutus) and Chinese cobra (Naja atra), respecitively. Following are the main contents and conclusions of this study:1. Enchriment of low-aboundance venom proteinsPart of low-abundance proteins during two-dimensional eletrophoresis might be ignored, the technology of low-abundance protein enchriment would improve this defect. In this study, the venom from adult G. brevicaudus was treated with buffers of different pH:3.8,7.2and9.0. Compared with original venom sample, the sample treated with pH7.2buffer showed four new bands with the molecular weight of120kDa,70kDa,26kDa and23kDa. Additionally, two more intensely stained bands with molecular weight of40kDa and18.4kDa were obversed in pH3.8and pH9.0buffer treated samples.2. Protein profiles and enzymatic activities of newborn and adult snake venoms from three speciesSDS-PAGE and2-DE analysis revealed that the venom from adult and newborn showed significant differents. There are more protein bands in venom from N. atra than other two venoms. Protein bands on the range>116.0kDa were preferentially expressed in adult venom from N. atra. However, more intensely-stained bands were observed in the range of45kDa-66.6kDa, in newborn venom. Therefore, the protein band in～20kDa was only been found in newborn venom. But66.2kDa didn't present in newborn venom which exsist in the adult venom.～40kDa,18.4kDa,16kDa only be found in the adult venom from D.acutus. But protein bands of23kDa and24kDa were newborn-specific. Newbon venom from G.brevicautus showed more protein bands in the range of45kDa-66.2kDa. Moreover, the bands of45kDa and25kDa in newborn venom were more intensely than adult. However, the band of～22kDa was more significant in adult venom. The analysis of Two-dimentional electrophoresis enlarged the csope of the one-dimentional analysis. One hundred and nity-nine spots were present in the newborn venom from G. Brevicaudus, one hundred and fifty-five spots were found in adult, so that46(29%) were present in both venoms. There are22were been found in both venoms from D. acutus. One hundred and tweven spots were present in the adult, one huandred and fourty-six in the newborn. One hundred and four spots were found in the adult venom from N. atra, eigty-five in the newborn, so that35(33%) were been found in both venom.Variations in venom composition and enzymatic activities had been found in newborn and adult G. brevicaudus, D. acutus and N. atra. The5'-nucleotidase activities were similar in venoms from newborn and adult G. brevicaudus, however, were showed remarkable differences in N. atra and D. acutus venoms, respectively. There were significant differences in protease and alkaline phosphatase activities in newborn and adult venoms of all species:protease activities were higher in adult venoms from these species, and alkaline phosphatase activities were lower in venoms from adult N. atra and D. acutus, but higher in adult G. brevicaudus. The L-Amino acid oxidase was showed similar activities in adult and newborn N. atra and G. brevicaudus venoms, and the activity only occurred in adult D. acutus. Acetylcholine esterase had only been found in N. atra venom. Newborn venom from D. acutus showed higher gelationlytic activity from zymography, and similar in newborn and adult G. brevicaudus venoms, but there was no significant gelationlytic activity in N. atra venom.3. Identification and relative quantitation of variable components in newborn and adult G. brevicaudus venoms by LC/MS/MS and iTRAQIn this study,2-DE, LC/MS/MS and iTRAQ (isobaric tags for relative and absolute quantitation, iTRAQ) technologies were applied for identification and relatively quantitation of variable components in newborn and adult G. brevicaudus venoms.2-DE analysis using linear IPG strips ranging from pH4-7. As the range narrows there is an increasing resolution of the proteins with that range the result of discrimination of additional spots that were not visualized in broader pⅠ range gels of new born and sdult venoms from G. brevicautus. In fact, the isoelectro focusing on narrower range IPG strips enabled the visualization of subtle differences in the venom proteomes. Particular of the sopts of p14-5.5and molecular mass of around66.2-116.0kDa. There are more spots present in the newborn venom, and these proteins are mainly P-Ⅲ class melloproteinases. So that P-Ⅲ class melloproteinases are more abundant in the newborn venom. On the other hand, the adult venom showed a remarkably distinct profiles of molecular mass of25kDa and pⅠ of6-7. These spots were much more abundant in the adult venom and were identified as P-Ⅰ class melloproteinases. Metalloproteinases were found in both of the G. brevicaudus venoms, and were much more aboundant in newborn venom than adult's. In contrast, serine proteinases were found more expressed in adult G. brevicaudus venom. Cysteine-rich secretory proteins were more abundant in newborn venom, while the amount of L-amino acid axidases and phospholipases A2were higher in adult venom.