Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts.
ABSTRACT Four novel antibacterial peptides, Leucrocin I-IV from Siamese crocodile white blood cell extracts were purified by reverse phase high performance liquid chromatography (RP-HPLC). Leucrocins exhibit strong antibacterial activity towards Staphylococcus epidermidis, Salmonella typhi and Vibrio cholerae. The peptides were 7-10 residues in length with different primary structure. The amino acid sequence of Leucrocin I is NGVQPKY with molecular mass around 806.99 Da and Leucrocin II is NAGSLLSGWG with molecular mass around 956.3 Da. Further, the interaction between peptides and bacterial membranes as part of their killing mechanism was studied by fluorescence and electron microscopy. The outer membrane and cytoplasmic membrane was the target of action of Leucrocins as assayed in model membrane by release of β-galactosidase due to the membrane permeabilization. Finally, the hemolytic effect was tested against human red blood cell. Leucrocin I, III and IV showed less toxicity against human red blood cells than Leucrocin II.
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ABSTRACT: Antioxidant peptides were isolated from the leukocyte extract of the Siamese crocodile, Crocodylus siamensis. Crocodile leukocyte was extracted by a combination of methods including freeze-thawing, acetic acid extraction and homogenization. The peptides in the leukocyte extract were purified by anion exchange chromatography and reversed phase-high performance liquid chromatography. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was used to evaluate the antioxidant activity of the elution peaks at each purification step. As a result, there were two purified peptides exhibiting strong antioxidant activity in reducing free radicals on DPPH molecules. The amino acid sequences of these peptides were determined by LC-MS/MS as TDVLGLPAK (912.5 Da) and DPNAALPAGPR (1,148.6 Da), and their IC50 values were 153.4 and 95.7 μM, respectively. The results of this study therefore indicate that leukocyte extract of C. siamensis contains peptides with antioxidant activity which could be used as a novel antioxidant.The Protein Journal 12/2013; · 1.13 Impact Factor
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ABSTRACT: Heteroscorpine-1 (HS-1) was identified as a member of the scorpine family. HS-1 shows insecticidal activities, exhibiting a low median lethal dose (LD50) in mealworm (Tenebrio molitor L.) and inhibitory activities against Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In this study, a recombinant HS-1 (rHS-1) was produced by overexpression in E. coli. A large yield of product was obtained. The structure of purified rHS-1 was confirmed through mass spectrometry. Both anti-crude venom and anti-rHS-1 antibodies specifically recognized rHS-1, suggesting its structural similarity. Reactivated rHS-1 caused roughening and blebbing of bacterial cell surfaces. It showed higher activity than that of pre-refolded protein. Antisera raised against a partially purified and mis- or unfolded peptide can inhibit relevant bioactivity.Biochemical Genetics 07/2014; · 0.94 Impact Factor
Article: Antimicrobial Peptides in Reptiles.[Show abstract] [Hide abstract]
ABSTRACT: Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development.Pharmaceuticals (Basel, Switzerland). 01/2014; 7(6):723-753.