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.
Similar content being viewed by others
Abbreviations
- ACN:
-
Acetonitrile
- DPPH:
-
1,1-Diphenyl-2-picrylhydrazyl
- hRBCs:
-
Human red blood cells
- IC50 :
-
Half maximal inhibitory concentration
- TFA:
-
Trifluoroacetic acid
References
Machlin LJ, Bendich A (1987) Free radical tissue damage: protective role of antioxidant nutrients. FASEB J 1:441–4452
Becker EM, Nissen LR, Skibsted LH (2004) Antioxidant evaluation protocols: food quality or health effects. Eur Food Res Technol 219:561–571
Middleton E Jr, Kandaswami C, Theoharides TC (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 52:673–751
Sampath Kumar NS, Nazeer RA, Jaiganesh R (2011) Purification and biochemical characterization of antioxidant peptide from horse mackerel (Magalaspis cordyla) viscera protein. Peptides 32:1496–1501
Sies H (1997) Oxidative stress: oxidants and antioxidants. Exp Physiol 82:291–295
Sharma S, Singh R, Rana S (2011) Bioactive peptides: a review. Int J Bioautomation 15:223–250
Hwang JY, Shyu YS, Wang YT, Hsu CK (2010) Antioxidative properties of protein hydrolysate from defatted peanut kernels treated with esperase. Food Sci Technol 43:285–290
Li XX, Han LJ, Chen LJ (2008) In vitro antioxidant activity of protein hydrolysates prepared from corn gluten meal. J Sci Food Agric 88:1660–1666
Megías C, Pedroche J, Yust MM, Girón-Calle J, Alaiz M, Millán F (2008) Production of copper chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin. Food Sci Technol 41:1973–1977
Je JY, Qian ZJ, Byun HG, Kim SK (2007) Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis. Process Biochem 42:840–846
Qian ZJ, Jung WK, Kim SK (2008) Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw. Bioresour Technol 99:1690–1698
Sakanaka S, Tachibana Y (2006) Active oxygen scavenging activity of egg-yolk protein hydrolysates and their effects on lipid oxidation in beef and tuna homogenates. Food Chem 95:243–249
Merchant ME, Pallansch M, Paulman RL, Wells JB, Nalca A, Ptak R (2005) Antiviral activity of serum from the American alligator (Alligator mississippiensis). Antiviral Res 66:35–38
Merchant ME, Roche C, Elsey RM, Prudhomme J (2003) Antibacterial properties of serum from the American alligator (Alligator mississippiensis). Comp Biochem Physiol B Biochem Mol Biol 136:505–513
Merchant ME, Leger N, Jerkins E, Mills K, Pallansch MB, Paulman RL, Ptak RG (2006) Broad spectrum antimicrobial activity of leukocyte extracts from the American alligator (Alligator mississippiensis). Vet Immunol Immunopathol 110:221–228
Preecharram S, Daduang S, Bunyatrachata W, Araki T, Thammasirirak S (2008) Antibacterial activity from Siamese crocodile (Crocodylus siamensis) serum. Afr J Biotechnol 7:3121–3128
Preecharram S, Jearranaiprepame P, Daduang S, Temsiripong Y, Somdee T, Fukamizo T, Svasti J, Araki T, Thammasirirak S (2010) Isolation and characterisation of crocosin, an antibacterial compound from crocodile (Crocodylus siamensis) plasma. Anim Sci J 81:393–401
Kommanee J, Preecharram S, Daduang S, Temsiripong Y, Dhiravisit A, Yamada Y, Thammasirirak S (2012) Antibacterial activity of plasma from crocodile (Crocodylus siamensis) against pathogenic bacteria. ACMA 11:22
Pata S, Yaraksa N, Daduang S, Temsiripong Y, Svasti J, Araki T, Thammasirirak S (2011) Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Dev Comp Immunol 35:545–553
Srihongthong S, Pakdeesuwan A, Daduang S, Araki T, Dhiravisit A, Thammasirirak S (2012) Complete amino acid sequence of globin chains and biological activity of fragmented crocodile hemoglobin (Crocodylus siamensis). Protein J 31:466–476
Jandaruang J, Siritapetawee J, Songsiriritthigul C, Daduang S, Dhiravisit A, Thumanu K, Krittanai C, Thammasirirak S (2012) The effects of temperature and pH on secondary structure and antioxidant activity of Crocodylus siamensis hemoglobin. Protein J 31:43–50
Rieger AM, Barreda DR (2011) Antimicrobial mechanisms of fish leukocytes. Dev Comp Immunol 35:1238–1245
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Arcan I, Yemenicioğlu A (2007) Antioxidant activity of protein extracts from heat-treated or thermally processed chickpeas and white beans. Food Chem 103:301–312
Huang W, Deng Q, Xie B, Shi J, Huang FH, Tian B, Huang Q, Xue S (2010) Purification and characterization of an antioxidant protein from Ginkgo biloba seeds. Food Res Int 43:86–94
Ningappa MB, Srinivas L (2008) Purification and characterization of ∼35 kDa antioxidant protein from curry leaves (Murraya koenigii L.). Toxicol In Vitro 22:699–709
Sarkar MK, Kinter M, Mazumder B, Sil PC (2009) Purification and characterization of a novel antioxidant protein molecule from Phyllanthus niruri. Food Chem 114:1405–1412
Smitha S, Dhananjaya BL, Dinesha R, Srinivas L (2009) Purification and characterization of a ∼34 kDa antioxidant protein (β-turmerin) from turmeric (Curcuma longa) waste grits. Biochim 91:1156–1162
Byun HG, Lee JK, Park HG, Jeon JK, Kim SK (2009) Antioxidant peptides isolated from the marine rotifer, Brachionus rotundiformis. Process Biochem 44:842–846
Chen N, Yang H, Sun Y, Niu J, Liu S (2012) Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates. Peptides 38:344–349
Kim SY, Je JY, Kim SK (2007) Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion. J Nutr Biochem 18:31–38
Liu Q, Kong B, Xiong YL, Xia X (2010) Antioxidant activity and functional properties of porcine plasma protein hydrolysate as influenced by the degree of hydrolysis. Food Chem 118:403–410
Tanzadehpanah H, Asoodeh A, Chamani J (2012) An antioxidant peptide derived from Ostrich (Struthio camelus) egg white protein hydrolysates. Food Res Int 49:105–111
Rajapakse N, Mendis E, Jung WK, Je JY, Kim SK (2005) Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Res Int 38:175–182
Wang WY, De Mejia EG (2005) A new frontier in soy bioactive peptides that may prevent age-related chronic diseases. Compr Rev Sci Food Saf 4:63–78
Chen HM, Muramoto K, Yamauchi F, Nokihara K (1996) Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. Agric Food Chem 44:2619–2813
Suetsuna K, Ukeda H, Ochi H (2000) Isolation and characterization of free radical scavenging activities peptides derived from casein. J Nutr Biochem 11:128–131
Acknowledgments
This study was supported by the National Research University Project of Thailand, Office of the Higher Education Commission, through the Food and Functional Food Research Cluster of Khon Kaen University and Thailand Research Fund (RMU5380045). Finally, we wish to acknowledge the support of the Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University, Thailand and to thank Sriracha Moda co., Ltd., Chon Buri, Thailand for supporting the project with crocodile blood samples.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Theansungnoen, T., Yaraksa, N., Daduang, S. et al. Purification and Characterization of Antioxidant Peptides from Leukocyte Extract of Crocodylus siamensis . Protein J 33, 24–31 (2014). https://doi.org/10.1007/s10930-013-9536-8
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10930-013-9536-8