alpha-chymotrypsin and 2-2--azobis(2-amidinopropane)

We investigated the effects of bioactive-peptides from hydrolysates of flounder fish muscle (FFM) on antioxidant activity. The hydrolysates were prepared by enzymatic reactions of FFM using eight commercial proteases such as papain, pepsin, trypsin, neutrase, alcalase, kojizyme, protamex, and α-chymotrypsin. The α-chymotrypsin hydrolysate showed the strongest antioxidant activity among the eight enzymatic hydrolysates. Further separation of the α-chymotrypsin hydrolysate was performed by ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. Consequently, two novel peptides with high antioxidant activity were purified, and their amino acid sequences were determined (Val-Cys-Ser-Val [VCSV] and Cys-Ala-Ala-Pro [CAAP], respectively). The two peptides showed good scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical (IC(50) values, 111.32 and 26.89 μM, respectively) and high cytoprotective activities against 2,2-azobis-(2-amidino-propane) dihydrochloride (AAPH) without cytotoxicity and scavenged total reactive oxygen species in Vero cells. In particular, apoptotic bodies produced by AAPH dose-dependently decreased following treatment with the CAAP peptide. These results revealed firstly the two peptides with strong antioxidative effects from FFM.

Topics: Amidines; Amino Acid Sequence; Animals; Antioxidants; Apoptosis; Chlorocebus aethiops; Chymotrypsin; Fish Proteins; Flounder; Free Radical Scavengers; Peptides; Protein Hydrolysates; Reactive Oxygen Species; Vero Cells

Insulysin (IDE) and neprilysin (NEP) were found to be inactivated by oxidation with hydrogen peroxide, an iron-ascorbate oxidation system, and by treatment with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). In each case reaction led to the introduction of protein carbonyl groups as judged by reaction with 2,4-dintrophenylhydrazine. IDE was inactivated by reaction with 4-hydroxy-2-nonenal (HNE) with the concomitant formation of protein adducts. NEP was not inactivated to a significant extent by HNE, but some HNE-adduct formation did occur. Prior reaction with hydrogen peroxide or AAPH led to enhanced formation of HNE adducts. Treatment of IDE with AAHP or hydrogen peroxide increased its susceptibility to proteolysis, while treatment of NEP with iron/ascorbate or hydrogen peroxide increased its susceptibility to proteolysis. Since IDE and NEP play a prominent role in the clearance of amyloid beta peptides, their oxidative inactivation and enhanced proteolysis can contribute to the onset and/or progression of Alzheimer's disease.

Topics: Aldehydes; Alzheimer Disease; Amidines; Amyloid beta-Peptides; Ascorbic Acid; Chlorides; Chymotrypsin; Ferric Compounds; Hydrogen Peroxide; Insulysin; Neprilysin; Oxidation-Reduction; Trypsin