Purification and Characterization of a Cysteine Protease Inhibitor from Chum Salmon ( Oncorhynchus keta) Plasma

Abstract

A cysteine protease inhibitor (CPI) in chum salmon ( Oncorhynchus keta) plasma (CSP) was detected after performing inhibitory activity staining against papain under nonreducing condition. The CPI was purified from CSP by affinity chromatography with a yield and purification ratio of 0.94% and 30.36-fold, respectively. CSP CPI had a molecular mass of 70 kDa based on the results of SDS-PAGE and Sephacryl S-100 gel filtration. CSP CPI was a glycoprotein based on the periodic acid-Schiff (PAS) staining of the SDS-PAGE gel and classified as a kininogen. CSP CPI was stable in the pH range of 6.0-9.0 with maximal stability at pH 7.0. CSP CPI presented thermal stability at temperatures below 50 degrees C and exhibited maximal activity at temperatures of 20-40 degrees C. CSP CPI was determined to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 105 nM.

Purification and characterization of &#x03B1;<inf>2</inf>-macroglobulin from chum salmon plasma

“…The inhibitory activity of purified 2 -M against papain and trypsin both significantly decreased under extreme acidic (pH < 4.0) and alkaline conditions (pH > 9.0) (Fig. 5), which was similar to the purified fish inhibitors in glassfish and rainbow trout plasma [19,20]. Because most endogenous cysteine and serine proteases are active at weak acidic pH with significant activity at around pH 7.0 [18], therefore, in theory the purified 2 -M could effectively inhibit the proteases and its application on gel-weakening prevention is favored.…”

Trypsin Inhibitory Activity and Gel‐Enhancing Effect of Sarcoplasmic Proteins from Common Carp

“…Nagashima and others (2004) reported that skin mucus of pufferfish, Takifugu pardalis, contained 2 trypsin inhibitors with molecular mass of 57 and 47 kDa. Li and others (2008) purified a cysteine proteinase inhibitor from chum salmon ( Oncorhynchus keta ) plasma, which presumably was a glycoprotein and classified as a kininogen. A cysteine proteinase inhibitor in glassfish ( Liparis tanakai ) eggs was classified as a member of the family I cystatins (Ustadi and others 2005).…”

“…This result indicated a relatively high thermal stability of common carp SP at 40 °C. Cysteine proteinase inhibitor from chum salmon ( O. keta ) plasma retained 70% of its inhibitory activity after being pre‐incubated at 50 °C for 30 min (Li and others 2008). …”

Natural Cathepsin Inhibitor to Inhibit Milkfish Deterioration During Chilling Storage

“…Dengan demikian maka ditetapkan bahwa suhu inkubasi terbaik adalah 80 ⁰ C dengan aktivitas inhibisi sebesar 93,06%. Hasil yang sama ditunjukkan oleh hasil penelitian Nurhayati et al (2013 b ) yang menunjukkan bahwa inhibitor enzim katepsin dari daging ikan patin dapat diekstrak dengan efektif pada suhu inkubasi 80 o C. Nurhayati et al (2013 a ) juga melaporkan bahwa inhibitor katepsin dari kulit ikan patin juga dapat diekstrak dengan baik menggunakan suhu 80 ⁰ C. Penelitian Ylonen et al (1999) dalam Li et al (2008) menyatakan bahwa pada suhu 80 ⁰ C, inhibitor protease jenis sistein dari ikan salmon atlantik mempunyai aktivitas inhibisi yang lebih stabil. Menurut Jiang (2000), pada suhu 60-70 ⁰ C terjadi aktivasi enzim katepsin, sedangkan pada suhu 80 ⁰ C aktivitas enzim telah menurun sehingga penghambatan yang efektif terjadi apabila suhu inkubasi telah mencapai 80 ⁰ C. Gambar 1.…”