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Isolation of two chymotrypsins from grass carp
1Department of Biochemistry, Chinese University of Hong Kong, Shatin, N.T., Hong Kong. b348738@mailserv.cuhk.edu.hk
Biochemistry and Molecular Biology International
|July 25, 1998
Summary
Researchers purified two chymotrypsins from grass carp hepatopancreas. Chymotrypsin I showed unique resistance to inhibitors and lower catalytic efficiency, suggesting it
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Chymotrypsins are serine proteases crucial for protein digestion.
- Investigating fish digestive enzymes provides insights into piscine physiology and evolution.
- Grass carp hepatopancreas is a rich source of digestive enzymes.
Purpose of the Study:
- To purify and characterize two distinct chymotrypsin enzymes from grass carp (Ctenopharyngodon idellus) hepatopancreas.
- To compare the biochemical properties, substrate specificity, and inhibitor susceptibility of the purified chymotrypsins.
- To determine the optimal activity conditions for the identified chymotrypsin variants.
Main Methods:
- Purification of chymotrypsin I and II from grass carp hepatopancreas using phenyl-Sepharose and Q-Sepharose chromatography.
- Determination of molecular weights using SDS-PAGE or similar techniques.
- Enzyme inhibition assays using specific inhibitors (PMSF, STI, TPCK, aprotinin).
- Enzyme activity assays using chymotrypsin-specific ester and amide substrates.
- Determination of optimal pH for enzyme activity.
Main Results:
- Two chymotrypsins, designated chymotrypsin I (28 kDa) and chymotrypsin II (27 kDa), were successfully purified.
- Both enzymes exhibited sensitivity to phenylmethylsulfonyl fluoride and soybean trypsin inhibitor.
- Chymotrypsin I demonstrated greater resistance to tosyl-L-phenylalanine chloromethyl ketone and aprotinin compared to chymotrypsin II.
- Chymotrypsin I displayed lower catalytic efficiency with specific substrates, indicating a less typical chymotrypsin profile.
- Optimal activity for both chymotrypsins was observed within the pH range of 7.0–8.5.
Conclusions:
- Grass carp hepatopancreas harbors at least two distinct chymotrypsin enzymes with differential biochemical characteristics.
- Chymotrypsin I represents a less conventional chymotrypsin variant with altered inhibitor susceptibility and catalytic function.
- The findings contribute to understanding the diversity and functional specialization of digestive proteases in fish.