alpha-chymotrypsin and divinyl-sulfone

Magnetic bead cellulose was prepared by a suspension method from the mixture of viscose and magnetite using thermal sol-gel transition and regeneration of cellulose. The prepared magnetic particles after their activation with divinyl sulfone were shown to be suitable magnetic carrier for immobilization of α-chymotrypsin and for its application in proteomic studies. The specific activity of the immobilized proteinase was high; its activity did not change in the course of storage. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH and temperature dependence of the activity, self-cleavage activity, and possibility of repeated use. α-Chymotrypsin immobilized to magnetic bead cellulose was used for the proteolytic digestion of porcine pepsin A and human gastric juice and a possibility of direct use of enzyme reaction products for matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis was shown.

Topics: Amino Acid Sequence; Animals; Cattle; Cellulose; Chymotrypsin; Enzymes, Immobilized; Gastric Juice; Humans; Magnets; Microspheres; Molecular Sequence Data; Pepsin A; Proteolysis; Solubility; Sulfones

Proteasome inhibition is a topic of great interest in anticancer research. The proteolytic activity of this multicatalytic complex relies on three subunits, β1, β2 and β5, containing a caspase-like, a trypsin-like and a chymotrypsin-like active site, respectively. Several studies have demonstrated that, of the three activities, the chymotrypsin-like activity was the most necessary for cell viability and protein processing. Thus, most efforts towards the development of proteasome inhibitors have focused on the selective inhibition of the β5 subunit active site. Herein, we report the design and synthesis of a series of conformationally constrained tripeptidyl vinyl sulfones were determined to be good inhibitors of the chymotrypsin-like activity of proteasome, with K(I) values in the sub-micromolar to micromolar range. These compounds were also tested against bovine pancreatic α-chymotrypsin and human cathepsin B and L, revealing a good selectivity for the target enzyme over these related enzymes.

Topics: Animals; Cathepsin B; Cathepsin L; Cattle; Chymotrypsin; Humans; Peptidomimetics; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sulfones