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Proteolysis of native proteins. Trapping of a reaction intermediate.

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  • Biology
  • Chemistry

Abstract

Formation of a high mass complex during proteolysis of MUP by trypsin. a, trypsin (50 μm) was incubated with MUP (65 μm) in 20 mm Hepes buffer, pH 7.5, for between 2 and 60 min. Wu C et al. J. Biol. Chem. 1999;274:1108-1115 ©1999 by American Society for Biochemistry and Molecular Biology Formation of a high mass complex during proteolysis of MUP by trypsin. a, trypsin (50 μm) was incubated with MUP (65 μm) in 20 mm Hepes buffer, pH 7.5, for between 2 and 60 min. At various times, samples were removed, and the reaction was terminated by addition of trichloroacetic acid to a final concentration of 10% (w/v). The proteins in the reaction mixture were then recovered by centrifugation, residual acid was washed away with diethyl ether, and the reaction mixture was analyzed on nonreducing SDS-PAGE.b, the analysis was conducted in essentially the same fashion as in a except that the time was fixed at 5 min and the concentrations of MUP or trypsin were varied. In the first experiment, trypsin was maintained at 50 μm, and MUP was varied from 16 to 60 μm. In the second experiment, MUP was maintained at 65 μm, and trypsin was varied from 12 to 45 μm. MUP′ refers, in this and subsequent figures, to the mixture of products of tryptic attack on MUP at Arg-8 and Arg-161.

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