Proteolytic digestion/cleavage

Fig. 10.4. Microperoxidase (MPOase) obtained by proteolytic digestion of cytochrome c. Cleavage by pepsin yields a fragment of the active site of the enzyme containing 11 amino acids (11-MPOase), whereas trypsin yields a fragment of 9 amino acids (9-MPOase). Combination of these digestions yields 8-MPOase. These MPOases have a peroxidatic activity several hundred times greater than the intact cytochrome c.

Proteolytic digestion is a very convenient means of locating signals which are strongly affected by a cleavage of the specific site by an enzyme, as demonstrated in Fig. 24.15. For instance, two peaks emerge in the difference spectrum (Fig. 24.15C) between intact (Fig. 24.15A) and papain-cleaved bR s... 

The values of the molecular weights calculated from the primary structures of Torpede a, B, y and a subunits from the nucleotide sequences of cloned cDNA [27, 28] are about 20% higher than those obtained by us from SEC/LALLS. We cannot clearly explain the discrepancy. However, as pointed out by Noda et al. [27], it is likely that a carboxy-terminal peptide is eliminated by post-transitional cleavage. Furthermore, we cannot entirely eliminate the possibility that partial proteolytic digestions may occur in the process of preparation of the receptor proteins. 

Fig. 5A, B Proteolytic cleavage analysed by A cross-correlation analysis B FRET on single-molecule-scale. In A, during the course of a specific proteolytic reaction GFP and DsRed get separated leading to gradually decreasing cross-correlation amplitudes Gx(0) determined in 40-s intervals, in B, alternatively, autocorrelation functions and photon counts per molecule in kHz (inset) for rsGFP and DsRed were determined in parallel during a proteolytic digest. Changes in fluorescence intensity of FRET donor and acceptor were detected immediately after enzyme addition, whereas autocorrelation G(0) values and fluorescent particle numbers remained constant. The monitored increase of rsGFP fluorescence corresponds to approximately 35% FRET in the intact substrate...

---