Peptide enzymatic cleavage

The enzymatic cleavage of esters is a vast and extensively reviewed area of chemistry. Recently, several new esters have been examined primarily for the preparation of peptides and glycopeptides. 

FIGURE 5.17 TEM micrographs of diphenylalanine peptide nanotubes self-assembled after the enzymatic cleavage, (a) TEM images of 22 prior to the enzymatic cleavage, (b and c) TEM images of 22 after the enzymatic cleavage. 

The choice of the appropriate time for the enzymatic cleavage is a critical point of sample preparation. All types of the reference proteinaceous binders (Section 6.2.3) were cleaved from 1 to 24 h at laboratory temperature 20 25 °C. The peptide peaks in the interval 900 2000 Da were registered below 900 Da the peaks of the matrix interfere and above 2000 Da the incompletely cleaved oligopeptides are present which makes interpretation difficult. The best result, i.e. the highest number of peaks, was obtained after digestion for 2 h at laboratory temperature [32]. 

PARACEST agents with a response to enzymatic activity have also been reported. Pagel et al. used a Tm1 DOTA monoamide complex containing a peptide chain which is hydrolyzed by the Caspase-3 enzyme (204,205). Following enzymatic cleavage, the PARACEST effect originating from the amide proton disappears due to the hydrolysis of the amide bond. 

Substitution of the l-amino acids with their D-amino acid configurational analogue, will enhance resistance to enzymatic cleavage of peptide bonds. The ll isomer of cyclo(Leu-Leu) hydrolyzes 3.5 times faster than the DL isomer in the presence of 0.5 moll HCl. This difference was explained by both steric shielding ui-trans isomer) and steric strain (ll- j isomer). ... 

Larsen et al. reported the enzymatic cleavage of a desB30 insuhn B-chain from a presequence (Lys(Boc))6. This spacer shifts the conformation of the growing peptide chain from a y9-structure to a random coil conformation and reduces pep-tide-chain aggregation, which otherwise causes serious synthetic problems. Nova-syn KA [18] was employed as a solid support, but unfortunately, no information about the enzyme used was reported [19]. 

Lowe et al. investigated the substrate selectivity of the proteases papain and chy-motrypsin using PEGA-bound combinatorial peptide hbraries (32) (Fig. 10.3) [24]. The quahtative extent of enzymatic cleavage of the resin-bound peptide in case of an accepted substrate strand was rapidly visualized by a significant reduction in the fluorescence of the beads visuahzed with a fluorescence microscope. Furthermore it was proven that substrate selectivity of the enzyme remain the same on the solid phase and in solution. 

The same enzymatic cleavage can then be applied to the unreduced enzyme. Pairs of peptide fragments remain linked by the S-S bridges. These crosslinked pairs can be separated, the disulfide bridges cleaved, and the resulting peptides identified, each as one of the already sequenced fragments. Mass spectrometry provides a rapid method for their identification.258... 

Enzymatic cleavage of the thioester bond (located either in the middle or at the C-terminus of the peptide) produces a free thiol compound that can be easily monitored by very sensitive assays (e.g., Ellman s reagent). 64 ... 

Once the protein s primary sequence has been determined, the location of disulfide bonds in the intact protein can be established by repeating a specific enzymatic cleavage on another sample of the same protein in which the disulfide bonds have not previously been cleaved. Separation of the resulting peptides shows the appearance of one new peptide and the disappearance of two other peptides, when compared with the enzymatic digestion product of the material whose disulfide bonds have first been chemically cleaved. In fact, these difference techniques are generally useful in the detection of sites of mutations in protein mole-... 

Heizmann, J., et al. 1996. Enzymatic cleavage of thymopoietin oligopeptides by pancreatic and intestinal brush-border enzymes. Peptides 17 1083. 

To facilitate sequencing analysis and enzymatic cleavage, reduction and alkylation of the peptides to open their potential disulfide bridges is recommended. Several alkyl groups can be used acrylamide, iodoacetamide, and 4-vinylpyridine (4-VP). S-pyridylethylation is preferred over the other alkyl groups when Edman degradation (PTH-4-pyridylethylated-Cys is commercially available) and MS (addition of 57 Da per alkylated cysteine residue) have to be performed. 

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