Cleavage of proteins

The objective of cleaving a protein at specific sites is to generate peptides of suitable size for mass spectrometry analysis. Two methods are commonly used to cleave proteins (1) hydrolysis with a suitable chemical reagent and (2) digestion 

Cleaving Agent Specificity Digestion Conditions buffer pH temperature (°C)] 

Degradation with Chemical Reagents A frequently used procedure is to react proteins at acidic pH with cyanogen bromide (CNBr), which cleaves the amide bond on the C-terminal side of methionine. This reaction converts methionine to homoserine, which under the acidic enviromnent exists in the lactone form  

Solution Cleavage of the peptide with Asp-N will produce a total of three peptides Tyr-Phe-Ser, Asp-Phe-Leu-Arg-Trp-Val-Gly-Met-Glu-Ala, and Asp-Phe-Gly-Thr-Lys-Ser-Asn-Ala-Leu. Further treatment of this mixture with trypsin will generate the following five peptides  

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Table 5.4 Enzymes used for the cleavage of proteins for sequence investigations and the corresponding amino acid residues at which they break the peptide backbone...

Several mechanisms have been proposed to explain HIAR the cleavage of protein-protein cross-links,3,6 the disruption of cross-links involving calcium ions,5 an increase in tissue permeability for antibodies, and the removal of trace amounts of paraffin in the tissues. However, recent studies have indicated that the fundamental mechanism of HIAR is based on the cleavage of protein-protein cross-links. 

Proteases (endopeptidases or proteinases) commonly used for specific cleavage of proteins are summarised in Table 6.2. Trypsin is almost always used as an enzyme of first choice it is highly specific and stable, has an appropriate pH-optimum and is commercially available in high purity and quality. When the results obtained are ambiguous, or the trypsin cannot be used for any other reason, a different protease can be easily chosen. In all experiments, described here, the trypsin cleavage was applied. 

Kim, K., Rhee, S.G., and Stadtman, E.R. (1985) Nonenzymatic cleavage of proteins by reactive oxygen species generated by dithiothreitol and iron./. Biol. Chem. 260, 15394-15397. 

The clotting factors are protein molecules. Activation mostly means proteolysis (cleavage of protein fragments) and, with the exception of fibrin, conversion into protein-hydrolyzing enzymes (proteases). Some activated factors require the presence of phospholipids (PL) and Ca + for their proteolytic activity. Conceivably, Ca + ions cause the adhesion of factor to a phospholipid surface, as depicted in C. Phospholipids are contained in platelet factor 3 (PF3), which is released from ag-Lullmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of license. 

Bond, J. S., and P. E. Butler, Intracellular proteases. Ann. Rev. Biochem. 56 333, 1987. An overview of the types of proteolytic enzymes that are found in cells and how they may function in biologically important cleavages of proteins. 

The selective enzymatic cleavage of proteins is critical to many biological processes. For example, the clotting of blood depends on the enzyme thrombin cleaving fibrinogen at specific points to produce fibrin, the protein that forms a clot. 

Neurotoxins, such as the clostridial neurotoxins responsible for tetanus and botulism. These are metallo-proteases that enter nerve cells and block neurotransmitter release via zinc-dependent cleavage of protein components of the neuroexocytosis apparatus. 

Where possible, enzymatic methods of cleavage should be used rather than chemical ones the latter do not generally proceed in good yield, and they are often accompanied by side reactions. Table 5-3 summarises a selection of reagents that have been used for chemical cleavage of proteins (Fontana and Gross 1986 Smith 1994b). 

Table 5-3. Reagents for specific chemical cleavage of proteins...

Smith, B. (1994b) Chemical Cleavage of Proteins, in Methods in Molecular Biology Vol. 32. Humana Press, Totowa, NJ. 

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