Chymotrypsin (Bovine)

Chymotypsin is a serine endopeptidase, which predominantly cleaves peptide bonds on the carboxy side of Tyrosine, Phenylalanine and Tryptophan.

Characteristics:

Chymotypsin is a serine endopeptidase, which predominantly cleaves peptide bonds on the carboxy side of Tyrosine, Phenylalanine and Tryptophan. In addition Chymotrypsin also catalyses hydrolysis at the carboxy side of Leucine, Methionine, Alanine, Aspartic and Glutamic acids, although at a much lower rate. It is therefore recommended to always use the shortest digestion time possible.

Chemical Modification:

Princeton Separations Sequencing grade, Modified Chymotrypsin is first treated with TPCK and then subjected to an extensive purification process to remove contaminating protease and chymotryptic autolysis by-products, which could affect the specificity of the digestion process. The highly purified enzyme is then modified chemically by a process developed by Princeton Separations scientists. As a result the modified enzyme is more resistant to autolytic inactivation and has improved stability. Thus the Princeton Separations Modified Chymotrypsin retains in excess of 80% of its activity after six hours at 30°C in reaction buffer, and in excess of 70% of its activity under the same conditions after 24 hours.

Application:

For fragmentation of protein, Modified Chymotrypsin is added to the protein at a ratio of 1/50 to 1/200, by weight, in a standard digestion buffer such as 50 mM Tris HCL pH 8.0 in 1mM CaCl2. The digestion mixture is allowed to incubate at 25°C - 30°C for 1 to 10 hours, but can be extended to 24 hours, the Modified Chymotrypsin, having at least that much extended lifetime of activity. It is however recommended to chose a ratio of enzyme to protein to allow for the shortest incubation time possible. This will reduce or eliminate the catalyzed hydrolysis of peptide bonds with non-aromatic amino acid residues.

Quality Control:

The Modified Chymotrypsin activity is monitored by assays developed in house which relate to its activity in sequencing applications. These assays, an amidase assay using Benzoyl Tyrosine para-Nitroanilide, and a protease assay using casein as substrate, were developed to replace the unreliable BTEE assay (Benzoyl Tyrosine Ethyl Ester) which is by contrast an esterase assay. The results are routinely compared with freshly prepared unmodified Chymotrypsin. Furthermore the enzyme protein digestion activity is also compared to Trypsin Activity and according to these measurements a Trypsin Activity Equivalent is calculated and reported. To check for specificity, an array of synthetic peptides is used.

Price and ordering information:

Product must be shipped 2 day air

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Procedure Guide for Chymotrypsin (Bovine)46.86 KB

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