Practical Protein Chemistry - A. Darbre 1989

Fragmentation of the polypeptide chain by enzymatic methods
Proteases with high specificity
Thrombin

One of the most specific and accessible Enzymes is thrombin, and it is quite surprising that it has not yet found Structure/182.html">Practical Application. This limited interest is likely due to the varying degrees of purity and activity among different commercial batches. Thrombin is manufactured by several companies, including Calbiochem, Miles Lab., and Sigma. A method for the further purification of commercial thrombin on SP-Sephadex G-50 has been described [106]. A detailed description of assays for activity and the identification of various molecular forms of thrombin is given in [64].

3.5.2.1. Specificity and Hydrolysis conditions.

Specificity. Thrombin-catalyzed hydrolysis occurs at the C-terminal peptide bond of Arginine residues, specifically at -Arg-X- sequences. In its native substrate, fibrinogen, this bond is -Arg-Gly-, whereas in other Proteins, the X residue may be Alanine, arginine, aspartic acid, Cysteine, or valine [64]. Hydrolysis at the -Arg-His- bond in calmodulin has also been observed [108]. Thrombin Treatment results in the Cleavage of only a very limited number of arginine peptide bonds, making it an ideal tool for generating large protein fragments intended for automated sequencing. In some cases, even under stringent conditions, thrombin hydrolyizes only a single peptide bond in a protein; in others, the extent of hydrolysis depends on the reaction time and Temperature. Relevant Examples are provided in [106].

Hydrolysis conditions. Thrombin is a Serine protease exhibiting maximum activity at pH 8.0. Hydrolysis is typically performed under conditions similar to those used for Trypsin, i.e., in 100 mM NH₄HCO₃ at 37 °C for 4–8 h, although these latter two parameters can vary widely. Thrombin activity is measured by the degree of fibrinogen clotting in NIH units (established by the U.S. National Institutes of Health) [64]. The Enzyme-to-substrate ratio also spans a broad range, from 1 : 100 to 1 : 5 [46, 70] and from 1 to 60 units/mg of substrate [59, 118]. Consequently, optimal hydrolysis conditions must be determined empirically for each specific case, for example, by monitoring the process via Electrophoresis. Relevant examples can be found in [71]. Thrombin is inhibited by DFP and PMSF.