Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
Proteases with high specificity
Submandibular gland protease

The enzyme was isolated from the mouse submandibular gland [86, 87]. Maximum activity is observed at pH 7.5–8.0. Detailed information on The properties of this enzyme can be found in [6]. It has been shown that this Serine protease is inhibited by DFP and is homologous to Thrombin [89, 90]. Similar results were published in [104]. The enzyme is commercially available from Pierce and Boehringer.

3.5.5.1. Specificity and Hydrolysis conditions.

Specificity. Studies on the enzyme's action on various synthetic and native substrates have demonstrated that it is specific for the C-terminal peptide bond of Arginine residues [88]. Using the hydrolysis of egg white Lysozyme and bovine Insulin as Examples, it was shown that hydrolysis occurs at specific arginine residues while leaving Lysine residues unaffected. Similar results were obtained during the hydrolysis of the immunoglobulin B-chain [113]. In this case, hydrolysis proceeds at the majority of arginine residues, with the exception of the resistant -Arg-Val- and -Arg-Arg- peptide bonds. Thus, in terms of its specificity, the enzyme is similar to Clostripain. However, when treating the phosphatidylcholine-binding protein from bovine Liver, Cleavage is observed at only one of the 10 arginine residues [69].

Hydrolysis conditions. Hydrolysis is carried out in 1% NH₄HCO₃ (pH 8.0) at 37 °C for various periods of time (up to 24 h) at an Enzyme-to-substrate ratio of 1 : 50. The hydrolysis is terminated by acidification with HCl to pH 1.5–2.0 [88].