Canonical serine proteinase inhibitors are distributed in a wide range of organisms from all kingdoms of life and play crucial role in various physiological mechanisms [1]. They interact from the canonical proteinase-inhibitor binding loop, where P1 residue has a predominant role (the residue at the P1 position contributing the carbonyl portion to the reactive-site peptide bond). These so-called canonical inhibitors bind to their cognate enzymes in the same manner as a good substrate, but are cleaved extremely slowly. Kazal-type inhibitors represent the most studied canonical proteinase inhibitors. Kazal inhibitors are extremely variable at their reactive sites. However, some regularity prevails such as the presence of lysine at position P1 indicating strong inhibition of trypsin [2].

The Kazal inhibitor has six cysteine residues engaged in disulfide bonds arranged as shown in the following schematic representation:

                       +------------------+
                       |                  |
                       *******************|***
        xxxxxxxxCxxxxxxCx#xxxxxCxxxxxxxxxxCxxCxxxxxxxxxxxxxxxxxC
                |              |             |                 |
                |              +-------------|-----------------+
                +----------------------------+

'C': conserved cysteine involved in a disulfide bond.
'#': active site residue.
'*': position of the pattern.

The structure of classical Kazal domains consists of a central α helix, which is inserted between two β-strands and a third that is toward the C-terminus (see for example <PDB:1OVO>)[3]. The reactive site P1 and the conformation of the reactive site loop is structurally highly conserved, similar to the canonical conformation of small serine proteinase inhibitors.

The proteins known to belong to this family are:

• Pancreatic secretory trypsin inhibitor (PSTI), whose physiological function is to prevent the trypsin-catalyzed premature activation of zymogens within the pancreas.
• Mammalian seminal acrosin inhibitors.
• Canidae and felidae submandibular gland double-headed protease inhibitors, which contain two Kazal-type domains, the first one inhibits trypsin and the second one elastase.
• A mouse prostatic secretory glycoprotein, induced by androgens, and which exhibits anti-trypsin activity.
• Avian ovomucoids, which consist of three Kazal-type domains.
• Chicken ovoinhibitor, which consists of seven Kazal-type domains.
• Bdellin B-3, a leech trypsin inhibitor.
• LDTI [4], a leech tryptase inhibitor.
• An eel peptide, which is probably a pancreatic serine proteinase inhibitor.
• An elastase inhibitor from a sea anemone.
• Rhodniin, a thrombin inhibitor from the insect Rhodnius prolixus [5]. This protein consists of two Kazal-type domains.
• Pig intestinal peptide PEC-60 [6]. This protein, while highly similar to other members of the Kazal family, does not seem to act as a protease inhibitor. Its exact biological function is not yet established, but it is known to inhibit the glucose-induced insulin secretion from perfused pancreas and to play a role in the immune system.

The pattern we developed to pick up Kazal-type inhibitors spans a region beginning with the second cysteine and ending with the fifth one. We also developed a profile that covers the entire Kazal domain.