TLCK (N-P-Tosyl-Lysine Chloromethyl Ketone) (Molecular Biology)

This reagent was developed by Shaw and co-workers as a specific inhibitor of trypsin (1). Its design was based on the principle of affinity labeling, which couples substrate specificity with chemical reactivity to generate a covalent bond between the reagent and a functional group at the active site of an enzyme. In this case, the lysine part of the reagent confers specificity for trypsin and the chloromethyl ketone provides chemical reactivity (Fig. 1). Although trypsin is a serine proteinase, the reagent does not interact with the active site serine residue but instead alkylates the histidine residue that is part of the catalytic triad. The reagent has essentially no effect on the activity of chymotrypsin or other serine proteinases and is often used to inactivate the small amounts of trypsin present in purified chymotrypsin preparations. It is also often combined with inhibitors of other types of proteinases to prevent unwanted proteolysis that might occur during the course of protein isolation from cell or tissue homogenates (2).

Figure 1. Interaction of TLCK (tosyl-lysyl-chloromethyl ketone, ie, L-1-chloro-3[4-tosylamido]-7-amino-2-heptanone) with the active-site histidine side chain of a trypsin-like serine proteinase.