For the first time, three different stefins, A, B and C, have been isolated from a single species. The complete amino acid sequence of bovine stefin A was determined. The inhibitor, with a calculated M r of 11,123, consists of 98 amino acid residues. Although it exhibits considerable similarity to human and rat stefin A, some significant differences in inhibition kinetics were found. Bovine stefin A bound tightly and rapidly to cathepsin L (k ass = 9.6·106 M −1·s −1, K i = 29 pM). The binding to cathepsin H was also rapid (k ass = 2.1·106 M −1·s −1), but weaker (K i = 0.4 nM) due to a higher dissociation rate constant. In contrast, the binding to cathepsin B was much slower (k ass = 1.4·105 M −1·s −1), but still tight (K i = 1.9 nM).
