Investigation of the active site of papain with fluorescent probes

Abstract

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) and 7-(2′-hydroxyethylthio)-NBD (obtained from NBD chloride and mercaptoethanol) undergo a reversible spectral change in alkaline solution that depends respectively on a single apparent pK_a 9.76 (at 25°C) and 8.81 (at 32°C). In acid solution however no spectral change was observed. NBD chloride reacts slowly with papain at pH7, but the rate of inhibition increases at lower pH and depends on an apparent pK_a of 3.7 (at 35°C), which has been tentatively assigned to the carboxyl group of aspartic acid-158. The spectral properties of NBD-papain indicate that the thiol group of cysteine-25 is the site of reaction. The intensity of the fluorescence-emission spectrum of NBD-papain depends on a single pK_a of 4.2 (at 26.7°C). The intensity of the fluorescence-emission spectrum of the mixed disulphide formed from papain and 7-(2′-mercaptoethylamino)-NBD (obtained from NBD chloride and cysteamine) depended on a single pK_a of 3.94 in water and 3.89 in aq. 19.2% (v/v) dioxan (at 27°C). This small change to lower pK_a value in a medium of lower dielectric constant is characteristic of a cationic acid. The only acid of this type in the active-site region is the conjugate acid of histidine-159.