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 pKa 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 pKa 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 pKa 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 pKa of 3.94 in water and 3.89 in aq. 19.2% (v/v) dioxan (at 27°C). This small change to lower pKa 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.