Acid phosphatase reaction with peroxyl radicals: Inactivation mechanism and behavior of the partially modified ensemble

Acid phosphatase (AP) is readily inactivated when exposed to the free radic als generated in the pyrolysis of 2,2'-azobis(2-amidinopropane) hydrochlori de (AAPH) under aerobic conditions. On average, a large number of tryptopha n groups are modified by each protein molecule that loses its catalytic act ivity. In spite of this, the enzyme inactivation takes place without induct ion times, a result that indicates either that damage is progressive or tha t damage of a critical target is needed to inactivate the enzyme (all-or-no thing mechanism). A Lineweaver-Burk plot of the enzyme activity measured at pH 4.8 is not compatible with an all-or-nothing mechanism, showing that af ter exposure of the native protein ensemble to the free radical source ther e are partially damaged molecules whose affinity for the substrate is widel y different from that of the native molecules. On the other hand, the parti ally damaged ensemble shows a normal Michaelis-Menten behavior when the act ivity is measured at pH 7.0, with only a reduced value of V-M, relative to that of the unmodified ensemble. These results show that the native protein and modified proteins that remain active constitute different populations, with different responses to pH changes. Comparative heat denaturation stud ies of the native and pretreated proteins support this proposal. (C) 2000 A cademic Press.