Free-radical-induced inactivation of lysozyme and carbonyl residue generation in protein are not necessarily associated

The 2,2'-azobis(2-amidinopropane) (AAPH)-induced inactivation and oxidative modification of lysozyme, as determined by the loss of tryptophan-associat ed fluorescence (TAF) and the increase in dinitrophenyl-hydrazine-reactive carbonyl groups (CO), were studied in the absence and in the presence of an tioxidants. AAPH induced a progressive inactivation of the enzyme and a par allel decrease of its TAF. Both changes were closely correlated (R-2 = 0.97 ); however, the inactivation was only partially associated with an increase in CO, The latter reached maximal values at times half those needed to att ain maximal losses in both lysozyme activity and TAF. A stoichiometric comp arison reveals that whereas over 74% of the enzyme molecules had lost their activity, only 5% exhibited an increment in CO. CO formation was affected differentially by boldine and trolox. Both antioxidants fully protected aga inst the early inactivation and loss of TAF; however, the increase in CO wa s completely unaffected by trolox. Exposure of lysozyme to Fe3+/ascorbate i nduced no loss of activity or TAF,, but it led to an accumulation of CO sim ilar to that induced by AAPH. Results indicate that CO formation and lysozy me inactivation are two mechanistically dissociable events and that changes in the former parameter can perfectly occur in the absence of changes in t he latter. (C) 2000 Academic Press.