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Fig. 10.4. Dimedone adduct formation and activity loss for the H2O2-treated C318A mutant of hBCATm. Samples (4.5 nmol each) were incubated with 1.5 mM (open circle) or 2 mM H2O2 (closedcircles) and 5 mM dimedone at 25°C before the addition of catalase, then assayed for BCAT activity and analysed by electrospray ionization mass spectrometry as described in Section 2. In (A), the transformed mass spectrometry data for the sample after 427 min of treatment are shown, with peaks of maximal abundance at 41,708 and 41,848 atomic mass units for the free and dimedone-adducted protein, respectively. In (B), the correlation between the fraction of dimedone adduct formed and the BCAT activity of the C318A samples from two independent experiments is illustrated, after treatment for 224, 427, 1,180, and 1,375 min (in order of increasing adduct formation; r = 0.936 and slope = -0.987).Reproduced from ref. (12). With permission from American Chemical Society.

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