Fig. 2
Analyses of 15d-PGJ2-hsEH CTD covalent complexes. a Purification of 15d-PGJ2-hsEH CTD covalent adducts. SDS-PAGE analysis of the BTS purification of the proteins when incubated with either buffer (control) or 15d-PGJ2 (MWM, molecular weight marker; FT, flow-through fraction; EL, elution fraction). All the proteins bound to the BTS resin in the control treatment with buffer alone. Upon treatment with 15d-PGJ2, a fraction of the WT, C423S and C522S enzymes was not retained by the resin and was collected in the flow-through. The C423S/C522S mutant interacted with the BTS in all conditions. Full uncropped gels are reported in Supplementary Fig. 2. b UV analyses of the covalent adducts. The figure reports the UV spectra of the BTS fractions collected either in the flow-through or in the elution fractions. The peaks at 250 and 330 nm confirmed 15d-PGJ2 adduction to the proteins collected in the BTS flow-through (from panel a). c Comparative enzymatic activity analysis. The enzymatic activity of hsEH covalent adducts and apoproteins were measured with a spectrofluorimetric assays. The comparison showed a significant reduction in hydrolytic activity for the covalent complexes (ND, non-determined; as the C423S/C522S did not generate any adduct). The asterisks refer to one-tailed homoscedastic t-test (apoprotein vs. adducts), and the significance is indicated as follows: *0.05 < p ≤ 0.005, **0.005 < p ≤ 0.0005, ***p < 0.0005. Data presented as average ± SEM of n = 6 WT, n = 6 C522, n = 5 C423S, and n = 5 C423S/C522S. (Source data available in Supplementary Data 2). d CD analyses. Comparison of the CD spectra of the apoproteins (WT, C423S and C522S) and the corresponding 15d-PGJ2 covalent adducts revealed a conformational rearrangement upon 15d-PGJ2 modification (see Table 1). Spectroscopic and enzymatic analyses of the other fractions collected in the purifications are reported in the Supplementary Fig. 3
