Figure 3
UV exposure alters the chemical composition of CRP in the absence and presence of DNA. Isolated CRP-cAMP complex (0.2 and 50 μM, respectively) was incubated in the dark, or exposed to UV light (λmax 310 nm, 30 nm band width, 6 W m−2, normoxia) for 15 min, before analysis using UPLC with pre-column derivatization and fluorescence detection (panels A,B) or LC-MS/MS (panels C–F). Panel A: Changes in amino acid composition determined by acid-hydrolysis and total amino acid analysis. Data are expressed as % modification of the indicated amino acids (positive values indicating loss, negative values indicating formation) relative to the UV exposure control. Mean ± SD from 3 independent experiments. Panel B: Material balance for Trp and Tyr residues determined by UPLC analysis with direct fluorescence detection on acid-hydrolysed UV-exposed CRP-cAMP complex. Levels of unmodified parent amino acid, formation of DOPA, the total of NFK and Kyn (as NFK is converted to Kyn during acid hydrolysis) and unknown products (difference to control values) are indicated. Mean ± SD from 3 independent experiments. Panel C: Extent of modification at different amino acids (Met, M; Pro, P; Ser, S; Tyr, Y; His, H; Trp, W) detected by LC-MS/MS for control CRP-cAMP, UV-exposed CRP-cAMP, and preformed CRP-cAMP-DNA complex exposed to UV. Modifications detected at individual sites (panels D–F) were summed and are expressed as a percentage of the total (native and modified) concentration of the amino acid detected. Panels D–F: Percentage extents of modification at individual amino acids in the CRP sequence (indicated on horizontal axis as position number in sequence). Panel D: control CRP-cAMP complex. Panel E: UV exposed CRP-cAMP. Panel F: CRP-cAMP exposed to UV in presence of DNA (as above). Modifications at the indicated residues are given as the change in m/z (−22, −10, +5, +16, +32, +42, +56) detected by MS for the modifications included in the data searches. The majority of the modifications correspond to m/z + 16 species assigned to the addition of one oxygen atom (formation of an alcohol at Pro and Tyr; generation of the sulfoxide from Met).
