FIGURE 4.

SDS-PAGE of Tpx inactivated by compounds 1–6. His-tagged Tpx was treated with the compounds in the presence or absence of DTT, and remaining free cysteines were modified by MAL-PEG (∼5 kDa) as described under “Experimental Procedures.” a, in the absence of DTT, Tpx did not react with MAL-PEG independent of prior incubation without (lane 2) or with compound 2 (lane 3). In the presence of DTT, but no inhibitor, both cysteines of Tpx reacted with MAL-PEG (Tpx-(MP)₂; lane 4). In contrast, if reduced Tpx was incubated with the compounds prior to MAL-PEG treatment, the mono-modified protein was the main product (Tpx-MP; lanes 5–10). b, reduced Tpx, untreated (lane 2) or treated with 2 (lane 3) (corresponding to lanes 4 and 6 in a), served as controls. Oxidized Tpx either untreated (lane 4) or pretreated with compounds 1–6 (lanes 5–10) revealed the unmodified protein as main species. c, reaction of Tpx mutants with compound 2. In the absence of DTT, reaction of C40S-Tpx with MAL-PEG resulted in the mono-modified protein as main species independent of a prior treatment with compound 2 (lanes 2 and 3). In contrast, in the case of C43S-Tpx, treatment with compound 2 prevented the subsequent reaction with MAL-PEG (lanes 6 and 7). For further details see the text. In lane 1 the protein marker was loaded. Depicted are representative gels of at least three independent analyses.