Figure 3.

Generation of an aldehyde group in peptide 2 upon in vitro translocation of ASA into microsomes. The [³⁵S]methionine-labeled peptide 2 coeluting with unlabeled P2* (see Fig. 2B) was assayed for the presence of an aldehyde group by reaction with DNP-hydrazine. (A and B) After incubation of the peptide with the reagent, P2* (I) and its DNP-hydrazone derivative (II) were separated by RP-HPLC and identified by mass spectrometry. Based on absorbance at 214 nm, 61% of the unlabeled P2* were recovered as hydrazone. The ³⁵S-labeled P2* was converted into the DNP-hydrazone with an efficiency of 57%. (C) RP-HPLC of the peptide incubated only with the solvent of DNP-hydrazine. (D) RP-HPLC of an incubation mixture lacking the peptide, thus showing the UV-signals associated with the reagent.