Peptide deformylase enzyme assays.

Peptide deformylase activity was measured in assay buffer containing 5 mM NAD⁺, 1 U of formate dehydrogenase, and 1 mM formyl-Met-Ala-Ser peptide. A partially anaerobic environment was maintained using a glucose oxidase and catalase system by adding 20 mM d-glucose, 0.5 U of glucose oxidase, and 700 U of catalase (23). The reaction was started by adding freshly prepared cell lysates to the mixture. PDF removes the formyl group from formyl-Met-Ala-Ser to generate formate. Formate is then oxidized to CO₂ and H₂O by formate dehydrogenase, with the reduction of NAD⁺ to NADH, which is monitored by an increase in the OD₃₄₀. The change in the absorbance per minute was converted to activity (micromoles per minute) units using a molar absorption coefficient of 6,220 M⁻¹ cm⁻¹, and the data were normalized to the protein amount.

To measure the effects of hydrogen peroxide (H₂O₂) or NO· on PDF activity ex vivo, lysates were prepared from zinc-treated AS192 or AS194, and equal amounts of the clarified lysates were incubated with 100 μM H₂O₂ or 200 μM DEA/NO for 10 min at room temperature (RT) before measuring the activity.