F 2500 fluorescence spectrophotometer

A slightly modified version of a published CP assay [11 (link), 14 (link)] utilized Boc-VLK-MCA as the substrate. Briefly, each reaction contained 50 μl of 100 mM succinate-borate (pH 4.0), 10 μl of 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 29 μl distilled water, and 1 μl of 10 mM Boc-VLK-MCA dissolved in DMSO. The reaction was initiated by adding 10 μl of an enzyme solution into 100 μl of the reaction mixture at 37°C. After a 30-min incubation period, the reaction was terminated by adding 2 ml of 1% (w/v) SDS in 100 mM sodium borate (pH 9.0). The fluorescence of the mixture was measured with an F-2500 fluorescence spectrophotometer (Hitachi High-Technologies, Tokyo, Japan; emission wavelength, 460 nm; excitation wavelength, 360 nm). The specific activity was expressed as the amount (mg) of purified enzyme that catalyzed the production of 1.0 μmol of 7-amino-4-methylcoumarin per hour at 37°C. The concentration of this product was determined by comparing its fluorescence intensity to a standard curve using authentic 7-amino-4-methylcoumarin. The synthetic fluorogenic peptides used in this study are listed in S1 Table.

Mitochondrial membrane potential was analyzed as per the manufacturer’s instructions. Briefly, 100 μL/well carbocyanine dye JC-1 was added to a 96-well plate after treatment with CRA (LD₅₀ = 0.71 μM) and incubated at 37 °C under light protection conditions for 10 min; the control was not exposed to CRA. An F-2500 Fluorescence Spectrophotometer, with λex of 529 nm and λem of 590 nm (251-0090, Hitachi High-Technologies Corporation, Tokyo, Japan), was used to determine the results.