Aging Aging

128 Barnett and Ioannides3.2. Measurement of Alkoxyresorufin Metabolism1. The reaction is carried out at 37°C. To a 3-mL fluorimetric cuvet (a 1.5-mLmicrofluorimetric cuvet may be used with appropriate adjustment of volumesgiven below) add the following reagents:1.935 mL of 0.1 M Tris-HCl buffer, pH 7.8, prewarmed to 37°C50 µL of microsomal suspension/cell homogenate*3 µL of 0.53 mM ethoxyresorufinor5 µL of 0.53 methoxyresorufinor5 µL of 1 mM pentoxyresorufin*If using cell homogenate (S9) then dicourmarol should be added to give a finalconcentration of 10 µM (substitute 10 µL of Tris buffer for 10 µL of dicoumarolstock solution).2. The cuvet is introduced into the spectrofluorometer and a baseline recorded priorto initiation of the reaction with 10 µL of NADPH solution.3. The reaction is monitored continuously until a measurable gradient is obtainedand an initial rate of reaction can be determined.4. Resorufin production from the alkoxyresorufin substrate can be calculated usingthe standard resorufin solution.5. A blank is prepared by replacing the microsomes or cell homogenate with 50 µLof Tris-buffer.6. Following the establishment of baseline fluorescence, at least three 10-µL samplesof the standard resorufin are introduced into the cuvet, noting the increase influorescence after each addition.3.3. Example Calculation1. Ten microliters of 0.1 mM resorufin caused an increase of 15.5 fluorescence units.2. Fifty microliters of sample A caused an increase of 1.2 fluorescence units per minute.3. Therefore 1 mL of sample A would cause 1000/50 × 1.2 unit increase per minuteor 24 units per minute.4. Ten microliters of 0.1 mM resorufin is equal to 1nmole of resorufin, therefore1 nmol of resorufin will cause a 15.5 unit increase in fluorescence.5. As such 1 mL of sample A produced 24/15.5 nmol resorufin per minute =1.5 nmol/min/mL.6. Having established the protein concentration in the microsomal suspension/cellhomogenate, the activity can be expressed as nmol/min/mg of protein.Many spectrofluorometers will perform these calculations directly followingthe calibration step with the resorufin standard.4. Notes1. A baseline cannot be established as the fluorescence is increasing at a steady rate.The alkoxyresorufin substrate may have become contaminated with NADPH or S9.Make up fresh substrate and ensure that a separate pipet is used for each addition.