Optimized Extraction and Quantification of Myeloperoxidase Activity from Mouse Organs

Anesthetized mice were transcardially perfused with 20 mL PBS to clear the intravascular compartment of blood cells. For analysis of whole tissue MPO activity, brains were harvested and homogenized by a mechanical homogenizer (Tissuemiser, Fisher Scientific, Waltham, MA) in 500 µl CTAB buffer (50 mM cetyltrimethylammonium bromide [CTAB, Sigma] in 50 mM potassium phosphate buffer at pH = 6), sonicated, and centrifuged at 15,000 g for 20 min. The supernatant was used for protein analysis with a BCA protein assay kit (Thermo Scientific, Waltham, MA) and for MPO activity assays. For separation of extra- (ECF) and intracellular protein fractions (ICF) we modified a method initially described for mouse brains [24] (link). We washed harvested organs (kidney, brain, liver, heart, spleen, and lungs) three times in PBS and incubated them for 2 hours in extraction buffer (0.32 M sucrose [Sigma], 1 mM CaCl₂ [Sigma], 10 U/ml Heparin [APP Pharmaceuticals, Schaumburg, IL] in Hanks Balanced Salt Solution [HBSS]). Then, organs were removed from the solution and processed in the same way as for whole tissue MPO activity to obtain the ICF. The extraction buffer containing the ECF was then centrifuged at 1000 g for 5 min to pellet any cellular debris, and the supernatant underwent protein precipitation by slow mixing with 4 parts ice-cold acetone (Fisher Scientific). This was performed in order to concentrate the very dilute extracellular fraction. The acetone-protein mixture was then incubated for 1 hour at −20°C, and proteins were precipitated by centrifugation at 3500 g for 15 min at 4°C. The supernatant was discarded, and the protein pellet was air-dried and resuspended in PBS for BCA and MPO activity assays.
Optimal protein precipitation conditions for MPO were tested by using purified human MPO (1.7 mg/ml; Lee Biosolutions, St. Louis, MO). 0.24, 0.12, 0.06 and 0.03 pmol MPO were precipitated with either acetone or ammonium sulfate as previously described [25] . Recovery of MPO after precipitation was compared to unprecipitated MPO activity using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP, AAT Bioquest, Sunnyvale, CA) as described below.