4mu iduronic acid

Mice were transcardially perfused with ice-cold PBS, and tissues were immediately dissected, snap frozen in liquid nitrogen, and stored under −80 °C until assay. The iduronidase activity assay was performed using a protocol previously described^{11 (link),28 (link)} with minor modifications. Tissues were homogenized in ice-cold T-PER protein extraction reagent (ThermoFisher Scientific, Cat. No. 78510) with protease inhibitor (Roche, Cat. No. 4693159001) using TissueLyser II (Qiagen). Supernatant was used to quantify total protein concentration using the BCA method (Pierce, Cat. No. 23225). No more than 80 μg of total protein was used in the enzymatic reaction (100 μL of total reaction volume), which includes sodium formate buffer, pH 3.5 (130 mM), D-saccharic acid 1,4-lactone monohydrate (0.42 mg/mL, Sigma-Aldrich, Cat. No. S0375), and 4MU-iduronic acid (0.12 mM, Gold Biotechnology, Cat. No. M-570-5). The reaction was incubated under 37°C for 24 to 48 hours, and quenched with glycine buffer, pH 10.8. The fluorescence of released 4MU (excitation wavelength: 365 nm; emission wavelength: 450 nm) was detected using a fluorescence plate reader (BioTek), and compared against a standard curve generated using 4MU (Sigma-Aldrich, Cat. No. M1381). The iduronidase specific activity was calculated as 4MU released (pmole) per milligram of total protein per hour.