U3S Activity Assay

UROPORPHYRINOGEN III SYNTHASE (U3S)

Cytosolic U3S Assay

• Mix 100 µL 20mM Tris pH 8.2, 260 µL 20mM KPi (potassium phosphate) pH 8.2 and 20 µL 0.5mg/mL rPBGD (recombinant porphobilinogen      deaminase).
• Mix well and incubate for at least 2 min at 37˚C.
• Add 20µL 2.2mM PBG (porphobilinogen) and incubate 120±2 sec at         37˚C.
• Add 20 µL sample previously diluted to 2 mg protein/mL with 20mM KPi   pH 8.2. (Use 2mg/mL BSA or bovine serum albumin as the zero       blank.)
• Incubate for 120±2 sec at 37˚C in the dark.
• Stop the reaction with 140 µL 6M HCL.
• Expose the mixture to UV light for 30 min or ambient light for 2h to oxidize all        porphyrinogen formed.
• Centrifuge at 16000xg for 10 min. Collect the supernatant.
• Quantify the porphyrins by UPLC (ultra performance liquid chromatography).

Replicates

Perform three reactions for each sample, a and b containing PBG, c with just the KPi buffer.

For each sample tube a,b,c - reagents were added at 30-sec staggered intervals, hence the 120±2 sec after the first addition of PBG.

For rhU3S protein

• dilute U3S stock to 0.5 mg/mL with buffer consisting of 10mm Tris pH7.5, 150mM NaCl, 10% glycerol and 1mM dithiothreitol.
• Use 2 µL of diluted stock in the assay above and 278µL KPi, not 260 µL.