In type 2, deiodinase (Dio2 or D2) assay, tissue homogenate is mixed with radiolabeled thyroxine and other cosubstrates. The resulting radiolabeled iodide is measured and used to determine the Dio2 activity. However, most of the protocol suggests to add 1 nM free unlabeled thyroxine (T4) in the reaction mastermix. If someone has performed this assay, can you tell me why it is used here?

Thank you.