a, Schematic summarizing in vitro thyroid-hormone synthesis and quantification via ELISA assays. b, T 4 assay calibration curves with added T 4 , under manufacturer-recommended and modified (T 4 synthesis as performed here) conditions. c, Validation of the T 4 ELISA assay. eTG presumably contains already-reacted tyrosine side chains. rTG produces T 4 . Addition of iodide is required for the reaction to occur. Lactoperoxidase is as active as TPO, taking the reduced 20% haem content in our TPO into account. Lysozyme (some tyrosines), FtsZ from S. aureus (no tyrosines) and T 3 produce no T 4 signal. d, Mutating residues in hormonogenic site D in a version of TG that is active only in site D shows that a conserved lysine residue is not important for the reaction. Adding an extra Ser-Asp before Y1310 has no effect, but the mutation D1309S abolished activity. e, Synthesis of T 4 from tyrosine copolymers as measured by the T 4 ELISA assay. Only a polymer in which tyrosines are spaced apart and preceded by Lys-Asp produces some T 4 . Activity is lower than in a single site of TG (or MBP, compare with Fig. 3). f, T 3 assay calibration curves with added T 3 under recommended and modified (as for T 4 ) conditions. g, No substantial T 3 production was detected from iodinated rTG or eTG from goitre. In c–e, g, bar plot and error bars indicate mean and s.d., n = 3.