Determination of serum biotinidase activity with biotinyl derivatives of iodotyramines as substrates

Abstract

We synthesized biotinylated mono- and di-iodotyramine and their radioactive counterparts and used these substances as substrates to estimate serum biotinidase activity in a radioassay system. The K_m values determined for mono- and di-iodobiotinyl derivatives were 15.8 and 25.9 μM, respectively, whereas, the maximum velocities of the enzymatic reaction were 27.0 and 8.7 nmol · min⁻¹ · mL⁻¹, respectively. Both substrates competed with biocytin for the same active site of the enzyme and the K_i values were 7.30 and 9.56 μM for the mono- and di-iodinated substrate, respectively. Higher assay sensitivity was obtained using [¹²⁵I]biotinyl-monoiodotyramine as substrate, and the values obtained were directly related with those determined with the wellestablished colorimetric method (r = 0.9377, n = 31). However, for routine use, the assay may be accomplished by diluting the radiotracer with biocytin instead of its “cold” counterpart, because it is a commercially available reagent. The values obtained in this case were very well correlated with those determined by the colorimetric assay as well (r = 0.9289, n = 31).