DETERMINATION OF SERUM BIOTINIDASE ACTIVITY WITH BIOTINYL DERIVATIVES OF IODOTYRAMINES AS SUBSTRATES

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 mu M, 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-l values were 7.30 and 9.56 mu M for the mono- and di-iodinated substrate, respectively. Higher assay sensitivity was obtained using [I-125]biotinyl-monoiodotyramine as substrate, and the values obtained were directly related with those determined with the well-established 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).