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

• Published in: Journal of pharmaceutical sciences 1993-12, Vol.82 (12), p.1228-1231
• Main Authors: Evangelatos, Stavros A., Kakabakos, Sotiris E., Evangelatos, Gregory P., Ithakissios, Dionyssis S.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.2600821209

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 Km 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 Ki 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).