Determination of serum biotinidase activity with biotinyl derivatives of lodotyramines 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 Km values determined for mono- and di-iodobiotinyl derivatives were 15.8 and 25.9 microM, 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 microM 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 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).
Evangelatos
S A
SA
Institute of Radioisotopes and Radiodiagnostic Products, Radioimmunochemistry Laboratory, N.C.S.R. Demokritos, Athens, Greece.
Kakabakos
S E
SE
Evangelatos
G P
GP
Ithakissios
D S
DS
D000581:Amidohydrolases / Q000097:blood*; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D043523:Biotinidase; D002700:Chloramines; D003124:Colorimetry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007700:Kinetics; D014105:Tosyl Compounds; D014439:Tyramine / Q000031:analogs & derivatives* / Q000378:metabolism
D002700:Chloramines; D007457:Iodine Radioisotopes; D014105:Tosyl Compounds; C016300:chloramine-T; D001710:Biotin; D000581:Amidohydrolases; D043523:Biotinidase; D014439:Tyramine