Human serum biotinidase is a thiol-type enzyme.
The effects of a disulfide reducing agent and sulfhydryl blocking agents on the biotinidase activity in human serum and on the purified biotinidase have been extensively studied by using a newly developed HPLC assay method. This HPLC method directly measures the product (p-aminobenzoate, PAB), and is not interfered with by sulfhydryl-reactive agents. Further, because the substrate solution of this HPLC assay method contains only substrate (biotin 4-amidobenzoate) and phosphate buffer, accurate studies on the effects of sulfhydryl blocking reagents on the purified enzyme could be performed. Biotinidase activities in human sera (n = 83) were always enhanced by 2-mercaptoethanol (ME). The optimum concentration was found to be 1 mM. The degree of activation was variable (100 to 400% of the original) depending on the serum sample. Sulfhydryl blocking reagents such as organic mercurials were tested on fresh serum and purified enzyme. Mercuric agents were found to inhibit the activity of fresh serum and purified enzyme at 0.05 and 0.005 mM, respectively. Sulfhydryl alkylating agents, N-ethylmaleimide (NEM) and dithiobis(2-nitro)benzoic acid (DTNB), inhibited 100 and 64% of the activity of the purified enzyme at 0.1 and 1.0 mM, respectively. However, lower concentrations (less than 5 nM) of organic mercurials and mercuric ion exhibited a slight enhancement (20-30%) of the activity of the purified enzyme. These results indicate the presence of an essential sulfhydryl residue at the active center. The enzyme contains 2.5 sulfhydryls per molecule, as determined by using Ellman's assay method. Serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) did not inhibit the enzyme activity at 0.05 mM or higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Hayakawa
K
K
Division of Metabolism, National Children's Medical Research Center, Tokyo.
Oizumi
J
J
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D043523:Biotinidase; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008623:Mercaptoethanol / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000097:blood*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008623:Mercaptoethanol; D000581:Amidohydrolases; D043523:Biotinidase