Purification of biotinidase from human plasma and its activity on biotinyl peptides.
Biotinidase catalyzes the hydrolysis of N epsilon-biotinyllysine (biocytin) to form biotin and free lysine. The enzyme has been purified 4800-fold from outdated human plasma and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of (76 +/- 2) X 10(3). The same molecular weight was found by molecular sieve chromatography under nondenaturing conditions, indicating biotinidase is a monomer. This value is in contrast to a molecular weight of 115 000 determined by Pispa [Pispa, J. (1965) Ann. Med. Exp. Biol. Fenn., Suppl. 5, 5-39] with an impure biotinidase. The Km for biocytin was 6.2 X 10(-6) M, and biotinidase was found to be sensitive to phenylmethanesulfonamide and iodoacetamide in agreement with earlier studies by Knappe and co-workers [Knappe, J., Brümmer, W., & Bierderbick, K. (1963) Biochem. Z. 338, 599-613], who suggested that serine hydroxyl groups and sulfhydryl groups are essential for enzymatic activity. The specificity of biotinidase was examined by using synthetic and natural biotinyl peptides isolated by specific proteolytic cleavage of the biotinyl subunit of transcarboxylase. It was found that the rate of hydrolysis of biocytin was 83-fold higher than that found for biotin-containing peptides 5-13 residues in length. Removal of methionine from either side of the conserved region around the biocytin did not greatly alter the rate of cleavage. Increasing the peptide to 65-123 residues in length decreased the rate 1200-fold compared to that of biocytin.(ABSTRACT TRUNCATED AT 250 WORDS)
D000581:Amidohydrolases / Q000097:blood* / Q000302:isolation & purification; D000818:Animals; D043523:Biotinidase; D001800:Blood Specimen Collection; D002250:Carbon Radioisotopes; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives; D008970:Molecular Weight; D013379:Substrate Specificity; D013552:Swine; D014316:Tritium
D002250:Carbon Radioisotopes; D014316:Tritium; D000581:Amidohydrolases; D043523:Biotinidase; C013411:biocytin; D008239:Lysine