Purification of biotinidase from human plasma and its activity on biotinyl peptides

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Abstract
Biotinidase catalyzes the hydrolysis of N.epsilon.-biotinyllysine (biocytin) to form biotin and free lysine. The enzyme was purified 4800-fold from outdated human plasma and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a MW of (76 .+-. 2) .times. 103. The same MW was found by molecular sieve chromatography under nondenaturing conditions, indicating biotinidase is a monomer. This value is in contrast to a MW of 115,000 determined by Pispa with an impure biotinidase. The Km for biocytin was 6.2 .times. 10-6 M, and biotinidase was sensitive to phenylmethanesulfonamide and iodoacetamide in agreement with earlier studies by Knappe and coworkers who suggested that serine hydroxyl groups and SH 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. 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. For these determinations, a procedure was developed for separating the released biotin from uncleaved biotinyl peptides and apopeptides (peptides with biotin removed) by using reverse-phase high-performance liquid chromatography. Evidently,the primary substrate of biotinidase in vivo is biocytin or very short biotinyl peptides.