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

Abstract

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 The enzyme has been purified 4800-fold from outdated human plasma and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of (76 +/- 2) X 10(3).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)

Biotinidase deficiency: “if you have to have an inherited metabolic disease, this is the one to have”

“…3,10,11 Normal serum or plasma biotinidase is a monomeric sialylated glycoprotein with a molecular mass of 76-77 kDa. The enzyme has at least nine isoforms (four major and five minor) between pH 4.15 and 4.35 as observed by isoelectric focusing.…”

“…Glycosylation of the protein could increase its mass by 13 to 19 kDa; the molecular mass of the glycosylated enzyme is estimated to be between 70 and 76 kDa, which is consistent with that of the reported glycosylated serum enzyme. 3,10,11,13 Most of the microheterogeneity observed on isoelectric focusing results from differences in the degree of sialylation.…”

“…The serum enzyme has a pH optimum of 5-6 when biocytin or biotinyl-p-aminobenzoate (artificial substrate) is the substrate. 1,3,11 Biocytin is cleaved more readily than larger biotinyl-peptides. 3 Biotinidase apparently does not cleave biotin from intact holocarboxylases.…”

“…1,3,11 Biocytin is cleaved more readily than larger biotinyl-peptides. 3 Biotinidase apparently does not cleave biotin from intact holocarboxylases. The biotinyl-binding site of biotinidase is specific for the ureido group of the biotinyl moiety of various substrates.…”

“…1,2 As a vitamin, biotin comes from the diet either in the free, non-protein-bound form or as small biotinylated peptides bound to protein. 3 Biotin in the free form can directly enter the biotin pool and be used by the four carboxylases that use it to convert them from the inactive to the active forms. 4 The carboxylases propionyl-CoA carboxylase and β-methylcrotonylCoA carboxylase are important for protein catabolism, pyruvate carboxylase is essential for gluconeogeneis, and acetyl CoA carboxylase is the first step in fatty acid synthesis.…”