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[44] Biotinidase in serum and tissues
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[44] B i o t i n i d a s e i n S e r u m a n d T i s s u e s By J E A N N E
HYMES, KRISTIN FLEISCHHAUER,
and
BARRY WOLF
Introduction Biotin, a water-soluble vitamin, is the coenzyme for four carboxylases in humans: acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and/3-methylcrotonyl-CoA carboxylase.~ The carboxyl group of biotin is covalently attached through an amide bond to the e-amino group of specific lysyl residues of these carboxylases by holocarboxylase synthetase. 2 The carboxylases are subsequently degraded proteolytically to biocytin (biotinyllysine) or small biotinyl peptides. Biotinidase then cleaves these molecules, releasing biotin, and thereby recycling the vitamin? There are two major types of inherited biotin-responsive disorders: early-onset multiple carboxylase deficiency, which is due to a deficiency of holocarboxylase synthetase, and late-onset multiple carboxylase deficiency, which is due to a deficiency of biotinidase in serum and other tissues. 4
Functions Hydrolytic Activity
Biotinidase (biotin-amide amidohydrolase, EC 3.5.1.12) hydrolyzes biocytin to biotin and lysine. Biotinidase is necessary for the endogenous recycling of the vitamin and probably for the liberation of the vitamin from dietary protein-bound sources. 3 Biotinidase specifically cleaves dbiotinylamides and esters, 5~ but does not cleave biotin from intact holocarboxylases. 7`s The rate of hydrolysis of biotin from natural and synthetic biotinylated peptides decreases with increasing size of the peptide. 7 The specific requirements for the hydrolysis of biotinyl substrates by biotinidase i j. M o s s and M. D. L a n e , Adv. Enzymol. 35, 321 (1971). 2 M. D. L a n e , D. L. Y o u n g , a n d F. L y n e n , ,1. Biol. Chem. 239, 2858 (1964). 3 B. Wolf, R. E. Grief, J. R. Secor M c V o y , and G. S. H e a r d , J. lnher. Metab. Dis. 8,(1),
53 (1985). 4 L. Sweetman, J. lnher. Metab. Dis. 4, 53 (1981). 5j. Pispa, Ann. Med. Exp. Biol. Fenn. 43(5), 1 (1965). ¢~J. Knappe, W. Brommer, and K. Biederbick, Biochem. Z. 228, 599 (1963). 7 D. V. Craft, N. H. Goss, N. Chandramouli, and H. G. Wood, Biochemistry 24, 2471 (1985). s j. Chauhan and K. Dakshinamurti, J. Biol. Chem. 261, 4268 (1986).
METHODS IN ENZYMOI_OGY. VOL 279
Copyright '!': 1997 by Academic Press All righls of reproduction in any form reserved. 0076 6N79/97 $25
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have been reviewed previously. 9 Because hydrolysis of biocytin by biotinidase requires acid pH and nonphysiological (micromolar) concentrations of biocytin, it has been suggested that hydrolysis is probably not the primary role of biotinidase unless the Km is decreased by a modifierJ ° When biotinylated derivatives are used either in vivo or in vitro, and biotinidase is present, it is important to determine if these derivatives are substrates for biotinidase. For example, deferodesaminolysylbiotin used for imaging and radiotherapy is converted to biotin and desaminolysyMeferoxamine by biotinidase. 11 In addition, biotinidase cleaves biotin that is attached through a spacer arm to erythrocytes. 12
Biotinyltrans~ferase Activi(v Biotinidase is biotinylated when incubated with biocytin, but not biotin. at pH 7 to 9. L~ Biotinidase can then transfer the biotin from the biotinyl acyl-enzyme to nonspecific nucleophiles such as hydroxylamine 5 or specific acceptors including histones.~4 Biotinidase biotinyltransferase activity (i.e., biotinylation of histones) occurs at physiological pH and at physiological (nano- to picomolar) concentrations of biocytin.
Lipoamidase Activity Human serum biotinidase hydrolyzes lipoyl-p-aminobenzoate (PABA), but the Km value is high (0.6 mM) relative to that for the hydrolysis of biocytin. J5 Lipoamidase activity in serum is due to biotinidase. ~5'~ Purified biotinidase from rat serum hydrolyzes both lipoyl-PABA and lipoyllysine at a concentration of about 1 raM. 17 Therefore, it is unlikely that serum biotinidase acts as a lipoamidase in viw).
Biotin-Binding Protein Chauhan and Dakshinamurti t° reported that biotinidase is the only protein in serum that binds tritiated biotin and that the enzyme contains two biotin-binding sites (Kd = 3 nM and Kd = 59 riM). Mock and Malik] s '~ B. Wolf, J. Hymes, and G. S. Heard, Methods Enzymol. 184, 103 (1990). ~oj. C h a u h a n and K. Dakshinamurti, Biochem..l. 256, 265 (1988). 11 S. F. Rosebrough, J. Pharnmcol. Exp. Ther. 265, 4(18 (1993). 1- M. Magnani, L. Chiarantini, and U. Mancini, Biotechnol. Appl. Biochem. 20, 335 (1994). /~ j. Hymes, K. Fleischhauer. and B. Wolf, Clin. Chim. Acre 233, 39 (1995). 14 j. Hymcs, K. Fleischhauer, and B. Wolf. Biochem. Mol. Med. 56, 76 (1995). /~ C. L. Garganta and B. Wolf, Clin. Chim. Acta 189, 313 (1990). L~,L. Nilsson and B. Kagedal, Biochem. J. 291, 545 (1993). t7 L. Nilsson and B. Kagedal, Eur..L Clin. Chem. Clin. Biochem. 32, 501 (1994). ts D. Mock and M. I. Malik, Ant. J. Clin. Nutr. 56, 427 (1992).
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also using tritiated biotin, found that most of the biotin in serum is not bound to protein. H y m e s e t al. ~3 have demonstrated that peroxidaseconjugated avidin reacts with biotinidase on transblot (after sodium dodecyl sulfate-polyacrylamide gel electrophoresis) when the enzyme is incubated at p H 7 to 9 in the presence of biocytin, but not biotin. Molecular Biology The c D N A that encodes normal human serum biotinidase has been cloned from a human liver c D N A library and has been sequenced. 1~)The gene is located on human c h r o m o s o m e 3p25. 2° The c D N A sequence contains two possible A T G initiator codons (numbered as bases 1 and 60) and an open reading frame of 1629 bp, relative to the first A T G codon and the termination codon at base 1629. This c D N A encodes a protein of 543 amino acids, including 41 amino acids of a putative signal peptide sequence. Starting at nucleotide 123, the sequence that encodes the N terminus of the mature serum biotinidase is in the same reading frame with both of the A T G codons. Either sequence (the entire 41-amino acid sequence or the shorter 21-amino acid sequence) is consistent with the motif for a secretory signal peptide. Biotinidase m R N A is a low-abundance message in all tissues examined, l~)
Physical Properties Isoforms
H u m a n serum biotinidase migrates as an c~ protein on serum electrophoresis.21-23 Isoelectric focusing (IEF) of serum from normal individuals reveals at least nine isoforms, four major and five minor, between p H 4.15 and 4.35. 24 H u m a n serum biotinidase migrates to the /3 region on electrophoresis after neuraminidase treatment, which indicates that the serum enzyme is extensively sialylated. 22 Most of the microheterogeneity found on IEF is due to differences in the degree of sialylation of the enzyme, because neuraminidase treatment reduces the n u m b e r of isoforms to one i,~ H. Cole, T. R. Rcynolds, G. B. Buck, J. M. Lockycr, T. Denson, J. E. Spcncc, J. Hymes, and B. Wolf, J. Biol. Chem. 269, 6566 (1994). 2o H. Cole, S. Wcremowicz, C. C. Morton, and B. Wolf, Genomics 22, 662 (1994). 2t M. Koivusalo and J. Pispa, Acta Physiol. Scand. 58, 13 (1963). 22 D. A. Weincr and B. Wolf, Ann. N.Y. Acad. Sci. 44"/, 435 (1985). :-~ B. Wolf, G. S. Heard, L. G. Jefferson, V. K. Proud, W. E. Nance, and K. A. Weissbecker, New EngL .l. Med. 313, 16 (1985). e4 p. S. Hart, J. Hymes, and B. Wolf, Clin. C7~irn. Acta 197, 257 (1991).
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major species ( p l 5.27) and three minor species (pI 5.01, 5.14, and 5.30). 24 On preparative IEF, serum biotinidase was detected at p I 4.0 and 4.4. ~(~ The isoform at p l 4.4 may be due to a cysteine adduct of biotinidase because it occurs when cysteine, but not mercaptoethanol, is used as a stabilizer.
Molecular Mass H u m a n serum biotinidase is a single glycosylated polypeplide with a molecular mass of about 66 to 76 kDa. 7~2s T r e a t m e n t of biotinidase with N-glycanase results in a lower molecular mass species of 60 kDa, indicating that biotinidase contains about 20% carbohydrate. ~-4The c D N A for mature human serum biotinidase contains 502 amino acid residues with a molecular mass of 56,771 Da. ~ It contains six potential glycosylation sites (Asn-XThr/Ser). Assuming varying degrees of glycosylation of all six sites, the molecular mass of the glycosylated enzyme is predicted to be between 70 and 80 kDa. Biotinidase from human serum is purified to homogeneity at around 20,000-fold purification, sw-25 One study identified two proteins in serum with biotinidase activity, a 76-kDa and a 110-kDa species, and suggested that the larger species was specific for biocytin. 26 These proteins were purified 349- and 11.2-fold, respectively. Moreover, the N-terminal amino acid sequence of both of these proteins did not correspond to the N terminus of biotinidase ascertained by others or to that predicted from the e D N A nucleotide sequence of biotinidase. ~'~The same investigators have reported that biotinidase cleaves enkephalins 27 and that biotinidase in human milk is capable of synthesizing biocytin in the presence of biotin and ATP. 2~
Heat Stability Biotinidase in human serum 2'~ and partially purified from hog serum and hog liver is stable both during the enzyme assay and during preincubation for up to 30 min at 370. 5 Denaturation of the porcine enzyme occurs after incubation for 30 rain at 60 °. H u m a n serum enzyme is relatively heat stable and is denatured 60% after incubation for 15 rain at 60 ° and 100% after incubation for 15 rain at 70°. ~'s° 2~ B. Wolf. J. B. Miller, J. Hymes. J. Secor McVoy. Y. lshikana, and E. Shapira, Clin. Chim. Acla 164, 27 (1987). 2(, j. Oizumi and K. Hayakawa, Clin. Chim. Acta 215, 63 (1993). 27 j. Oizumi and K. Hayakawa, Biochim. Biophys. Acta 10"/4, 433 (1991). "~ J. Oizumi and K. Hayakawa, J. Chronmtogr. 612, 156 (1993). ~ B. Wolf, unpublished results (1986). ~ K. Hayakawa and J. Oizumi, Clin. Chim. Acta 178, 95 (1988).
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Catalytic Properties
pH Dependence Hydrolysis of biocytin by human serum is maximal at pH 4.5 to 6. s Hydrolysis of biotinyl-p-aminobenzoate ( B P A B A ) has a broader pH profile with considerable activity from pH 5 to 8 and maximal activity from p H 5.5 to 6.5. Biotinyltransferase activity of human serum biotinidase occurs at pH 7 to 9 and is undetectable below pH 7.14 A broad pH range has also been reported for biotinidase from rat plasma and liver using B P A B A as the substrate. ~7 B P A B A is hydrolyzed by biotinidase from hog liver and serum at pH 6.5 and from hog kidney at pH 6.0 at the same rate as is biocytin. 5(' Dialysis of hog serum biotinidase in acetate buffer below pH 5.0 and in potassium phosphate or Tris-malate buffers above pH 6.0 results in loss of enzyme activity. 5 Purified human serum biotinidase can be stored at 4 ° in 0.1 M phosphate buffer, pH 6.0, containing 1 m M 2-mercaptoethanol and 1 m M E D T A for 1 month without loss of activity, s
Acyl-Enzyme Formation The following is evidence for biotinylacylbiotinidase formation during catalysis: (1) biotinylhydroxamate is formed when biotinidase is incubated in the presence of high concentrations of hydroxylamine with either B P A B A or biocytin as substrateS'~; (2) biotinidase exhibits a considerably higher Kj for the same inhibitor at pH 7 than at pH 5.5 (i.e., the lack of inhibition by biotin at pH 7 may be due to slower dissociation of biotin from the acyl-enzyme, resulting in less available, inhibitable enzymeS'~); (3) biotinylated biotinidase can be detected by avidin reactivity on transblot after incubation at pH 7 to 9 in the presence of biocytin, but not biotin~3; (4) biotinylated biotinidase is not detected in the presence of high concentration of mercaptoethanol or hydroxylamine (reagents known to cleave thioesters), suggesting that when biocytin is cleaved, biotin forms an acylenzyme (thioester) with the cysteine in the active site of the enzyme; and (5) biotinidase transfers biotin to histones at pH 7 to 9 in the presence of biocytin, but not biotin. 14 Mercaptoethanol (0.1-0.2 mM) or dithiothreitol (1-2 mM) protects or stabilizes biotinidase. 5'7's'3~ Hydrolytic activity of biotinidase in human serum is enhanced by addition of mercaptoethanol. 32 This enhanced activity may be due to the protection of the cysteine in the active site or to the 31K. Hayakawa and J. Oizumi, Clin. (~71im. A c t a 168, 109 (1987). ~? K. Hayakawa and J. Oizumi, J. Biochem. 103, 773 (1988).
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increased turnover of biotinylated biotinidase that results from removal of biotin from the acyl-enzyme by mercaptoethanol, thereby allowing more substrate to be cleavedJ a
Kinetics Kinetics for hydrolysis of various related substrates by biotinidase and for its inhibition has been reviewed previously2 The following are the most important findings. The Km value for B P A B A hydrolysis by human serum biotinidase is 10 to 34/zM ~'33 and that for biocytin is 5 to 7.8/xM. s21 The Km values of B P A B A and biocytin for human plasma biotinidase are 10 and 6.2/xM, respectively] The Km value for B P A B A hydrolysis by biotinidase in crude rat liver homogenate is 15/xM. 17 The Km of biocytin for biotinyltransferase activity has not been determined precisely, but the transfer of biotin to histones can be detected at picomolar concentrations of biocytin. ~4 The degree of biotinylation of histones is greater when biocytin is the substrate than when B P A B A is used.
Inhibition Biotin is a competitive inhibitor of biotinidase at acidic pH when either biocytin or B P A B A is the substrate. At pH 7.4, biotin is not inhibitory when B P A B A is the substrate. 5 Biotinidase activity is not competitively inhibited by dl-dethiobiotim /-biotin, 1-1ysine, e-aminocaproic acid, fatty acids, d-norbiotin, d-biotin sulfone, or PABA. 5's Sulfhydryl inhibitors inactivate biotinidase. Biotinidase from hog liver and serum is inactivated completely by 0.l m M p-chloromercuribenzoate 5 and the enzymes in human plasma 7 and hog kidney ~' are inactivated completely by 10/~M p-chloromercuribenzoate. 5 Monoiodoacetate also inhibits enzyme activity, whereas N-ethylmaleimide or arsenite does not. s Partially purified biotinidases from Lactobacillus casei and Streptococcus fiwcalis are not inhibited by 0.1 m M p-chloromercuribenzoate. <~4 Evidence for the presence of a serine-hydroxyl group in or near the active site of biotinidase is equivocal. ~ Tissue Distribution a n d Cellular Localization In animals, including humans, the highest specific activity of biotinidase is in serum. 5"21 Activities are high in liver, kidney, and adrenal glands, and ~ B. Wolf, R. E. Grier, R. J. Allen, S. I. Goodman, and C. L. Kien, Clin. Chim. Acta 131, 273 (1983). 34 M. Koivusalo, C. Elorriaga, Y. Kaziro. and S. Ochoa, J. Biol. Chem. 238, 1038 (1963).
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low in brain. 5:7'35 B i o t i n i d a s e is also p r e s e n t in s e c r e t o r y cells, i n c l u d i n g fibroblasts a n d l e u k o c y t e s , and in p a n c r e a t i c juice a n d z y m o g e n granules. 3<37 B i o t i n i d a s e activity has b e e n d e t e c t e d in the u r i n e of h u m a n s . 3s B e c a u s e b i o t i n i d a s e activity in b l o o d c o r r e l a t e s p o s i t i v e l y with the conc e n t r a t i o n of a l b u m i n , b i o t i n i d a s e p r o b a b l y o r i g i n a t e s f r o m l i v e r ) ') Subcellular f r a c t i o n a t i o n studies of rat h e p a t o c y t e s suggest t h a t the e n z y m e is e n r i c h e d in t h e m i c r o s o m a l f r a c t i o n and, to a lesser extent, in the l y s o s o m a l fraction, s37 Studies of s u b c e l l u l a r fractions of rat liver confirm these resuits] 7 M i c r o s o m a l b i o t i n i d a s e is localized in the r o u g h e n d o p l a s m i c reticulum.
Biotinidase Deficiency C h i l d r e n with b i o t i n i d a s e deficiency exhibit n e u r o l o g i c a n d c u t a n e o u s a b n o r m a l i t i e s in a s s o c i a t i o n with k e t o l a c t i c acidosis, o r g a n i c aciduria, a n d m i l d h y p e r a m m o n e m i a . 4° T h e r e is c o n s i d e r a b l e v a r i a b i l i t y in the clinical f e a t u r e s of a f f e c t e d children. S y m p t o m a t i c c h i l d r e n have i m p r o v e d m a r k edly a f t e r t r e a t m e n t with 5 - 2 0 m g of oral biotin p e r day. B e c a u s e s y m p t o m s can b e p r e v e n t e d by biotin t r e a t m e n t , m a n y states h a v e i n c l u d e d s c r e e n i n g for b i o t i n i d a s e deficiency in t h e i r n e w b o r n s c r e e n i n g p r o g r a m s . B i o t i n i d a s e deficiency has b e e n d i v i d e d into p r o f o u n d e n z y m e deficiency (less t h a n 10% of m e a n n o r m a l s e r u m b i o t i n i d a s e activity) and p a r t i a l e n z y m e deficiency ( 1 0 - 3 0 % o f m e a n n o r m a l s e r u m activity). 4~ P r o f o u n d deficiency includes c h i l d r e n w h o are u n t r e a t e d a n d b e c o m e s y m p t o m a t i c and those w h o are d e t e c t e d b y n e w b o r n screening. P a r t i a l b i o t i n i d a s e deficiency includes c h i l d r e n w h o are d e t e c t e d by n e w b o r n screening. If stressed with an infection o r s t a r v a t i o n a small n u m b e r of p a r t i a l b i o t i n i d a s e - d e f i cient c h i l d r e n m a y b e c o m e s y m p t o m a t i c . I n d i v i d u a l s with p r o f o u n d b i o t i n i d a s e deficiency can be classified into nine distinct b i o c h e m i c a l p h e n o t y p e s on the basis of the p r e s e n c e of crossr e a c t i n g m a t e r i a l ( C R M ) to a n t i b o d y p r e p a r e d against h u m a n s e r u m bio3~S. F. Suchy. J. R. McVoy Secor, and B. Wolf, Neurology 35, 1510 (1985). > B. Wolf, G. S. Heard, J. S. McVoy. and H. M. Raetz, J. lnher. Metab. Dis. 7(2), 121 (1984). ~7G. S. Heard, R. E. Grier, D. L. Weiner, J. R. Secor McVoy, and B. Wolf, Ann. N. K Aead. Sci. 447, 259 (1985). ~s C. De Felice, K. Hayakawa, T. Tanaka, and E. Teremiewl, Nephron 70, 115 (1995). >) D. L. Weiner, R. E. Grier. P. Watkins, G, S. Heard, and B. WolL Am. J. Hum. Genel. 34, 56A (1983). 4o B. Wolf, in "'The Metabolic and Molecular Bases of Inherited Disease" (C. R. Scriver, A. L. Beaudet. W. S. Sly, and D. Valle, eds.), p. 3151. McGraw-Hill, New York, 1995. 41 j. R. McVoy, H. L. Levy, M. LawleL M. S. Schmidt, D. D. Eberts, P. S. Hart, D. D. PeUit, M. G. Blitzer, and B. Wolf, .I. Pedialr. 116, 78 (1990).
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tinidase. P h e n o t y p e s differ in n u m b e r of isoforms and distribution and f r e q u e n c y of the isoforms. 42 Individuals with partial biotinidase deficiency are similarly classified into six distinct biochemical p h e n o t y p e s ? 3 Biotinyltransferase activity has been d e t e r m i n e d in sera from patients with p r o f o u n d deficiency of biotinyl hydrolytic activityJ 4 Cross-reacting material to anti-biotinidase was also d e t e r m i n e d in this group. N o n e of the sera f r o m s y m p t o m a t i c children have biotinyltransferase activity, whereas 67% of the sera with C R M and n o n e of the sera without C R M from children detected by n e w b o r n screening have transferase activity. A c o m m o n 7-bp deletion and 3-bp insertion in the putative leader sequence of the biotinidase gene were d e m o n s t r a t e d in at least 1 allele of 10 of 20 s y m p t o m a t i c children with p r o f o u n d biotinidase deficiency? 4 This m u t a t i o n results in a frameshift and the p r e m a t u r e termination of the translated enzyme, resulting in p r o f o u n d biotinidase deficiency. A s s a y s of B i o t i n i d a s e H y d r o l y t i c Activity Natural and artificial substrates have been used in various assays of biotinidase activity; these include s e c o n d a r y enzymatic, 4~ radiometric, 4~4v colorimetric, ~''33and fluorometric 4~-5° measurements. S o m e m e t h o d s require derivatization 4s or c h r o m a t o g r a p h i c separation of the reaction products. 464'~ Serum and tissues, such as liver with high biotinidase activity, can be m e a s u r e d using the colorimetric assay described below. 33 Biotinidase activity can also be m e a s u r e d in cultured ceils, such as hepatocytes and fibroblasts, by this method. Biotinidase activity in tissues, such as peripheral b l o o d leukocytes and brain, requires a m o r e sensitive m e t h o d , such as the radioassay described below. 47 A h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) m e t h o d for measuring biotinidase activity using biotinyl-6-aminoquinoline as substrate has b e e n developed. 51 A n H P L C radioassay uses b i o t i n y l m o n o [ 12sI]iodotyramine and biotinyldi[~25I]iodotyramine as substrates, s2 ~2p. S. Hart, J. Hymes, and B. Wolf, Am. ,L Hum. Genet. 50, 126 (1992). 4.', p. S. Hart, J. Hymcs, and B. Wolf, Pediatr. Res. 31, 261 (1992). ~ R. J. Pomponio, T. R. Reynolds, H. Cole, G. A. Buck, and B. Wolf, Nature Genet. ll, 96 (1995). 4>D. L. Weiner, R. E. Grier, and B. Wolf, J. lnher. Metal). Dis. 8, 10l (1985). 4, L. E. Thuy, B. Ziclinska, L. Sweetman, and W. L. Nyhan, Ann. N. 1/. Acad. Sci. 447, 434 (1985). 4: B. Wolf and J. R. Secor McVoy, Clin. Chim. Acta 135, 275 (1984). ~s H. Ebrahim and K. Dakshinamurti, Anal. Biochem. 154, 282 (1986). 4~K. Hayakawa and J. Oizumi, J. Chromatogr. 383, 148 (1986). so H. Wastell, G. Dale, and K. Bartlett, Anal. Biochem. 140, 69 (1984). ~ K. Hayakawa, K. Yoshikawa, J. Oizumi, and K. Yamauchi, J. Chromatogr. 617, 29 (1993). ~-~E. Livaniou, A. K. Roboti, S. E. Kakabakos, J. Nyalala, G. 1". Evangelatos. and D. S. llhakissio, J. Chromatogr. B Biomed. Appl. 656, 215 (1994).
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The simplest and most commonly used method for measuring biotinidase activity uses B P A B A as substrate. The two methods described as follows are based on measurement of the P A B A released from B P A B A by the hydrolytic action of biotinidase. The first method is a modification of the method of Knappe et al. and Wolf et al. 33 and is usually used for the diagnosis of biotinidase deficiency. A procedure based on this method, for semiquantitatively measuring biotinidase activity in blood-soaked filter paper disks, is used to screen newborn infants for biotinidase deficiency.23'53 The second method, that of Wolf and Secor McVoy, 47 is 100-fold more sensitive than the colorimetric assay and can measure biotinidase activity in tissues, such as leukocytes and amniotic cells, that have low enzyme activities.
Colorimetric Assay of Biotinidase Activity in S e r u m Principle
The hydrolysis of B P A B A by biotinidase results in production of biotin and PABA. The primary aromatic amine, PABA, is released, diazotized, and coupled to a naphthol reagent producing purple color, which is quantitared spectrophotometrically at 546 nm. Specimen Requirements
This method is applicable to specimens that contain relatively high enzyme activity, including serum or plasma, and homogenates of liver, kidney, and adrenal glands. Serum should be separated from whole blood within 1 hr after collection and should be stored at 70 °. Long-term storage of serum samples at 20 ° is not recommended because biotinidase activity decreases with time. One to 2 ml of serum or plasma is required for testing. Heavy metals, such as copper and zinc, inhibit biotinidase activity. 5 Therefore, 1-10 m M E D T A is included in storage buffers to prevent this inhibition. Anionic detergents, such as sodium dodecyl sulfate (SDS), denature biotinidase, whereas low concentrations (0.1 to 0.5%) of nonionic detergents, such as Triton X-100 or Nonidet P-40, can be used without loss of activity. ~ Triton X-100 was included in the incubation buffer when biotinidase activity was determined by autoanalyzer. 54 ~3 G. S. Heard. B. Wolf, L. G. Jefferson, K. A. Weissbecker, W. E. Nance, Jr., J. R. Sccor McVoy, A. Napolitano, P. L. Mitchell, F. W. Lambert, and A. S. Linycar, J. Pediatr. 108, 40 (1986). 54 K. A. Weissbccker. H. D. Grucmer, G. S. Heard, and W. G. Miller. Clin. Chem. 35, 831 (1989).
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Reagents Potassium phosphate buffer (KPB; 50 raM, pH 6.0): Combine 0.70 g of K2HPO4 and 2.98 g of KH2PO4, bring to a final volume of 500 ml with H P L C water, and store at 0-5 ° Substrate buffer (0.05 m M biotinyl-PABA): Add 9.64 mg of biotin-4amidobenzoic acid (Sigma, St. Louis, MO) dissolved in 0.1-0.2 ml of 1 M sodium bicarbonate and 8.41 mg of disodium E D T A to 500 ml of H P L C water and stir until the E D T A is in solution. Store at 0-5 °
Standard solution (0.2 m M P A B A standard): Dilute a 2 m M P A B A stock solution with KPB; dissolve 13.71 mg of analytically pure P A B A (potassium salt) in 50 ml of 50 m M KPB. Aliquots of the stock can be stored indefinitely at 70 ° . The standard solution is stable at 0 - 5 ° for 1-2 months Trichloroacetic acid solution (TCA; 30%, w/v): Add 30 ml of T C A solution (6.1 N) to 70 ml of H P L C water and store at room temperature Sodium nitrite solution (0.1%, w/v): Prepare fresh daily: bring 0.1 g of sodium nitrite (crystalline) to a final volume of 100 ml with H P L C water A m m o n i u m sulfamate solution (0.5%, w/v): Bring 0.5 g of a m m o n i u m sulfamate (ACS reagent) to a final volume of 100 ml with H P L C water and store at room temperature N-1-Naphthylethylenediamine dihydrochloride solution ( N E D D ; 0.1%, w/v): This solution (0.1 g of N E D D per 100 ml of H P L C water) is light sensitive and should be stored in a dark bottle at room t e m p e r a t u r e
Calibration The standard must be included in each assay performed. Enzyme activity is calculated on the basis of the absorbance of this standard.
Quality Control Controls for biotinidase testing are not commercially available. A normal serum control is obtained from an individual ascertained to have normal biotinidase activity. A control with low biotinidase activity can be prepared by incubating serum from an individual with normal enzyme activity at 37 ° for 72 hr. This partially inactivated enzyme can then be stored at - 7 0 °.
Procedure Serum samples should be assayed for enzyme activity in duplicate and an additional sample, which does not contain substrate buffer, should be
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BIOTIN AND DERIVATIVES
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included to determine background absorbance and to detect the presence of substances that can interfere with color development. 1. Pipette 1.9 ml of substrate buffer into subject and control sample tubes and pipette 1.9 ml of KPB into subject and control background tubes. Pipette 1.9 ml of KPB into the tube used for the P A B A standard. Pipette 2 ml of KPB into a tube that is designated as the standard blank. The tubes are incubated at 37 ° for 10 min. 2. Pipette 0.1 ml of the standard, and 0.1 ml of subject and control serum into appropriate sample and background tubes and gently vortex each tube. 3. Incubate the tubes in a water bath at 37 ° for 30 min and then pipette 0.2 ml of 30% T C A into each tube. Vortex and centrifuge the tubes for 10 min at room temperature at 700 g. 4. Pipette 1.5 ml of the supernatant from each tube into clean test tubes. 5. Pipette 0.5 ml of water into each tube and vortex. 6. Pipette the following color-developing reagents: 0.2 ml of 0.1% sodium nitrite, 0.2 ml of 0.5% a m m o n i u m sulfamate, and 0.2 ml of 0.1% N E D D , consecutively into each tube at 3-min intervals and vortex after each addition. 7. Allow color development to proceed for 10 min and then measure the absorbance of the solution in each tube at 546 nm within 30 min.
Calculation of Activity Biotinidase activity is expressed as nanomoles of P A B A liberated per minute per milliliter of serum. The standard contains 20 nmol of P A B A and the activity of the subject sample is calculated using the following equation: (Abs ..... p,~ - Activity
....... d) × 20 nmol × 10 (conversion factor) (Absurd - Absb~ckg........l) × 30 min
AbSbackg
The conversion factor expresses the final activity per milliliter of serum. The incubation time is 30 min. The mean biotinidase activity in normal human serum is 7.1 nmol/min/ml serum with a range from 4.4 to 12.0 nmol/min/ml serum. Individuals with profound biotinidase deficiency have activities that are less than 10% of mean normal activity (<0.7 nmol of P A B A / m i n / m l serum); individuals with partial biotinidase deficiency have activities that are 10-30% of mean normal activity (0.7-2.1 nmol of P A B A / min/ml serum): and obligate heterozygotes for biotinidase deficiency have activities that are 30-50% of mean normal activity (2.2-5.2 nmol of P A B A / min/ml serum).
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Radioassay of Biotinidase Activity in Serum, Cells, and Tissues
Principle Biotinidase is assayed by measuring the hydrolysis of N-(d-biotinyl)[14C-carboxyl]PABA. The [14C]PABA released is separated from unhydrolyzed radioactive substrate by reacting it with avidin and precipitating the biotin-avidin and BPABA-avidin complexes with bentonite. The [~4C]PABA remaining in the supernatant is determined by a liquid scintillation counter.
Specimen Requirement Serum is diluted to 1% (v/v) with KPB. Extracts of leukocytesY fibroblasts, and other tissues are prepared by homogenizing in KPB in the presence of 0.1% (v/v) Triton X - 1 0 0 . 47 Extracts should contain 2-3 mg of protein per milliliter.
Reagents Potassium phosphate buffer (KPB; 200 raM, pH 6.0): Bring 2.39 g of KH2PO4 and 0.56 g of K2HPO4 to 100 ml with HPLC water N-(d-Biotinyl)-[~4C-carboxyl]PABA solution (2.3 mCi/mmol): Synthesize as previously described ~ and make 3.6 mM by adding cold biotinyl-PABA p-hydroxymercuribenzoate (PHMB; 2 raM): Dissolve 14.41 mg of PHMB in 0.1 ml of 1 N NaOH and dilute with HPLC water in 100 ml. PHMB inhibits enzyme activity Avidin (1%, w/v): Prepare fresh daily 10 mg of avidin per milliliter of KPB Bentonite (6.67%, w/v): Suspend 0.67 g of bentonite in 10 ml of KPB. Make fresh daily and vortex thoroughly before use
Procedu re Each sample is assayed in duplicate and an identical sample with inhibitor is prepared as a background sample. One standard with avidin and one that does not contain avidin are run per assay. Tubes are prepared as follows: Sample: Add 0.018 ml of KPB and 0.1 ml of cell supernatant (or diluted serum) to duplicate sample tubes. Background sample: Add 0.018 ml of PMBA and 0.1 ml of cell supernatant (or diluted serum) to each back-
~ B. Wolf and L. E. Rosenberg, J. Clin. Invest. 62, 931 (1978).
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BIOTIN AND DERIVATIVES
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ground tube. Standard with avidin: Add 0.118 ml of KPB to a tube. Standard without avidin: Add 0.118 ml of KPB to a tube. 1. After all of the tubes have been prepared, place the tubes in a water bath at 37 ° for 10 min. 2. Add 0.015 ml of [14C]biotinyl-PABA to each tube. 3. Incubate the tubes for 4 hr at 37 ° (2 hr for serum). 4. After the incubation, add 0.018 ml of 2 m M H M B A to all sample tubes and 0.018 ml of KPB to all background tubes. A d d 0.018 ml of 2 m M H M B A to both standard tubes. Gently vortex all tubes. 5. Add 0.025 ml of 1% avidin to all tubes, except for the standard tube without avidin. To this tube, add 0.025 ml of KPB. 6. Vortex all tubes and incubate at room temperature for 30 min. 7. Add 0.075 ml of 6.67% bentonite to all tubes. Vortex and incubate at room t e m p e r a t u r e for 15 rain. 8. Centrifuge the tubes in a microcentrifuge at approximately 2000 g for 4 min. 9. Transfer 0.15 ml of the supernatant from each tube into individual scintillation vials. Add 8 ml of scintillant to each vial. 10. Determine the radioactivity in each vial in a scintillation counter.
Calculation of" Enzyme Activity The activity in each sample is calculated according to the following formula: Activity =
(cpm ......i~1~- cpm ......p~t, la,k) × assay volume Cpmsubstrate )< time (in min) × volume counted × mg protein
in which cpm . . . . plc and cpm . . . . plcblank are the counts per minute (cpm) associated with the sample and sample blank tubes, respectively, and cpmsub~m,~c is the counts per minute per picomole of radioactive substrate (typically about 1500 cpm). Volumes (in milliliters) are of the total stopped-reaction mixture in the numerator and of the aliquot counted in the denominator. Incubation time is in minutes.
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[44] B i o t i n i d a s e i n S e r u m a n d T i s s u e s By J E A N N E
HYMES, KRISTIN FLEISCHHAUER,
and
BARRY WOLF
Introduction Biotin, a water-soluble vitamin, is the coenzyme for four carboxylases in humans: acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and/3-methylcrotonyl-CoA carboxylase.~ The carboxyl group of biotin is covalently attached through an amide bond to the e-amino group of specific lysyl residues of these carboxylases by holocarboxylase synthetase. 2 The carboxylases are subsequently degraded proteolytically to biocytin (biotinyllysine) or small biotinyl peptides. Biotinidase then cleaves these molecules, releasing biotin, and thereby recycling the vitamin? There are two major types of inherited biotin-responsive disorders: early-onset multiple carboxylase deficiency, which is due to a deficiency of holocarboxylase synthetase, and late-onset multiple carboxylase deficiency, which is due to a deficiency of biotinidase in serum and other tissues. 4
Functions Hydrolytic Activity
Biotinidase (biotin-amide amidohydrolase, EC 3.5.1.12) hydrolyzes biocytin to biotin and lysine. Biotinidase is necessary for the endogenous recycling of the vitamin and probably for the liberation of the vitamin from dietary protein-bound sources. 3 Biotinidase specifically cleaves dbiotinylamides and esters, 5~ but does not cleave biotin from intact holocarboxylases. 7`s The rate of hydrolysis of biotin from natural and synthetic biotinylated peptides decreases with increasing size of the peptide. 7 The specific requirements for the hydrolysis of biotinyl substrates by biotinidase i j. M o s s and M. D. L a n e , Adv. Enzymol. 35, 321 (1971). 2 M. D. L a n e , D. L. Y o u n g , a n d F. L y n e n , ,1. Biol. Chem. 239, 2858 (1964). 3 B. Wolf, R. E. Grief, J. R. Secor M c V o y , and G. S. H e a r d , J. lnher. Metab. Dis. 8,(1),
53 (1985). 4 L. Sweetman, J. lnher. Metab. Dis. 4, 53 (1981). 5j. Pispa, Ann. Med. Exp. Biol. Fenn. 43(5), 1 (1965). ¢~J. Knappe, W. Brommer, and K. Biederbick, Biochem. Z. 228, 599 (1963). 7 D. V. Craft, N. H. Goss, N. Chandramouli, and H. G. Wood, Biochemistry 24, 2471 (1985). s j. Chauhan and K. Dakshinamurti, J. Biol. Chem. 261, 4268 (1986).
METHODS IN ENZYMOI_OGY. VOL 279
Copyright '!': 1997 by Academic Press All righls of reproduction in any form reserved. 0076 6N79/97 $25
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have been reviewed previously. 9 Because hydrolysis of biocytin by biotinidase requires acid pH and nonphysiological (micromolar) concentrations of biocytin, it has been suggested that hydrolysis is probably not the primary role of biotinidase unless the Km is decreased by a modifierJ ° When biotinylated derivatives are used either in vivo or in vitro, and biotinidase is present, it is important to determine if these derivatives are substrates for biotinidase. For example, deferodesaminolysylbiotin used for imaging and radiotherapy is converted to biotin and desaminolysyMeferoxamine by biotinidase. 11 In addition, biotinidase cleaves biotin that is attached through a spacer arm to erythrocytes. 12
Biotinyltrans~ferase Activi(v Biotinidase is biotinylated when incubated with biocytin, but not biotin. at pH 7 to 9. L~ Biotinidase can then transfer the biotin from the biotinyl acyl-enzyme to nonspecific nucleophiles such as hydroxylamine 5 or specific acceptors including histones.~4 Biotinidase biotinyltransferase activity (i.e., biotinylation of histones) occurs at physiological pH and at physiological (nano- to picomolar) concentrations of biocytin.
Lipoamidase Activity Human serum biotinidase hydrolyzes lipoyl-p-aminobenzoate (PABA), but the Km value is high (0.6 mM) relative to that for the hydrolysis of biocytin. J5 Lipoamidase activity in serum is due to biotinidase. ~5'~ Purified biotinidase from rat serum hydrolyzes both lipoyl-PABA and lipoyllysine at a concentration of about 1 raM. 17 Therefore, it is unlikely that serum biotinidase acts as a lipoamidase in viw).
Biotin-Binding Protein Chauhan and Dakshinamurti t° reported that biotinidase is the only protein in serum that binds tritiated biotin and that the enzyme contains two biotin-binding sites (Kd = 3 nM and Kd = 59 riM). Mock and Malik] s '~ B. Wolf, J. Hymes, and G. S. Heard, Methods Enzymol. 184, 103 (1990). ~oj. C h a u h a n and K. Dakshinamurti, Biochem..l. 256, 265 (1988). 11 S. F. Rosebrough, J. Pharnmcol. Exp. Ther. 265, 4(18 (1993). 1- M. Magnani, L. Chiarantini, and U. Mancini, Biotechnol. Appl. Biochem. 20, 335 (1994). /~ j. Hymes, K. Fleischhauer. and B. Wolf, Clin. Chim. Acre 233, 39 (1995). 14 j. Hymcs, K. Fleischhauer, and B. Wolf. Biochem. Mol. Med. 56, 76 (1995). /~ C. L. Garganta and B. Wolf, Clin. Chim. Acta 189, 313 (1990). L~,L. Nilsson and B. Kagedal, Biochem. J. 291, 545 (1993). t7 L. Nilsson and B. Kagedal, Eur..L Clin. Chem. Clin. Biochem. 32, 501 (1994). ts D. Mock and M. I. Malik, Ant. J. Clin. Nutr. 56, 427 (1992).
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also using tritiated biotin, found that most of the biotin in serum is not bound to protein. H y m e s e t al. ~3 have demonstrated that peroxidaseconjugated avidin reacts with biotinidase on transblot (after sodium dodecyl sulfate-polyacrylamide gel electrophoresis) when the enzyme is incubated at p H 7 to 9 in the presence of biocytin, but not biotin. Molecular Biology The c D N A that encodes normal human serum biotinidase has been cloned from a human liver c D N A library and has been sequenced. 1~)The gene is located on human c h r o m o s o m e 3p25. 2° The c D N A sequence contains two possible A T G initiator codons (numbered as bases 1 and 60) and an open reading frame of 1629 bp, relative to the first A T G codon and the termination codon at base 1629. This c D N A encodes a protein of 543 amino acids, including 41 amino acids of a putative signal peptide sequence. Starting at nucleotide 123, the sequence that encodes the N terminus of the mature serum biotinidase is in the same reading frame with both of the A T G codons. Either sequence (the entire 41-amino acid sequence or the shorter 21-amino acid sequence) is consistent with the motif for a secretory signal peptide. Biotinidase m R N A is a low-abundance message in all tissues examined, l~)
Physical Properties Isoforms
H u m a n serum biotinidase migrates as an c~ protein on serum electrophoresis.21-23 Isoelectric focusing (IEF) of serum from normal individuals reveals at least nine isoforms, four major and five minor, between p H 4.15 and 4.35. 24 H u m a n serum biotinidase migrates to the /3 region on electrophoresis after neuraminidase treatment, which indicates that the serum enzyme is extensively sialylated. 22 Most of the microheterogeneity found on IEF is due to differences in the degree of sialylation of the enzyme, because neuraminidase treatment reduces the n u m b e r of isoforms to one i,~ H. Cole, T. R. Rcynolds, G. B. Buck, J. M. Lockycr, T. Denson, J. E. Spcncc, J. Hymes, and B. Wolf, J. Biol. Chem. 269, 6566 (1994). 2o H. Cole, S. Wcremowicz, C. C. Morton, and B. Wolf, Genomics 22, 662 (1994). 2t M. Koivusalo and J. Pispa, Acta Physiol. Scand. 58, 13 (1963). 22 D. A. Weincr and B. Wolf, Ann. N.Y. Acad. Sci. 44"/, 435 (1985). :-~ B. Wolf, G. S. Heard, L. G. Jefferson, V. K. Proud, W. E. Nance, and K. A. Weissbecker, New EngL .l. Med. 313, 16 (1985). e4 p. S. Hart, J. Hymes, and B. Wolf, Clin. C7~irn. Acta 197, 257 (1991).
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B1OTINIDASE IN SERUM AND TISSUES
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major species ( p l 5.27) and three minor species (pI 5.01, 5.14, and 5.30). 24 On preparative IEF, serum biotinidase was detected at p I 4.0 and 4.4. ~(~ The isoform at p l 4.4 may be due to a cysteine adduct of biotinidase because it occurs when cysteine, but not mercaptoethanol, is used as a stabilizer.
Molecular Mass H u m a n serum biotinidase is a single glycosylated polypeplide with a molecular mass of about 66 to 76 kDa. 7~2s T r e a t m e n t of biotinidase with N-glycanase results in a lower molecular mass species of 60 kDa, indicating that biotinidase contains about 20% carbohydrate. ~-4The c D N A for mature human serum biotinidase contains 502 amino acid residues with a molecular mass of 56,771 Da. ~ It contains six potential glycosylation sites (Asn-XThr/Ser). Assuming varying degrees of glycosylation of all six sites, the molecular mass of the glycosylated enzyme is predicted to be between 70 and 80 kDa. Biotinidase from human serum is purified to homogeneity at around 20,000-fold purification, sw-25 One study identified two proteins in serum with biotinidase activity, a 76-kDa and a 110-kDa species, and suggested that the larger species was specific for biocytin. 26 These proteins were purified 349- and 11.2-fold, respectively. Moreover, the N-terminal amino acid sequence of both of these proteins did not correspond to the N terminus of biotinidase ascertained by others or to that predicted from the e D N A nucleotide sequence of biotinidase. ~'~The same investigators have reported that biotinidase cleaves enkephalins 27 and that biotinidase in human milk is capable of synthesizing biocytin in the presence of biotin and ATP. 2~
Heat Stability Biotinidase in human serum 2'~ and partially purified from hog serum and hog liver is stable both during the enzyme assay and during preincubation for up to 30 min at 370. 5 Denaturation of the porcine enzyme occurs after incubation for 30 rain at 60 °. H u m a n serum enzyme is relatively heat stable and is denatured 60% after incubation for 15 rain at 60 ° and 100% after incubation for 15 rain at 70°. ~'s° 2~ B. Wolf. J. B. Miller, J. Hymes. J. Secor McVoy. Y. lshikana, and E. Shapira, Clin. Chim. Acla 164, 27 (1987). 2(, j. Oizumi and K. Hayakawa, Clin. Chim. Acta 215, 63 (1993). 27 j. Oizumi and K. Hayakawa, Biochim. Biophys. Acta 10"/4, 433 (1991). "~ J. Oizumi and K. Hayakawa, J. Chronmtogr. 612, 156 (1993). ~ B. Wolf, unpublished results (1986). ~ K. Hayakawa and J. Oizumi, Clin. Chim. Acta 178, 95 (1988).
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Catalytic Properties
pH Dependence Hydrolysis of biocytin by human serum is maximal at pH 4.5 to 6. s Hydrolysis of biotinyl-p-aminobenzoate ( B P A B A ) has a broader pH profile with considerable activity from pH 5 to 8 and maximal activity from p H 5.5 to 6.5. Biotinyltransferase activity of human serum biotinidase occurs at pH 7 to 9 and is undetectable below pH 7.14 A broad pH range has also been reported for biotinidase from rat plasma and liver using B P A B A as the substrate. ~7 B P A B A is hydrolyzed by biotinidase from hog liver and serum at pH 6.5 and from hog kidney at pH 6.0 at the same rate as is biocytin. 5(' Dialysis of hog serum biotinidase in acetate buffer below pH 5.0 and in potassium phosphate or Tris-malate buffers above pH 6.0 results in loss of enzyme activity. 5 Purified human serum biotinidase can be stored at 4 ° in 0.1 M phosphate buffer, pH 6.0, containing 1 m M 2-mercaptoethanol and 1 m M E D T A for 1 month without loss of activity, s
Acyl-Enzyme Formation The following is evidence for biotinylacylbiotinidase formation during catalysis: (1) biotinylhydroxamate is formed when biotinidase is incubated in the presence of high concentrations of hydroxylamine with either B P A B A or biocytin as substrateS'~; (2) biotinidase exhibits a considerably higher Kj for the same inhibitor at pH 7 than at pH 5.5 (i.e., the lack of inhibition by biotin at pH 7 may be due to slower dissociation of biotin from the acyl-enzyme, resulting in less available, inhibitable enzymeS'~); (3) biotinylated biotinidase can be detected by avidin reactivity on transblot after incubation at pH 7 to 9 in the presence of biocytin, but not biotin~3; (4) biotinylated biotinidase is not detected in the presence of high concentration of mercaptoethanol or hydroxylamine (reagents known to cleave thioesters), suggesting that when biocytin is cleaved, biotin forms an acylenzyme (thioester) with the cysteine in the active site of the enzyme; and (5) biotinidase transfers biotin to histones at pH 7 to 9 in the presence of biocytin, but not biotin. 14 Mercaptoethanol (0.1-0.2 mM) or dithiothreitol (1-2 mM) protects or stabilizes biotinidase. 5'7's'3~ Hydrolytic activity of biotinidase in human serum is enhanced by addition of mercaptoethanol. 32 This enhanced activity may be due to the protection of the cysteine in the active site or to the 31K. Hayakawa and J. Oizumi, Clin. (~71im. A c t a 168, 109 (1987). ~? K. Hayakawa and J. Oizumi, J. Biochem. 103, 773 (1988).
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increased turnover of biotinylated biotinidase that results from removal of biotin from the acyl-enzyme by mercaptoethanol, thereby allowing more substrate to be cleavedJ a
Kinetics Kinetics for hydrolysis of various related substrates by biotinidase and for its inhibition has been reviewed previously2 The following are the most important findings. The Km value for B P A B A hydrolysis by human serum biotinidase is 10 to 34/zM ~'33 and that for biocytin is 5 to 7.8/xM. s21 The Km values of B P A B A and biocytin for human plasma biotinidase are 10 and 6.2/xM, respectively] The Km value for B P A B A hydrolysis by biotinidase in crude rat liver homogenate is 15/xM. 17 The Km of biocytin for biotinyltransferase activity has not been determined precisely, but the transfer of biotin to histones can be detected at picomolar concentrations of biocytin. ~4 The degree of biotinylation of histones is greater when biocytin is the substrate than when B P A B A is used.
Inhibition Biotin is a competitive inhibitor of biotinidase at acidic pH when either biocytin or B P A B A is the substrate. At pH 7.4, biotin is not inhibitory when B P A B A is the substrate. 5 Biotinidase activity is not competitively inhibited by dl-dethiobiotim /-biotin, 1-1ysine, e-aminocaproic acid, fatty acids, d-norbiotin, d-biotin sulfone, or PABA. 5's Sulfhydryl inhibitors inactivate biotinidase. Biotinidase from hog liver and serum is inactivated completely by 0.l m M p-chloromercuribenzoate 5 and the enzymes in human plasma 7 and hog kidney ~' are inactivated completely by 10/~M p-chloromercuribenzoate. 5 Monoiodoacetate also inhibits enzyme activity, whereas N-ethylmaleimide or arsenite does not. s Partially purified biotinidases from Lactobacillus casei and Streptococcus fiwcalis are not inhibited by 0.1 m M p-chloromercuribenzoate. <~4 Evidence for the presence of a serine-hydroxyl group in or near the active site of biotinidase is equivocal. ~ Tissue Distribution a n d Cellular Localization In animals, including humans, the highest specific activity of biotinidase is in serum. 5"21 Activities are high in liver, kidney, and adrenal glands, and ~ B. Wolf, R. E. Grier, R. J. Allen, S. I. Goodman, and C. L. Kien, Clin. Chim. Acta 131, 273 (1983). 34 M. Koivusalo, C. Elorriaga, Y. Kaziro. and S. Ochoa, J. Biol. Chem. 238, 1038 (1963).
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low in brain. 5:7'35 B i o t i n i d a s e is also p r e s e n t in s e c r e t o r y cells, i n c l u d i n g fibroblasts a n d l e u k o c y t e s , and in p a n c r e a t i c juice a n d z y m o g e n granules. 3<37 B i o t i n i d a s e activity has b e e n d e t e c t e d in the u r i n e of h u m a n s . 3s B e c a u s e b i o t i n i d a s e activity in b l o o d c o r r e l a t e s p o s i t i v e l y with the conc e n t r a t i o n of a l b u m i n , b i o t i n i d a s e p r o b a b l y o r i g i n a t e s f r o m l i v e r ) ') Subcellular f r a c t i o n a t i o n studies of rat h e p a t o c y t e s suggest t h a t the e n z y m e is e n r i c h e d in t h e m i c r o s o m a l f r a c t i o n and, to a lesser extent, in the l y s o s o m a l fraction, s37 Studies of s u b c e l l u l a r fractions of rat liver confirm these resuits] 7 M i c r o s o m a l b i o t i n i d a s e is localized in the r o u g h e n d o p l a s m i c reticulum.
Biotinidase Deficiency C h i l d r e n with b i o t i n i d a s e deficiency exhibit n e u r o l o g i c a n d c u t a n e o u s a b n o r m a l i t i e s in a s s o c i a t i o n with k e t o l a c t i c acidosis, o r g a n i c aciduria, a n d m i l d h y p e r a m m o n e m i a . 4° T h e r e is c o n s i d e r a b l e v a r i a b i l i t y in the clinical f e a t u r e s of a f f e c t e d children. S y m p t o m a t i c c h i l d r e n have i m p r o v e d m a r k edly a f t e r t r e a t m e n t with 5 - 2 0 m g of oral biotin p e r day. B e c a u s e s y m p t o m s can b e p r e v e n t e d by biotin t r e a t m e n t , m a n y states h a v e i n c l u d e d s c r e e n i n g for b i o t i n i d a s e deficiency in t h e i r n e w b o r n s c r e e n i n g p r o g r a m s . B i o t i n i d a s e deficiency has b e e n d i v i d e d into p r o f o u n d e n z y m e deficiency (less t h a n 10% of m e a n n o r m a l s e r u m b i o t i n i d a s e activity) and p a r t i a l e n z y m e deficiency ( 1 0 - 3 0 % o f m e a n n o r m a l s e r u m activity). 4~ P r o f o u n d deficiency includes c h i l d r e n w h o are u n t r e a t e d a n d b e c o m e s y m p t o m a t i c and those w h o are d e t e c t e d b y n e w b o r n screening. P a r t i a l b i o t i n i d a s e deficiency includes c h i l d r e n w h o are d e t e c t e d by n e w b o r n screening. If stressed with an infection o r s t a r v a t i o n a small n u m b e r of p a r t i a l b i o t i n i d a s e - d e f i cient c h i l d r e n m a y b e c o m e s y m p t o m a t i c . I n d i v i d u a l s with p r o f o u n d b i o t i n i d a s e deficiency can be classified into nine distinct b i o c h e m i c a l p h e n o t y p e s on the basis of the p r e s e n c e of crossr e a c t i n g m a t e r i a l ( C R M ) to a n t i b o d y p r e p a r e d against h u m a n s e r u m bio3~S. F. Suchy. J. R. McVoy Secor, and B. Wolf, Neurology 35, 1510 (1985). > B. Wolf, G. S. Heard, J. S. McVoy. and H. M. Raetz, J. lnher. Metab. Dis. 7(2), 121 (1984). ~7G. S. Heard, R. E. Grier, D. L. Weiner, J. R. Secor McVoy, and B. Wolf, Ann. N. K Aead. Sci. 447, 259 (1985). ~s C. De Felice, K. Hayakawa, T. Tanaka, and E. Teremiewl, Nephron 70, 115 (1995). >) D. L. Weiner, R. E. Grier. P. Watkins, G, S. Heard, and B. WolL Am. J. Hum. Genel. 34, 56A (1983). 4o B. Wolf, in "'The Metabolic and Molecular Bases of Inherited Disease" (C. R. Scriver, A. L. Beaudet. W. S. Sly, and D. Valle, eds.), p. 3151. McGraw-Hill, New York, 1995. 41 j. R. McVoy, H. L. Levy, M. LawleL M. S. Schmidt, D. D. Eberts, P. S. Hart, D. D. PeUit, M. G. Blitzer, and B. Wolf, .I. Pedialr. 116, 78 (1990).
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tinidase. P h e n o t y p e s differ in n u m b e r of isoforms and distribution and f r e q u e n c y of the isoforms. 42 Individuals with partial biotinidase deficiency are similarly classified into six distinct biochemical p h e n o t y p e s ? 3 Biotinyltransferase activity has been d e t e r m i n e d in sera from patients with p r o f o u n d deficiency of biotinyl hydrolytic activityJ 4 Cross-reacting material to anti-biotinidase was also d e t e r m i n e d in this group. N o n e of the sera f r o m s y m p t o m a t i c children have biotinyltransferase activity, whereas 67% of the sera with C R M and n o n e of the sera without C R M from children detected by n e w b o r n screening have transferase activity. A c o m m o n 7-bp deletion and 3-bp insertion in the putative leader sequence of the biotinidase gene were d e m o n s t r a t e d in at least 1 allele of 10 of 20 s y m p t o m a t i c children with p r o f o u n d biotinidase deficiency? 4 This m u t a t i o n results in a frameshift and the p r e m a t u r e termination of the translated enzyme, resulting in p r o f o u n d biotinidase deficiency. A s s a y s of B i o t i n i d a s e H y d r o l y t i c Activity Natural and artificial substrates have been used in various assays of biotinidase activity; these include s e c o n d a r y enzymatic, 4~ radiometric, 4~4v colorimetric, ~''33and fluorometric 4~-5° measurements. S o m e m e t h o d s require derivatization 4s or c h r o m a t o g r a p h i c separation of the reaction products. 464'~ Serum and tissues, such as liver with high biotinidase activity, can be m e a s u r e d using the colorimetric assay described below. 33 Biotinidase activity can also be m e a s u r e d in cultured ceils, such as hepatocytes and fibroblasts, by this method. Biotinidase activity in tissues, such as peripheral b l o o d leukocytes and brain, requires a m o r e sensitive m e t h o d , such as the radioassay described below. 47 A h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) m e t h o d for measuring biotinidase activity using biotinyl-6-aminoquinoline as substrate has b e e n developed. 51 A n H P L C radioassay uses b i o t i n y l m o n o [ 12sI]iodotyramine and biotinyldi[~25I]iodotyramine as substrates, s2 ~2p. S. Hart, J. Hymes, and B. Wolf, Am. ,L Hum. Genet. 50, 126 (1992). 4.', p. S. Hart, J. Hymcs, and B. Wolf, Pediatr. Res. 31, 261 (1992). ~ R. J. Pomponio, T. R. Reynolds, H. Cole, G. A. Buck, and B. Wolf, Nature Genet. ll, 96 (1995). 4>D. L. Weiner, R. E. Grier, and B. Wolf, J. lnher. Metal). Dis. 8, 10l (1985). 4, L. E. Thuy, B. Ziclinska, L. Sweetman, and W. L. Nyhan, Ann. N. 1/. Acad. Sci. 447, 434 (1985). 4: B. Wolf and J. R. Secor McVoy, Clin. Chim. Acta 135, 275 (1984). ~s H. Ebrahim and K. Dakshinamurti, Anal. Biochem. 154, 282 (1986). 4~K. Hayakawa and J. Oizumi, J. Chromatogr. 383, 148 (1986). so H. Wastell, G. Dale, and K. Bartlett, Anal. Biochem. 140, 69 (1984). ~ K. Hayakawa, K. Yoshikawa, J. Oizumi, and K. Yamauchi, J. Chromatogr. 617, 29 (1993). ~-~E. Livaniou, A. K. Roboti, S. E. Kakabakos, J. Nyalala, G. 1". Evangelatos. and D. S. llhakissio, J. Chromatogr. B Biomed. Appl. 656, 215 (1994).
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The simplest and most commonly used method for measuring biotinidase activity uses B P A B A as substrate. The two methods described as follows are based on measurement of the P A B A released from B P A B A by the hydrolytic action of biotinidase. The first method is a modification of the method of Knappe et al. and Wolf et al. 33 and is usually used for the diagnosis of biotinidase deficiency. A procedure based on this method, for semiquantitatively measuring biotinidase activity in blood-soaked filter paper disks, is used to screen newborn infants for biotinidase deficiency.23'53 The second method, that of Wolf and Secor McVoy, 47 is 100-fold more sensitive than the colorimetric assay and can measure biotinidase activity in tissues, such as leukocytes and amniotic cells, that have low enzyme activities.
Colorimetric Assay of Biotinidase Activity in S e r u m Principle
The hydrolysis of B P A B A by biotinidase results in production of biotin and PABA. The primary aromatic amine, PABA, is released, diazotized, and coupled to a naphthol reagent producing purple color, which is quantitared spectrophotometrically at 546 nm. Specimen Requirements
This method is applicable to specimens that contain relatively high enzyme activity, including serum or plasma, and homogenates of liver, kidney, and adrenal glands. Serum should be separated from whole blood within 1 hr after collection and should be stored at 70 °. Long-term storage of serum samples at 20 ° is not recommended because biotinidase activity decreases with time. One to 2 ml of serum or plasma is required for testing. Heavy metals, such as copper and zinc, inhibit biotinidase activity. 5 Therefore, 1-10 m M E D T A is included in storage buffers to prevent this inhibition. Anionic detergents, such as sodium dodecyl sulfate (SDS), denature biotinidase, whereas low concentrations (0.1 to 0.5%) of nonionic detergents, such as Triton X-100 or Nonidet P-40, can be used without loss of activity. ~ Triton X-100 was included in the incubation buffer when biotinidase activity was determined by autoanalyzer. 54 ~3 G. S. Heard. B. Wolf, L. G. Jefferson, K. A. Weissbecker, W. E. Nance, Jr., J. R. Sccor McVoy, A. Napolitano, P. L. Mitchell, F. W. Lambert, and A. S. Linycar, J. Pediatr. 108, 40 (1986). 54 K. A. Weissbccker. H. D. Grucmer, G. S. Heard, and W. G. Miller. Clin. Chem. 35, 831 (1989).
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Reagents Potassium phosphate buffer (KPB; 50 raM, pH 6.0): Combine 0.70 g of K2HPO4 and 2.98 g of KH2PO4, bring to a final volume of 500 ml with H P L C water, and store at 0-5 ° Substrate buffer (0.05 m M biotinyl-PABA): Add 9.64 mg of biotin-4amidobenzoic acid (Sigma, St. Louis, MO) dissolved in 0.1-0.2 ml of 1 M sodium bicarbonate and 8.41 mg of disodium E D T A to 500 ml of H P L C water and stir until the E D T A is in solution. Store at 0-5 °
Standard solution (0.2 m M P A B A standard): Dilute a 2 m M P A B A stock solution with KPB; dissolve 13.71 mg of analytically pure P A B A (potassium salt) in 50 ml of 50 m M KPB. Aliquots of the stock can be stored indefinitely at 70 ° . The standard solution is stable at 0 - 5 ° for 1-2 months Trichloroacetic acid solution (TCA; 30%, w/v): Add 30 ml of T C A solution (6.1 N) to 70 ml of H P L C water and store at room temperature Sodium nitrite solution (0.1%, w/v): Prepare fresh daily: bring 0.1 g of sodium nitrite (crystalline) to a final volume of 100 ml with H P L C water A m m o n i u m sulfamate solution (0.5%, w/v): Bring 0.5 g of a m m o n i u m sulfamate (ACS reagent) to a final volume of 100 ml with H P L C water and store at room temperature N-1-Naphthylethylenediamine dihydrochloride solution ( N E D D ; 0.1%, w/v): This solution (0.1 g of N E D D per 100 ml of H P L C water) is light sensitive and should be stored in a dark bottle at room t e m p e r a t u r e
Calibration The standard must be included in each assay performed. Enzyme activity is calculated on the basis of the absorbance of this standard.
Quality Control Controls for biotinidase testing are not commercially available. A normal serum control is obtained from an individual ascertained to have normal biotinidase activity. A control with low biotinidase activity can be prepared by incubating serum from an individual with normal enzyme activity at 37 ° for 72 hr. This partially inactivated enzyme can then be stored at - 7 0 °.
Procedure Serum samples should be assayed for enzyme activity in duplicate and an additional sample, which does not contain substrate buffer, should be
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included to determine background absorbance and to detect the presence of substances that can interfere with color development. 1. Pipette 1.9 ml of substrate buffer into subject and control sample tubes and pipette 1.9 ml of KPB into subject and control background tubes. Pipette 1.9 ml of KPB into the tube used for the P A B A standard. Pipette 2 ml of KPB into a tube that is designated as the standard blank. The tubes are incubated at 37 ° for 10 min. 2. Pipette 0.1 ml of the standard, and 0.1 ml of subject and control serum into appropriate sample and background tubes and gently vortex each tube. 3. Incubate the tubes in a water bath at 37 ° for 30 min and then pipette 0.2 ml of 30% T C A into each tube. Vortex and centrifuge the tubes for 10 min at room temperature at 700 g. 4. Pipette 1.5 ml of the supernatant from each tube into clean test tubes. 5. Pipette 0.5 ml of water into each tube and vortex. 6. Pipette the following color-developing reagents: 0.2 ml of 0.1% sodium nitrite, 0.2 ml of 0.5% a m m o n i u m sulfamate, and 0.2 ml of 0.1% N E D D , consecutively into each tube at 3-min intervals and vortex after each addition. 7. Allow color development to proceed for 10 min and then measure the absorbance of the solution in each tube at 546 nm within 30 min.
Calculation of Activity Biotinidase activity is expressed as nanomoles of P A B A liberated per minute per milliliter of serum. The standard contains 20 nmol of P A B A and the activity of the subject sample is calculated using the following equation: (Abs ..... p,~ - Activity
....... d) × 20 nmol × 10 (conversion factor) (Absurd - Absb~ckg........l) × 30 min
AbSbackg
The conversion factor expresses the final activity per milliliter of serum. The incubation time is 30 min. The mean biotinidase activity in normal human serum is 7.1 nmol/min/ml serum with a range from 4.4 to 12.0 nmol/min/ml serum. Individuals with profound biotinidase deficiency have activities that are less than 10% of mean normal activity (<0.7 nmol of P A B A / m i n / m l serum); individuals with partial biotinidase deficiency have activities that are 10-30% of mean normal activity (0.7-2.1 nmol of P A B A / min/ml serum): and obligate heterozygotes for biotinidase deficiency have activities that are 30-50% of mean normal activity (2.2-5.2 nmol of P A B A / min/ml serum).
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Radioassay of Biotinidase Activity in Serum, Cells, and Tissues
Principle Biotinidase is assayed by measuring the hydrolysis of N-(d-biotinyl)[14C-carboxyl]PABA. The [14C]PABA released is separated from unhydrolyzed radioactive substrate by reacting it with avidin and precipitating the biotin-avidin and BPABA-avidin complexes with bentonite. The [~4C]PABA remaining in the supernatant is determined by a liquid scintillation counter.
Specimen Requirement Serum is diluted to 1% (v/v) with KPB. Extracts of leukocytesY fibroblasts, and other tissues are prepared by homogenizing in KPB in the presence of 0.1% (v/v) Triton X - 1 0 0 . 47 Extracts should contain 2-3 mg of protein per milliliter.
Reagents Potassium phosphate buffer (KPB; 200 raM, pH 6.0): Bring 2.39 g of KH2PO4 and 0.56 g of K2HPO4 to 100 ml with HPLC water N-(d-Biotinyl)-[~4C-carboxyl]PABA solution (2.3 mCi/mmol): Synthesize as previously described ~ and make 3.6 mM by adding cold biotinyl-PABA p-hydroxymercuribenzoate (PHMB; 2 raM): Dissolve 14.41 mg of PHMB in 0.1 ml of 1 N NaOH and dilute with HPLC water in 100 ml. PHMB inhibits enzyme activity Avidin (1%, w/v): Prepare fresh daily 10 mg of avidin per milliliter of KPB Bentonite (6.67%, w/v): Suspend 0.67 g of bentonite in 10 ml of KPB. Make fresh daily and vortex thoroughly before use
Procedu re Each sample is assayed in duplicate and an identical sample with inhibitor is prepared as a background sample. One standard with avidin and one that does not contain avidin are run per assay. Tubes are prepared as follows: Sample: Add 0.018 ml of KPB and 0.1 ml of cell supernatant (or diluted serum) to duplicate sample tubes. Background sample: Add 0.018 ml of PMBA and 0.1 ml of cell supernatant (or diluted serum) to each back-
~ B. Wolf and L. E. Rosenberg, J. Clin. Invest. 62, 931 (1978).
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ground tube. Standard with avidin: Add 0.118 ml of KPB to a tube. Standard without avidin: Add 0.118 ml of KPB to a tube. 1. After all of the tubes have been prepared, place the tubes in a water bath at 37 ° for 10 min. 2. Add 0.015 ml of [14C]biotinyl-PABA to each tube. 3. Incubate the tubes for 4 hr at 37 ° (2 hr for serum). 4. After the incubation, add 0.018 ml of 2 m M H M B A to all sample tubes and 0.018 ml of KPB to all background tubes. A d d 0.018 ml of 2 m M H M B A to both standard tubes. Gently vortex all tubes. 5. Add 0.025 ml of 1% avidin to all tubes, except for the standard tube without avidin. To this tube, add 0.025 ml of KPB. 6. Vortex all tubes and incubate at room temperature for 30 min. 7. Add 0.075 ml of 6.67% bentonite to all tubes. Vortex and incubate at room t e m p e r a t u r e for 15 rain. 8. Centrifuge the tubes in a microcentrifuge at approximately 2000 g for 4 min. 9. Transfer 0.15 ml of the supernatant from each tube into individual scintillation vials. Add 8 ml of scintillant to each vial. 10. Determine the radioactivity in each vial in a scintillation counter.
Calculation of" Enzyme Activity The activity in each sample is calculated according to the following formula: Activity =
(cpm ......i~1~- cpm ......p~t, la,k) × assay volume Cpmsubstrate )< time (in min) × volume counted × mg protein
in which cpm . . . . plc and cpm . . . . plcblank are the counts per minute (cpm) associated with the sample and sample blank tubes, respectively, and cpmsub~m,~c is the counts per minute per picomole of radioactive substrate (typically about 1500 cpm). Volumes (in milliliters) are of the total stopped-reaction mixture in the numerator and of the aliquot counted in the denominator. Incubation time is in minutes.