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Thermostability of human serum biotinidase activity
95
Clinica Chimica Acta, 178 (1988) 95-100 Elsevier
CCA 04307
Short communication
Thermostability
of human serum biotinidase activity Kou Hayakawa and Jun Oizumi
Naiional Children’s Medical Research Center, Diuisron of Metabolism,
Tokyo (Japan)
(Received 19 April 1988; revision received 14 July 1988, accepted 12 August 1988)
Key words: Biotinidase; Thermal inactivation; HPLC assay
Introduction Each enzyme has its own characteristic thermal stability at the physiological temperature range (20-37 o C) and at a high temperature range (50-60 ’ C). Knowledge of the thermal stability of the enzyme is essential for practical purposes, i.e. manipulation or handling. Thermal stability at the physiological temperature range is essential for studying the effects of endo- or exo-type glycosidase or protease on the enzyme activity. Thermostability must also be known at high temperatures for utilizing in biotinidase purification [1,2] or in stopping the enzyme reaction [3]. Purified (21200-fold) human serum biotinidase lost 60% of its original activity after heating at 60 o C for 15 min, as recently reported by Chauhan and Dakshinamurti [4] using Knappe’s calorimetric method. Partially purified hog serum biotinidase (1910-fold) completely lost activity after heating at 60°C for 30 min as reported by Pispa [2]. Bacterial biotinidase of Streptococcu.s fuecafis (strain lOC1) [3] is reportedly heat labile; 92% of the original activity was lost after treatment at 60 o C for 10 min. In this report, we attempted to clarify these conflicting results in a systematic way by a newly developed HPLC biotinidase assay method [5]. We found that biotinidase activity in human native serum was completely inactivated by heat treatment at 60 o C for 15 min. However, simple dilution of the fresh serum with a neutral phosphate buffer or dialysis against the phosphate buffer increased the heat stability of biotinidase activity in fresh serum; 27% and 50% of enzyme activity, respectively, remained after heat treatment at 60 o C for 15 min. Purified human serum biotinidase was relatively heat resistant; 40% of enzyme activity remained after heat treatment at 60 “C for 15 min. This finding is in accordance with the result of Chauhan and Dakshinamurti [4].
Correspondence to: K. Hayakawa, National Children’s Medical Research Center, Division of Metabolism, 3-35-31 Taishido, Setagaya-Ku, Tokyo 154, Japan.
0009-8981/88/$03.50
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
96
Materials and methods Chemicals and reagents Biotinyl 4-aminobenzoate was purchased from Sigma Chemical Co., St. Louis, MO, USA. 2-Mercaptoethanol was from Kanto Chemical Co., Tokyo, Japan. DEAE-Sephacel, Sephacryl S-300 were from Pharmacia Fine Chemical Co., Uppsala, Sweden, Affi-Gel Blue was from Bio-Rad Laboratories, Richmond, CA. Nonidet P-40 was from Nakarai Chem., Kyoto, Japan. Methanol (HPLC grade) was from Wako Pure Chemical Co., Osaka, Japan. Fresh Serum specimens Hospital, Tokyo, Japan.
serum
was kindly
supplied
by the National
Children’s
Purification of serum biotinidase Human serum biotinidase was purified essentially according to the method described recently by Craft et al. [6] and Chauhan and Dakshinamurti [4], using 1 liter of human serum, except for the additional step of isoelectric focusing (IEF) separation. This procedure was published recently by us [7]. The enzyme fraction after Sephacryl S-300 (1200-fold) was further processed by an Affi-Gel Blue affinity column and isoelectric focusing (IEF) gel electrophoresis. The flow-through fraction from the Affi-Gel Blue column (0.05 mol/l sodium phosphate buffer; P-buffer, at pH 7.0) contained biotinidase activity. The enzyme fraction was further purified by the IEF-PAGE (pH 2.5-6.5, containing 1% Nonidet P-40) using the LKB Ultrophor thin-layer-type IEF-PAGE system. The summary of the purification is presented in Table I. The final specific activity was increased by a factor of 30000 (serum; 0.133 nmol/min per mg, final enzyme fraction; 3!900 nmol/min/mg). Biotinidase assay Biotinidase activity was assayed either by the method of Knappe et al. [l] or by our own [5]. The HPLC method measures the product p-aminobenzoate (PAB) directly by its intrinsic fluorescence (Ex. 276 nm, Em. 340
TABLE
I
Summary of purification Step
Fraction
of human serum biotinidase Volume
Protein
(ml)
(mg)
1000 335
67400 22759
100
237
35
50
32
20
Purification (-fold)
(W)
8 964 8785
1 3
100 98
8722
277
97
171
8498
1286
94
334
6613
2511
74
3900
1731
30000
19
Spec act (nmol/min
Total activity (nmoI/min)
per mg) Serum Ammonium sulfate DEAESephacel Sephacryl s-300 Affi-Gel Blue IEF-PAGE
12
0.44
0.133 0.386 36.8
Yield
91
nm). Knappe’s purification.
method
was
mainly
used
as a qualitative
measure
during
the
Total protein concentration was determined by the method Protein measurement of Lowry et al. [8], using bovine serum albumin as a standard protein. was assessed by incubating 0.1 ml of enzyme Heat stability test Thermostability solution (fresh serum and purified enzyme) at a temperature fixed at 50 or 60 ’ C for 0, 5, and 15 min in a water bath. Following incubation, an aliquot (0.02 ml) was pipetted into 0.08 ml of substrate solution (0.1 mg/ml of biotinyl 4-aminobenzoate in 0.1 mol/l P-buffer, pH 6.4). The reaction mixture was incubated at 37°C for 30 min. After stopping the reaction by adding 0.20 ml of methanol, the mixture was centrifuged at 1500 X g for 5 min to precipitate the enzyme. A portion (0.01 ml) of the clear supematant was injected into the HPLC system, and the liberated product (PAB) was measured [5]. The HPLC system used was Waters Model 600 combined with a fluorimetric detector (Hitachi F-3000). Results Freshly collected human sera (0.1 ml) were incubated at 50 o C and 60 o C for 2, 5, 10, and 15 mm in a water bath with shaking. After heating, serum was assayed for biotinidase activity, and residual enzyme activity was illustrated (Fig. 1). Native serum biotinidase activity was inactivated 66% and 100% after heat treatment of 50°C and 60°C for 15 min, respectively. Five sera from different individuals (biotinidase activity; 8.08, 6.81, 4.69, 1.44, 1.06 nmol/min per ml) were also tested,
0
I 5 Incubation
60°C IO Time
15 (min
1
Fig. 1. Effect of heat pretreatment on biotinidase activity in native serum. Serum sample of spec act of 0.133 nmol/min per mg (100% activity) was heated, and determined the remaining activity at 37 o C as described in ‘Materials and Methods’. Each point is the average of triplicate measurements. SD (unbiased form) was calculated and indicated.
98
and all five specimens completely lost the activity after heat treatment at 60°C for 15 min. These results indicate that biotinidase activity in native human serum is relatively heat labile. Then, serum was two-fold diluted with 0.1 mol/l P-buffer (pH 6.0) and treated at 60°C for 15 min. It was found that 73% of the biotinidase activity was lost. A simple dilution of the serum by a neutral buffer was found to slightly increase the heat stability of the enzyme activity from 0% residual activity (nondilute serum) to 27% residual activity (2-fold diluted serum). As already indicated by Chauhan and Dakshinamurti [4], human serum biotinidase contains a considerable amount of cysteine residues (3.17 residues/100 residues). Also, the change in electrophoretical pattern with the addition of 2mercaptoethanol suggests the presence of interstructural disulfide bonds, because of the molecular weight change (3000 daltons) of the enzyme [4]. In general, disulfide bonds may play an important role in heat stability, as pointed out by Baldwin and Eisenberg [9]. In serum, many small molecular weight sulfhydryl active compounds are present, which might interfere with heat stability of the enzyme. We extensively dialyzed the serum against 0.1 mol/l P-buffer (pH 6.0) at 4°C overnight in order to eliminate the compounds which may affect the disulfide bond. Dialysis itself did not affect the specific activity of the enzyme at all. However, dialyzed serum exhibited only 50% inactivation by heating at 60 o C for 15 min, whereas native serum enzyme was 100% inactivated by the same procedure. Dialysis of the serum induced the apparent heat resistance against at this temperature. Since the condition of the SH group may affect the heat stability of the enzyme, we dialyzed serum with 1 mmol/l of 2-mercaptoethanol. The reagent had no apparent effect on heat stability. A summary of these experiments is presented in Table II. We purified human serum biotinidase by a factor of 30000 using the procedure described in ‘Materials and Methods’. This purified enzyme was tested for heat stability. The results are illustrated in Fig. 2. The heat stability on this enzyme is
TABLE
II
Residual activity after heat treatment of 60’ C for 15 min a Serum specimen
Residual act (8)
Fresh serum Diluted serum with P-buffer b Dialyzed serum without 2-ME c Dialyzed serum with 2-ME ’
o+o 27+3 5016 51+8
Procedure for heat treatment and enzyme assay method are as described in ‘Materials and Methods’. Specific activity of fresh serum (0.133 nmoI/min per mg) was taken as 100%. Each values are average of 5 determinations (with SD). Fresh serum (0.133 nmol/min per mg) was diluted two-fold with 0.1 mol/l sodium phosphate buffer (pH 6.0) and assayed as above. Fresh serum (10 ml) was dialyzed against 5 1of 0.1 moI/I sodium phosphate buffer (pH 6.0) at 4O C for 15 h in the presence or absence of 1 mmol/l 2-mercaptoethanol (2-ME), and assayed as above. After dialysis, no change in specific activity was detected in the absence of 2-ME, i.e. 0.133 nmol/min per mg. However, in the presence of 2-ME, a two-fold increase in specific activity was observed (0.266 nmol/min per mg). Thus, these specific activities were taken as 100%.
99
Incubation
Time
t min )
Fig. 2. Heat stability of purified enzyme. The enzyme was purified to 30000-fold (3900 nmol/min per mg) as described in ‘Materials and Methods’. Enzyme activity without heating (3900 nmol/min per mg) was considered as lOO$. Each point is the average of triplicate measurements. SD (unbiased form) calculated and indicated. Other conditions are as described in ‘Materials and Methods’.
comparable to the result of Chauhan and Dakshinamurti [4]; 40% of residual activity was demonstrated using the 30000-fold purified enzyme. Discussion Because the thermal unfolding or denaturation of proteins usually occurs between 50° C and 60 o C at neutral pH [9], and since Koivusalo et al. [3] and Chauhan and Dakshinamurti [4] described the characteristics of thermostability of biotinidase at 50 o C and 60 o C for 10 min and 15 min, respectively, we also tested at 50°C and 60°C. We found a complete inactivation of biotinidase activity in fresh serum at 60°C for 15 min. Dialysis and a simple dilution of serum increased heat stability. The heat stability of purified human serum biotinidase demonstrated 40% residual activity after heat treatment at 60 OC for 15 min. The reason for increased resistance to heat treatment on serum biotinidase is not known at this time. However, some factor(s) in fresh serum apparently induced heat lability on the enzyme. This is in contrast to the heat lability of bacterial biotinidase, where 92% inactivation occurred after heat treatment at 6O“C for 10 min [3]. This type of study might shed light on the mechanism of inborn errors of metabolism caused by mutated enzymes [lO,ll]. Acknowledgement This work was supported Japan.
by a grant from the Ministry
of Health
and Welfare
of
100
References 1 Knappe J, Brtimmer W, Biederbick K. Reinigung and Eigenschaften der Biotinidase aus Schweinenieren und L.actobacillus cmei. Biochem Z 1963;388;599-613. 2 Pispa J. Animal biotinidase. Ann Med Exp Biol Fenn 1965;SuppI 5:5-39. 3 KoivusaIo M, Elorriaga C, Kaziro Y, Ochoa S. Bacterial biotinidase. J Biol Chem 1963;238:1038-1042. 4 Chauhan J, Dakshinamurti K. Purification and characterization of human serum biotinidase. J Biol Chem 1986;261:4268-4275. 5 Hayakawa K, Oizumi J. Determination of biotinidase activity by liquid chromatography with fluorimetric detection. J Chromatogr 1986;383:148-152. 6 Craft DV, Goss NH, Chandramouri N, Wood HG. Purification of biotinidase from human plasma and its activity on biotinylpeptides. Biochemistry 1985;24:2471-2476. 7 Hayakawa K, Oizumi J. Effects of surfactants on human serum biotinidase. Clin Chim Acta 1987;168:109-111. 8 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 9 Baldwin RL, Eisenberg D. Protein stability. In: Oxender DL, Fox CF, eds. Protein engineering. New York: A.R. Liss, 1987;127-148. 10 Wolf B, Hsia YE, Rosenberg LE. Biochemical differences between mutant propionyl-CoA carboxylases from two complementation groups. Am J Hum Genet 1978;30:455-464. 11 Hsia YE, Scully KJ, Rosenberg LE. Human propionyl CoA carboxylase: some properties of the partially purified enzyme in fibroblasts from control and patients with propionic acidemia. Pediat Res 1979;13:746751.
Clinica Chimica Acta, 178 (1988) 95-100 Elsevier
CCA 04307
Short communication
Thermostability
of human serum biotinidase activity Kou Hayakawa and Jun Oizumi
Naiional Children’s Medical Research Center, Diuisron of Metabolism,
Tokyo (Japan)
(Received 19 April 1988; revision received 14 July 1988, accepted 12 August 1988)
Key words: Biotinidase; Thermal inactivation; HPLC assay
Introduction Each enzyme has its own characteristic thermal stability at the physiological temperature range (20-37 o C) and at a high temperature range (50-60 ’ C). Knowledge of the thermal stability of the enzyme is essential for practical purposes, i.e. manipulation or handling. Thermal stability at the physiological temperature range is essential for studying the effects of endo- or exo-type glycosidase or protease on the enzyme activity. Thermostability must also be known at high temperatures for utilizing in biotinidase purification [1,2] or in stopping the enzyme reaction [3]. Purified (21200-fold) human serum biotinidase lost 60% of its original activity after heating at 60 o C for 15 min, as recently reported by Chauhan and Dakshinamurti [4] using Knappe’s calorimetric method. Partially purified hog serum biotinidase (1910-fold) completely lost activity after heating at 60°C for 30 min as reported by Pispa [2]. Bacterial biotinidase of Streptococcu.s fuecafis (strain lOC1) [3] is reportedly heat labile; 92% of the original activity was lost after treatment at 60 o C for 10 min. In this report, we attempted to clarify these conflicting results in a systematic way by a newly developed HPLC biotinidase assay method [5]. We found that biotinidase activity in human native serum was completely inactivated by heat treatment at 60 o C for 15 min. However, simple dilution of the fresh serum with a neutral phosphate buffer or dialysis against the phosphate buffer increased the heat stability of biotinidase activity in fresh serum; 27% and 50% of enzyme activity, respectively, remained after heat treatment at 60 o C for 15 min. Purified human serum biotinidase was relatively heat resistant; 40% of enzyme activity remained after heat treatment at 60 “C for 15 min. This finding is in accordance with the result of Chauhan and Dakshinamurti [4].
Correspondence to: K. Hayakawa, National Children’s Medical Research Center, Division of Metabolism, 3-35-31 Taishido, Setagaya-Ku, Tokyo 154, Japan.
0009-8981/88/$03.50
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
96
Materials and methods Chemicals and reagents Biotinyl 4-aminobenzoate was purchased from Sigma Chemical Co., St. Louis, MO, USA. 2-Mercaptoethanol was from Kanto Chemical Co., Tokyo, Japan. DEAE-Sephacel, Sephacryl S-300 were from Pharmacia Fine Chemical Co., Uppsala, Sweden, Affi-Gel Blue was from Bio-Rad Laboratories, Richmond, CA. Nonidet P-40 was from Nakarai Chem., Kyoto, Japan. Methanol (HPLC grade) was from Wako Pure Chemical Co., Osaka, Japan. Fresh Serum specimens Hospital, Tokyo, Japan.
serum
was kindly
supplied
by the National
Children’s
Purification of serum biotinidase Human serum biotinidase was purified essentially according to the method described recently by Craft et al. [6] and Chauhan and Dakshinamurti [4], using 1 liter of human serum, except for the additional step of isoelectric focusing (IEF) separation. This procedure was published recently by us [7]. The enzyme fraction after Sephacryl S-300 (1200-fold) was further processed by an Affi-Gel Blue affinity column and isoelectric focusing (IEF) gel electrophoresis. The flow-through fraction from the Affi-Gel Blue column (0.05 mol/l sodium phosphate buffer; P-buffer, at pH 7.0) contained biotinidase activity. The enzyme fraction was further purified by the IEF-PAGE (pH 2.5-6.5, containing 1% Nonidet P-40) using the LKB Ultrophor thin-layer-type IEF-PAGE system. The summary of the purification is presented in Table I. The final specific activity was increased by a factor of 30000 (serum; 0.133 nmol/min per mg, final enzyme fraction; 3!900 nmol/min/mg). Biotinidase assay Biotinidase activity was assayed either by the method of Knappe et al. [l] or by our own [5]. The HPLC method measures the product p-aminobenzoate (PAB) directly by its intrinsic fluorescence (Ex. 276 nm, Em. 340
TABLE
I
Summary of purification Step
Fraction
of human serum biotinidase Volume
Protein
(ml)
(mg)
1000 335
67400 22759
100
237
35
50
32
20
Purification (-fold)
(W)
8 964 8785
1 3
100 98
8722
277
97
171
8498
1286
94
334
6613
2511
74
3900
1731
30000
19
Spec act (nmol/min
Total activity (nmoI/min)
per mg) Serum Ammonium sulfate DEAESephacel Sephacryl s-300 Affi-Gel Blue IEF-PAGE
12
0.44
0.133 0.386 36.8
Yield
91
nm). Knappe’s purification.
method
was
mainly
used
as a qualitative
measure
during
the
Total protein concentration was determined by the method Protein measurement of Lowry et al. [8], using bovine serum albumin as a standard protein. was assessed by incubating 0.1 ml of enzyme Heat stability test Thermostability solution (fresh serum and purified enzyme) at a temperature fixed at 50 or 60 ’ C for 0, 5, and 15 min in a water bath. Following incubation, an aliquot (0.02 ml) was pipetted into 0.08 ml of substrate solution (0.1 mg/ml of biotinyl 4-aminobenzoate in 0.1 mol/l P-buffer, pH 6.4). The reaction mixture was incubated at 37°C for 30 min. After stopping the reaction by adding 0.20 ml of methanol, the mixture was centrifuged at 1500 X g for 5 min to precipitate the enzyme. A portion (0.01 ml) of the clear supematant was injected into the HPLC system, and the liberated product (PAB) was measured [5]. The HPLC system used was Waters Model 600 combined with a fluorimetric detector (Hitachi F-3000). Results Freshly collected human sera (0.1 ml) were incubated at 50 o C and 60 o C for 2, 5, 10, and 15 mm in a water bath with shaking. After heating, serum was assayed for biotinidase activity, and residual enzyme activity was illustrated (Fig. 1). Native serum biotinidase activity was inactivated 66% and 100% after heat treatment of 50°C and 60°C for 15 min, respectively. Five sera from different individuals (biotinidase activity; 8.08, 6.81, 4.69, 1.44, 1.06 nmol/min per ml) were also tested,
0
I 5 Incubation
60°C IO Time
15 (min
1
Fig. 1. Effect of heat pretreatment on biotinidase activity in native serum. Serum sample of spec act of 0.133 nmol/min per mg (100% activity) was heated, and determined the remaining activity at 37 o C as described in ‘Materials and Methods’. Each point is the average of triplicate measurements. SD (unbiased form) was calculated and indicated.
98
and all five specimens completely lost the activity after heat treatment at 60°C for 15 min. These results indicate that biotinidase activity in native human serum is relatively heat labile. Then, serum was two-fold diluted with 0.1 mol/l P-buffer (pH 6.0) and treated at 60°C for 15 min. It was found that 73% of the biotinidase activity was lost. A simple dilution of the serum by a neutral buffer was found to slightly increase the heat stability of the enzyme activity from 0% residual activity (nondilute serum) to 27% residual activity (2-fold diluted serum). As already indicated by Chauhan and Dakshinamurti [4], human serum biotinidase contains a considerable amount of cysteine residues (3.17 residues/100 residues). Also, the change in electrophoretical pattern with the addition of 2mercaptoethanol suggests the presence of interstructural disulfide bonds, because of the molecular weight change (3000 daltons) of the enzyme [4]. In general, disulfide bonds may play an important role in heat stability, as pointed out by Baldwin and Eisenberg [9]. In serum, many small molecular weight sulfhydryl active compounds are present, which might interfere with heat stability of the enzyme. We extensively dialyzed the serum against 0.1 mol/l P-buffer (pH 6.0) at 4°C overnight in order to eliminate the compounds which may affect the disulfide bond. Dialysis itself did not affect the specific activity of the enzyme at all. However, dialyzed serum exhibited only 50% inactivation by heating at 60 o C for 15 min, whereas native serum enzyme was 100% inactivated by the same procedure. Dialysis of the serum induced the apparent heat resistance against at this temperature. Since the condition of the SH group may affect the heat stability of the enzyme, we dialyzed serum with 1 mmol/l of 2-mercaptoethanol. The reagent had no apparent effect on heat stability. A summary of these experiments is presented in Table II. We purified human serum biotinidase by a factor of 30000 using the procedure described in ‘Materials and Methods’. This purified enzyme was tested for heat stability. The results are illustrated in Fig. 2. The heat stability on this enzyme is
TABLE
II
Residual activity after heat treatment of 60’ C for 15 min a Serum specimen
Residual act (8)
Fresh serum Diluted serum with P-buffer b Dialyzed serum without 2-ME c Dialyzed serum with 2-ME ’
o+o 27+3 5016 51+8
Procedure for heat treatment and enzyme assay method are as described in ‘Materials and Methods’. Specific activity of fresh serum (0.133 nmoI/min per mg) was taken as 100%. Each values are average of 5 determinations (with SD). Fresh serum (0.133 nmol/min per mg) was diluted two-fold with 0.1 mol/l sodium phosphate buffer (pH 6.0) and assayed as above. Fresh serum (10 ml) was dialyzed against 5 1of 0.1 moI/I sodium phosphate buffer (pH 6.0) at 4O C for 15 h in the presence or absence of 1 mmol/l 2-mercaptoethanol (2-ME), and assayed as above. After dialysis, no change in specific activity was detected in the absence of 2-ME, i.e. 0.133 nmol/min per mg. However, in the presence of 2-ME, a two-fold increase in specific activity was observed (0.266 nmol/min per mg). Thus, these specific activities were taken as 100%.
99
Incubation
Time
t min )
Fig. 2. Heat stability of purified enzyme. The enzyme was purified to 30000-fold (3900 nmol/min per mg) as described in ‘Materials and Methods’. Enzyme activity without heating (3900 nmol/min per mg) was considered as lOO$. Each point is the average of triplicate measurements. SD (unbiased form) calculated and indicated. Other conditions are as described in ‘Materials and Methods’.
comparable to the result of Chauhan and Dakshinamurti [4]; 40% of residual activity was demonstrated using the 30000-fold purified enzyme. Discussion Because the thermal unfolding or denaturation of proteins usually occurs between 50° C and 60 o C at neutral pH [9], and since Koivusalo et al. [3] and Chauhan and Dakshinamurti [4] described the characteristics of thermostability of biotinidase at 50 o C and 60 o C for 10 min and 15 min, respectively, we also tested at 50°C and 60°C. We found a complete inactivation of biotinidase activity in fresh serum at 60°C for 15 min. Dialysis and a simple dilution of serum increased heat stability. The heat stability of purified human serum biotinidase demonstrated 40% residual activity after heat treatment at 60 OC for 15 min. The reason for increased resistance to heat treatment on serum biotinidase is not known at this time. However, some factor(s) in fresh serum apparently induced heat lability on the enzyme. This is in contrast to the heat lability of bacterial biotinidase, where 92% inactivation occurred after heat treatment at 6O“C for 10 min [3]. This type of study might shed light on the mechanism of inborn errors of metabolism caused by mutated enzymes [lO,ll]. Acknowledgement This work was supported Japan.
by a grant from the Ministry
of Health
and Welfare
of
100
References 1 Knappe J, Brtimmer W, Biederbick K. Reinigung and Eigenschaften der Biotinidase aus Schweinenieren und L.actobacillus cmei. Biochem Z 1963;388;599-613. 2 Pispa J. Animal biotinidase. Ann Med Exp Biol Fenn 1965;SuppI 5:5-39. 3 KoivusaIo M, Elorriaga C, Kaziro Y, Ochoa S. Bacterial biotinidase. J Biol Chem 1963;238:1038-1042. 4 Chauhan J, Dakshinamurti K. Purification and characterization of human serum biotinidase. J Biol Chem 1986;261:4268-4275. 5 Hayakawa K, Oizumi J. Determination of biotinidase activity by liquid chromatography with fluorimetric detection. J Chromatogr 1986;383:148-152. 6 Craft DV, Goss NH, Chandramouri N, Wood HG. Purification of biotinidase from human plasma and its activity on biotinylpeptides. Biochemistry 1985;24:2471-2476. 7 Hayakawa K, Oizumi J. Effects of surfactants on human serum biotinidase. Clin Chim Acta 1987;168:109-111. 8 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 9 Baldwin RL, Eisenberg D. Protein stability. In: Oxender DL, Fox CF, eds. Protein engineering. New York: A.R. Liss, 1987;127-148. 10 Wolf B, Hsia YE, Rosenberg LE. Biochemical differences between mutant propionyl-CoA carboxylases from two complementation groups. Am J Hum Genet 1978;30:455-464. 11 Hsia YE, Scully KJ, Rosenberg LE. Human propionyl CoA carboxylase: some properties of the partially purified enzyme in fibroblasts from control and patients with propionic acidemia. Pediat Res 1979;13:746751.