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[116] β-Hexosaminidase A from human placenta
[I16]
~-HEXOSAMINIDASE A FROM H U M A N PLACF.NTA
[116] ]~-Hcxosaminidase A from Human
857
Placenta
By WXLLIAM G. JOHNSOn, GEORGE MOOK, and ROSCOE O. BRADY R-N-Acetylhexosaminide -{- HsO --* R-OH -}- N-Acetylhexosamine Assay Methods
Principle. Enzyme activity causing the release of strongly fluorescent 4-methylumbelliferone from its nonfluorescent fl-hexosaminide is designated fl-hexosaminidase and is quantified by fluorometry at basic pH. Both the fl-glucosaminide and the fl-galactosaminide derivatives of 4methylumbelliferone are cleaved by hexosaminidase A. The fl-glucosaminide has been used more frequently as a substrate and is used here; however, the fl-galactosaminide is probably more convenient for work with tissue. Reagents 4-Methylumbelliferyl-2-acetamido-2-deoxy-fl-D-glucopyranoside (4 MU-fl-N-aeetylglucosaminide) (Koch-Light Laboratories) Citrate-phosphate buffer, "0.15 M," pH 4.41 4-Methylumbelliferone (4-MU, K + K Laboratories, Inc.) Glycine, 0.2 M in I-I~O Sodium hydroxide, 0.2 M in H20
Procedure. A substrate solution of 4 MU-fl-glucosaminide, 5 mM (19 mg in 10 ml H20) is prepared. An incubation mixture contains 10 ~l of enzyme extract, 150 ~l of citrate-phosphate buffer "150 mM," pH 4.4 (made by mixing 22.2 ml of 0.2 M disodium phosphate and 27.8 ml of 0.1 M citric acid solution1), and 50 /A of substrate solution to a total volume of 0.2I ml. The blank, containing the same amounts of buffer and substrate but water instead of enzyme extract, is treated identically to the sample. An enzyme blank is usually unnecessary in the fluorometric assay. After incubation at 37 ° for up to 15 minutes, the reaction is stopped by adding 0.8 ml glycine--sodium hydroxide buffer, 0.1 M, pH 10.7 (made by mixing equal volumes of NaOH 0.2 M and glycine 0.2 M). Tenfold dilution is performed, if necessary by mixing 0.1 ml of samples and blanks with 0.9 ml of the pH 10.7 buffer. Further dilution should not be attempted. Instead very active enzyme extracts should be diluted with the citrate-phosphate buffer pH 4.4 before addition to the incubation mixture. The fluorescence is measured and the 'G. Gomori, Vol. 1 [16].
858
DEGRADATION OF COMPLEX CARBOHYDRATES
[116]
result compared with the fluorescence of known amounts of 4-MU. In this laboratory an Eppendorf fluorometer was used with a primary filter transmitting the Hg line, 366 nm, and a double barrier secondary filter transmitting 430-470 nm. A liquid standard (quinine alkaloid, 10 #g/ml in 0.1 N 1-I2S04) and quartz euvettes were used throughout. Purification of Placental Hexosaminidase A
Reagents Monosodium phosphate Disodium phosphate Ammonium sulfate Sephadex G-200 (Pharmaeia) DEAE Sephadex A-25 (Pharmacia) Sodium chloride Citric acid Sodium hydroxide CM Sephadex C-25 (Pharmaeia)
Extraction. Fresh human placentas are placed on ice immediately after birth. They are processed within 12 hours after delivery. All subsequent procedures are carried out at cold room temperature (3°). Placentas are perfused with normal saline (0.9~ NaCI in H20) to remove clots. Then 2500 g is dissected free from fibrous tissue and homogenized in portions in a Waring blendor (two 1-minute runs) in five volumes of sodium phosphate buffer 25 mM, pH 6. 2 The homogehate is centrifuged at 10,000 g for 20 minutes, and the supernatant is retained. Ammonium Sul]ate Precipitation. Ammonium sulfate fractionation of the solution is performed (using 70.5 g of (NH4)~SO4 per 100 ml of solution as 100% saturation), and the fraction which precipitated between 25% and 55% is collected by centrifugation at 10,000 g for 30 minutes and resuspended in sodium phosphate buffer 25 mM pH 6 to a total volume of 800 ml. This highly concentrated protein solution is dialyzed against sodium phosphate buffer 25 mM, pH 6, for 48 hours. Sephadex G-~.O0. The retentate is centrifuged at 50,000 g for 1 hour, and the supernatant is applied to an upward flowing column of Sephadex G-200 (Pharmacia column, 10 × 100 cm) equilibrated with the same phosphate buffer. Fractions are collected (25 ml) and analyzed for flhexosaminidase. A single fl-hexosaminidase peak is found; fractions con2G. Gomori, Vol. I [16].
[115]
fl-HEXOSAMINInASE& FROM HUMAN PL&CENTA
859
taining 90•o of the total assayed activity are pooled and retained. Pooled fractions from two such runs are combined, concentrated togethe~ to a volume of 300 ml (Amicon system with PM-10 ultrafilters), and centrifuged at 30,000 g for 20 minutes, the supernatant is reapplied to a freshly packed column of Sephadex G-200 in identical fashion. Fractions containing 9 0 ~ of the total assayed fl-hexosaminidase are collected and concentrated in the same way to a volume of 300 ml. D,EAE-Sephadex. Sodium phosphate buffer (8 liters) 25 mM, pH 6, is titrated to pH 6.00 _ 0.02 at 25 °. DEAE Sephadex A-25 (about 150 g) is swollen in 2 liters of this buffer and titrated to pH 6.00 ± 0.02 (25°). The gel suspension is washed twice with fresh buffer and retitrated each time. A Pharmacia column (5 × 60 cm) is packed with this gel and washed with 2 liters of the buffer. Combined fractions from two runs of the preceding step (300 ml) are titrated to pH 6.00 ± 0.02 at 25 ° and applied slowly (downward flow) to the column; the column is washed with 500 ml of the phosphate buffer and eluted with a linear NaC1 gradient, 0-600 mM (in the same phosphate buffer), total volume 2 liters (Kontes gradient maker). Fractions are collected (25 ml), and two well-separated peaks of fl-hexosaminidase are found on assay. The first peak, in the material which does not stick to the column, corresponds to fl-hexosaminidase B3 The second peak is eluted with the salt gradient and corresponds to fl-hexosaminidase A. Fractions containing 80% of the fl-hexosaminidase A are pooled, concentrated to 30-50 ml (Amicon system, PM-10 membranes), and dialyzed overnight against the sodium phosphate buffer 25 mM pH 6. CM-Sephadex. Citrate-phosphate buffer, "30 mM," pH 4.8 is prepared by mixing 24.8 ml disodium phosphate 0.2 M and 252 ml citric acid 0.1 M, diluted 5-fold, and titrated to pH 4.80 ± 0.02 (25 °) with citric acid 1 M. CM Sephadex C-25 is swollen in this buffer, titrated to pH 4.80 ± 0.02 (25°), washed twice with fresh buffer, retitrated, and packed into a column (0.9 × 20-cm, Pharmacia). The column is washed with the same buffer (50 ml). The protein sample of fl-hexosaminidase A (30 ml) is titrated to pH 4.80 ± 0.02 (25 °) and applied slowly (downward flow) to the column; the column is washed with 50 ml of the citrate-phosphate buffer and eluted with a linear NaC1 gradient 0-600 mM (prepared in the same citrate-phosphate buffer), total volume 200 ml. Fractions of 3.5 ml are collected. At this pH the fl-hexosaminidase is bound to the column and eluted by the salt gradient in a single peak. The peak fractions are pooled, immediately retitrated to pH 6.0, and dialyzed against sodium phosphate buffer 25 mM pH 6. s D. Robinson and J. L. Stifling, Biochem. J. 107, 321 (1968).
860
[116]
DEGRADATION OF COMPLEX CARBOHYDRATES
TABLE I PURIFICATION OF ~-HExosAMINIDASE FROM HUMAN PLACENTAa
Total protein (g)
Fraction Fresh placental tissue, 10kg Ammonium sulfate concentrate Sephadex G-200, 2 passes DEAE4Sephadex~ CM-Sephadex
.
Activity Total activity ~ (0-hexos(0-hexosaminidase aminidase ~moles/mg mmoles/hour) protein/hour) .
.
.
.
73.6
260
2.6
112
43
55 39
306 6960
0.18 0.0056
Purification factor Yield (-fold) (%)
3.5
1
100
12.3
43
158 3590
38 27
° The values reported are average values from several preparative runs. b Fractions (2) and (3) contain a mixture of ~-hexosaminidase isozymes A and B, and specific activities, purification factors, and yields of enzyme activities, listed are totals for the mixture. Fractions (4) and (5) contain 0-hexosaminidase A only; enzyme activities, specific activities purification factors, and yields listed are for this enzyme only. All ~-hexosaminidase assays used 4-MU-$-glucosaminide at 1.25 mM concentration. c Hexosaminidase A is separated from hexosaminidase B by this step and constitutes 55% of the total ~-hexosaminidase recovered at this stage. A typical purification is summarized in Table I. Substrate K . and Vm~ are compared in Table I I . Properties of the Purified E n z y m e
pH Optimum. The. p H - a c t i v i t y curve of fl-hexosaminidase A is very broad peaking at in the region of p H 4.2-4.4. Ef]ect of Detergents. fl-hexosaminidase A shows marked inhibition by anionic detergents but not by neutral or cationic detergents (Table I I I ) . Some stimulation of activity is seen with cationic detergents. Inhibitors. Table I I I shows inhibitors of fl-hexosaminidase A. Nature o] the Reaction. Hexosaminidase A catalyzes the hydrolytic TABLE II Km AND Vmax OF PLACENTAL ~-HExosAMINIDASE A
Substrate
K. (raM)
Vm~x (mmoles/mg protein/hour)
4-MU-0-N-acetylgincosaminide 4-MU-0-N-acetylgalactosaminide
1.4 0.88
84 3.8
[115]
~-HEXOSAMINIDASE A FROM HUMAN PLACENTA
861
TABLE I I I INHIBITORS OF HEXOSAMINIDASEA; SUBSTRATE:4-MU-~GLUCOSAMINIDE
1.25 mM Compound
Concentration
Effect (%)
N-Acetylgalactosamine N-Acetylglucosamine Galactosamine Glucosamine Galacturonic acid p-Chloromercurisulfonate HgC12 p-Nitrophenol Sodium cholate Sodium taurocholate Triton X-100 Cetylpyridium chloride Lecithin Cetyltrimethylammonium bromide
2.5 mM 2.5 mM 2.5 mM 2.5 mM 2.5 mM 2.5 mM 2.5 mM 2.5 mM 5 mg/ml 5 mg/ml 5 mg/ml 5 mM 5 mM 5 mM
- 75 -- 35 - 25 0 - 44 -95 - 97 - 99 -69 -66 0 -23 + 14 + 16
c l e a v a g e of t e r m i n a l l y l i n k e d f l - N - a c e t y l h e x o s a m i n e . T h e e n z y m e is p r e s e n t in a wide v a r i e t y of h u m a n tissues i n c l u d i n g liver, spleen, k i d n e y , serum, w h i t e blood cells, b r a i n , a n d p l a c e n t a . A b s e n c e of h e x o s a m i n i d a s e A, a h u m a n genetic d e f e c t c a r r i e d as a n a u t o s o m a l recessive t r a i t , is a s s o c i a t e d w i t h a c c u m u l a t i o n of g a n g l i o s i d e GM~ (with s m a l l e r a m o u n t s of a s i a l o - G M 2 ) a n d l e t h a l b r a i n disease in T a y - S a e h s disease. ~,5 A s s a y of t h i s e n z y m e p e r m i t s a c c u r a t e d i a g n o s i s of p a t i e n t s , 4 d e t e c t i o n of carriers, ~ a n d r e l i a b l e genetic counseling for t h i s disorder.
' S. Okada and J. 0'Brien, Science 165, 6 ~ (1969). E. H. Kolodny, R. O. Brady, and B. W. Volk, Biochem. Biophys. Res. Commun. 37, 526 (1969). 6j. O'Brien, S. Okada, A. Chen, and D. Fillerup, N. Engl. J. Med. 283, 15 (1970).
~-HEXOSAMINIDASE A FROM H U M A N PLACF.NTA
[116] ]~-Hcxosaminidase A from Human
857
Placenta
By WXLLIAM G. JOHNSOn, GEORGE MOOK, and ROSCOE O. BRADY R-N-Acetylhexosaminide -{- HsO --* R-OH -}- N-Acetylhexosamine Assay Methods
Principle. Enzyme activity causing the release of strongly fluorescent 4-methylumbelliferone from its nonfluorescent fl-hexosaminide is designated fl-hexosaminidase and is quantified by fluorometry at basic pH. Both the fl-glucosaminide and the fl-galactosaminide derivatives of 4methylumbelliferone are cleaved by hexosaminidase A. The fl-glucosaminide has been used more frequently as a substrate and is used here; however, the fl-galactosaminide is probably more convenient for work with tissue. Reagents 4-Methylumbelliferyl-2-acetamido-2-deoxy-fl-D-glucopyranoside (4 MU-fl-N-aeetylglucosaminide) (Koch-Light Laboratories) Citrate-phosphate buffer, "0.15 M," pH 4.41 4-Methylumbelliferone (4-MU, K + K Laboratories, Inc.) Glycine, 0.2 M in I-I~O Sodium hydroxide, 0.2 M in H20
Procedure. A substrate solution of 4 MU-fl-glucosaminide, 5 mM (19 mg in 10 ml H20) is prepared. An incubation mixture contains 10 ~l of enzyme extract, 150 ~l of citrate-phosphate buffer "150 mM," pH 4.4 (made by mixing 22.2 ml of 0.2 M disodium phosphate and 27.8 ml of 0.1 M citric acid solution1), and 50 /A of substrate solution to a total volume of 0.2I ml. The blank, containing the same amounts of buffer and substrate but water instead of enzyme extract, is treated identically to the sample. An enzyme blank is usually unnecessary in the fluorometric assay. After incubation at 37 ° for up to 15 minutes, the reaction is stopped by adding 0.8 ml glycine--sodium hydroxide buffer, 0.1 M, pH 10.7 (made by mixing equal volumes of NaOH 0.2 M and glycine 0.2 M). Tenfold dilution is performed, if necessary by mixing 0.1 ml of samples and blanks with 0.9 ml of the pH 10.7 buffer. Further dilution should not be attempted. Instead very active enzyme extracts should be diluted with the citrate-phosphate buffer pH 4.4 before addition to the incubation mixture. The fluorescence is measured and the 'G. Gomori, Vol. 1 [16].
858
DEGRADATION OF COMPLEX CARBOHYDRATES
[116]
result compared with the fluorescence of known amounts of 4-MU. In this laboratory an Eppendorf fluorometer was used with a primary filter transmitting the Hg line, 366 nm, and a double barrier secondary filter transmitting 430-470 nm. A liquid standard (quinine alkaloid, 10 #g/ml in 0.1 N 1-I2S04) and quartz euvettes were used throughout. Purification of Placental Hexosaminidase A
Reagents Monosodium phosphate Disodium phosphate Ammonium sulfate Sephadex G-200 (Pharmaeia) DEAE Sephadex A-25 (Pharmacia) Sodium chloride Citric acid Sodium hydroxide CM Sephadex C-25 (Pharmaeia)
Extraction. Fresh human placentas are placed on ice immediately after birth. They are processed within 12 hours after delivery. All subsequent procedures are carried out at cold room temperature (3°). Placentas are perfused with normal saline (0.9~ NaCI in H20) to remove clots. Then 2500 g is dissected free from fibrous tissue and homogenized in portions in a Waring blendor (two 1-minute runs) in five volumes of sodium phosphate buffer 25 mM, pH 6. 2 The homogehate is centrifuged at 10,000 g for 20 minutes, and the supernatant is retained. Ammonium Sul]ate Precipitation. Ammonium sulfate fractionation of the solution is performed (using 70.5 g of (NH4)~SO4 per 100 ml of solution as 100% saturation), and the fraction which precipitated between 25% and 55% is collected by centrifugation at 10,000 g for 30 minutes and resuspended in sodium phosphate buffer 25 mM pH 6 to a total volume of 800 ml. This highly concentrated protein solution is dialyzed against sodium phosphate buffer 25 mM, pH 6, for 48 hours. Sephadex G-~.O0. The retentate is centrifuged at 50,000 g for 1 hour, and the supernatant is applied to an upward flowing column of Sephadex G-200 (Pharmacia column, 10 × 100 cm) equilibrated with the same phosphate buffer. Fractions are collected (25 ml) and analyzed for flhexosaminidase. A single fl-hexosaminidase peak is found; fractions con2G. Gomori, Vol. I [16].
[115]
fl-HEXOSAMINInASE& FROM HUMAN PL&CENTA
859
taining 90•o of the total assayed activity are pooled and retained. Pooled fractions from two such runs are combined, concentrated togethe~ to a volume of 300 ml (Amicon system with PM-10 ultrafilters), and centrifuged at 30,000 g for 20 minutes, the supernatant is reapplied to a freshly packed column of Sephadex G-200 in identical fashion. Fractions containing 9 0 ~ of the total assayed fl-hexosaminidase are collected and concentrated in the same way to a volume of 300 ml. D,EAE-Sephadex. Sodium phosphate buffer (8 liters) 25 mM, pH 6, is titrated to pH 6.00 _ 0.02 at 25 °. DEAE Sephadex A-25 (about 150 g) is swollen in 2 liters of this buffer and titrated to pH 6.00 ± 0.02 (25°). The gel suspension is washed twice with fresh buffer and retitrated each time. A Pharmacia column (5 × 60 cm) is packed with this gel and washed with 2 liters of the buffer. Combined fractions from two runs of the preceding step (300 ml) are titrated to pH 6.00 ± 0.02 at 25 ° and applied slowly (downward flow) to the column; the column is washed with 500 ml of the phosphate buffer and eluted with a linear NaC1 gradient, 0-600 mM (in the same phosphate buffer), total volume 2 liters (Kontes gradient maker). Fractions are collected (25 ml), and two well-separated peaks of fl-hexosaminidase are found on assay. The first peak, in the material which does not stick to the column, corresponds to fl-hexosaminidase B3 The second peak is eluted with the salt gradient and corresponds to fl-hexosaminidase A. Fractions containing 80% of the fl-hexosaminidase A are pooled, concentrated to 30-50 ml (Amicon system, PM-10 membranes), and dialyzed overnight against the sodium phosphate buffer 25 mM pH 6. CM-Sephadex. Citrate-phosphate buffer, "30 mM," pH 4.8 is prepared by mixing 24.8 ml disodium phosphate 0.2 M and 252 ml citric acid 0.1 M, diluted 5-fold, and titrated to pH 4.80 ± 0.02 (25 °) with citric acid 1 M. CM Sephadex C-25 is swollen in this buffer, titrated to pH 4.80 ± 0.02 (25°), washed twice with fresh buffer, retitrated, and packed into a column (0.9 × 20-cm, Pharmacia). The column is washed with the same buffer (50 ml). The protein sample of fl-hexosaminidase A (30 ml) is titrated to pH 4.80 ± 0.02 (25 °) and applied slowly (downward flow) to the column; the column is washed with 50 ml of the citrate-phosphate buffer and eluted with a linear NaC1 gradient 0-600 mM (prepared in the same citrate-phosphate buffer), total volume 200 ml. Fractions of 3.5 ml are collected. At this pH the fl-hexosaminidase is bound to the column and eluted by the salt gradient in a single peak. The peak fractions are pooled, immediately retitrated to pH 6.0, and dialyzed against sodium phosphate buffer 25 mM pH 6. s D. Robinson and J. L. Stifling, Biochem. J. 107, 321 (1968).
860
[116]
DEGRADATION OF COMPLEX CARBOHYDRATES
TABLE I PURIFICATION OF ~-HExosAMINIDASE FROM HUMAN PLACENTAa
Total protein (g)
Fraction Fresh placental tissue, 10kg Ammonium sulfate concentrate Sephadex G-200, 2 passes DEAE4Sephadex~ CM-Sephadex
.
Activity Total activity ~ (0-hexos(0-hexosaminidase aminidase ~moles/mg mmoles/hour) protein/hour) .
.
.
.
73.6
260
2.6
112
43
55 39
306 6960
0.18 0.0056
Purification factor Yield (-fold) (%)
3.5
1
100
12.3
43
158 3590
38 27
° The values reported are average values from several preparative runs. b Fractions (2) and (3) contain a mixture of ~-hexosaminidase isozymes A and B, and specific activities, purification factors, and yields of enzyme activities, listed are totals for the mixture. Fractions (4) and (5) contain 0-hexosaminidase A only; enzyme activities, specific activities purification factors, and yields listed are for this enzyme only. All ~-hexosaminidase assays used 4-MU-$-glucosaminide at 1.25 mM concentration. c Hexosaminidase A is separated from hexosaminidase B by this step and constitutes 55% of the total ~-hexosaminidase recovered at this stage. A typical purification is summarized in Table I. Substrate K . and Vm~ are compared in Table I I . Properties of the Purified E n z y m e
pH Optimum. The. p H - a c t i v i t y curve of fl-hexosaminidase A is very broad peaking at in the region of p H 4.2-4.4. Ef]ect of Detergents. fl-hexosaminidase A shows marked inhibition by anionic detergents but not by neutral or cationic detergents (Table I I I ) . Some stimulation of activity is seen with cationic detergents. Inhibitors. Table I I I shows inhibitors of fl-hexosaminidase A. Nature o] the Reaction. Hexosaminidase A catalyzes the hydrolytic TABLE II Km AND Vmax OF PLACENTAL ~-HExosAMINIDASE A
Substrate
K. (raM)
Vm~x (mmoles/mg protein/hour)
4-MU-0-N-acetylgincosaminide 4-MU-0-N-acetylgalactosaminide
1.4 0.88
84 3.8
[115]
~-HEXOSAMINIDASE A FROM HUMAN PLACENTA
861
TABLE I I I INHIBITORS OF HEXOSAMINIDASEA; SUBSTRATE:4-MU-~GLUCOSAMINIDE
1.25 mM Compound
Concentration
Effect (%)
N-Acetylgalactosamine N-Acetylglucosamine Galactosamine Glucosamine Galacturonic acid p-Chloromercurisulfonate HgC12 p-Nitrophenol Sodium cholate Sodium taurocholate Triton X-100 Cetylpyridium chloride Lecithin Cetyltrimethylammonium bromide
2.5 mM 2.5 mM 2.5 mM 2.5 mM 2.5 mM 2.5 mM 2.5 mM 2.5 mM 5 mg/ml 5 mg/ml 5 mg/ml 5 mM 5 mM 5 mM
- 75 -- 35 - 25 0 - 44 -95 - 97 - 99 -69 -66 0 -23 + 14 + 16
c l e a v a g e of t e r m i n a l l y l i n k e d f l - N - a c e t y l h e x o s a m i n e . T h e e n z y m e is p r e s e n t in a wide v a r i e t y of h u m a n tissues i n c l u d i n g liver, spleen, k i d n e y , serum, w h i t e blood cells, b r a i n , a n d p l a c e n t a . A b s e n c e of h e x o s a m i n i d a s e A, a h u m a n genetic d e f e c t c a r r i e d as a n a u t o s o m a l recessive t r a i t , is a s s o c i a t e d w i t h a c c u m u l a t i o n of g a n g l i o s i d e GM~ (with s m a l l e r a m o u n t s of a s i a l o - G M 2 ) a n d l e t h a l b r a i n disease in T a y - S a e h s disease. ~,5 A s s a y of t h i s e n z y m e p e r m i t s a c c u r a t e d i a g n o s i s of p a t i e n t s , 4 d e t e c t i o n of carriers, ~ a n d r e l i a b l e genetic counseling for t h i s disorder.
' S. Okada and J. 0'Brien, Science 165, 6 ~ (1969). E. H. Kolodny, R. O. Brady, and B. W. Volk, Biochem. Biophys. Res. Commun. 37, 526 (1969). 6j. O'Brien, S. Okada, A. Chen, and D. Fillerup, N. Engl. J. Med. 283, 15 (1970).