VOL. XVII (1975)
BIOTECHNOLOGY AND BIOENGINEERING
Glycosylation of Proteins: A New Method of Ensyme Stabilization Conjugat.es between different polysaccharides and different proteins have been prepared by the cyanogen bromide method. .4lthough the enzymes which were modified in this way suffered a marked loss in specific activity, the stability towards thermal and proteolytic inactivation was significantly improved. I t is suggested that optimalization of the method with respect to the coupling procedure and the carbohydrate components used might lead to an economic method of enzyme stabilization.
EXPERIMENTAL PROCEDURE The conjugates prepared are listed in Table I . The lysozyme and chymotrypsin derivatives were made according to the method of Salter, Ford, and Scott,' whereby the carbohydrate is attached to free amino and carbox71 groups. The other conjugates were prepared by the method of Kdgedal and Akerstr@m*using free amino groups only. The polysaccharides were dextran T10 and FITC-dextran-3 with a molecular weight of 10,000 and 3,000 D, respectively. Both were obtained from Pharmacia, Sweden. Starch dextrin was obtained from Hopkin and Williams, England, and the fraction passing through a dialysis membrane (av mol wt 6,000 D ) was used for our experiments. Lysozyme was assayed by measuring the lysis of bacterial cell walls,3 P-glucosidase by the hydrolysis of nitrophenol glucoside,' the proteases by the caseinolytic method of Kunitz,6 and the extent of proteolysis of nonenzyme proteins was TABLE I Protein-Polysaccharide Preparations
Preparation
Relative specific enzyme activity ("/o)
Carbohydrate content (76, w h )
~~
Lysozyme-TI0 Lysozyme-T10-A Lysozyrne-T10-B Chymotrypsin-T10 8-glucosidase-FITC dextran 3 @-glucosidase-TlO Casein Aextrin Serum albumin-dextrin
40 16 24 .5.5 30 32
73 90-
2.5 3.5
*The carbohydrate content was obtained directly from the quantities used in the coupling procedure as no subsequent fractionation was carried out. 1391
@ 1975 by John Wiley & Sons, Inc.
25mM citratephosphate (pH 6.2) S5rnMCaCl2 25mM c i t r a t e phosphate (pH 6 . 2 ) +5mMCaC12 25mM citratephosphate (pH 6.2) +5mMCaC1, lOOmM phosphate (pH 7.5) lOOmM phosphate (pH 7.5)
0.05 0.20
0.05 0.20
0.50
0.70
Lysozyme-T10-A chymotrypsin
Lysozyme-T 10-B
Chymotrypsin
Chymotrypsin-T10
Lysozyme
+ chymotrypsin
+
+ chymotrypsin
lysozyme
chymotrypsin
lysozyme
lysozyme
lysozyme
Enzyme assay
100
100
100
100
100
0
72
52
40
52
13
1
60
41
32
45
0
50
29
28
42
0
40
17
27
40
0
Incubation time (days) 2 3 4
Buffer
Protein concn (mg/ml)
0.05 0.20
Contents
Remaining enzyme activity (%)
Incubation mixture8
TABLE I1 Proteolytic Inactivation of Protein-Carbohydrate Conjugates and of the Free Proteins
-
-
25
40
0
5
Casein 0.365
lOOmM phosphate (pH 7.5) lOOmM phosphate (pH 7.5) lOOmM phosphate (pH 7.5)
6.60 0.01
2.70 0.02 2.70 0.02 0.170
0.300
0.500
lOOmM phosphate (pH 7.5)
6.60 0.01
*All incubations were carried out a t 37°C except for the mixtures with serum albumin which were incubated a t 50°C.
+ trypsin Serum albumin + trypsin Serum albumin-dextrin + trypsin
Casein-dextrin
+ trypsin
Proteolytic degradation during 1 hr (absorbance a t 280 nm)
w
W
W
w
z
0
7
5
tr!
2M
c3 0
u)
Z
0
=!
Q P
5
c
kz2
Q
0.5
1.0
2.0
0.5
1. 0
2.0
Lysozyme
Lysosyme
Lysosyme-T 10
Lysozyme-T10
Lysozyme-T10
Protein concn (mg/ml)
Lysozyme
Contents
Incubation mixture
25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2)
Buffer
100
100
100
100
100
100
Temperature ("C)
100
100
100
100
100
100
1 -
99
80
65
20
36
38
Incubation time (min) 15 30
-
40
Remaining enzyme activity (yo)
TABLE I11 Thermal Inactivation of Enzyme-Carbohydrate Conjugates and of Free Enzymes
0.5
0.5
0.1
0.1
0.1
Lysozyme
Lysozy me-T10
@-glucosidase
8-glucosidase-T10
@-glucosidaseFITC dextran 3
lOOmM acetate (PH 5.5) lOOmM acetate (PH 5 . 5 ) l00mM acetate (pH ,5.5)
+
25mM citratephosphate (pH 6.2) 0.01% (w/w) SDS
+
25mM citratephosphate (pH 6.2) 0.01% (w/w) SDS
100 100 100
60 60
100
100
60
100
100
-
-
-
-
-
-
50
0
79
82
41
-
-
m
x
cl
0
cl
1396 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVII (1975) determined by measuring the absorbance a t 280 nm of the supernatant after precipitating undegraded protein with a solution of trichloroacetic acid.5 The percent relative specific enzyme activity was calculated on the basis of the protein content of the preparations and relates to the activity of the nonconjugated enzymes. Lysozyme-T10-A and -B were obtained by chromatography on Sephadex G-100 subsequent to the coupling procedure in order to obtain fractions varying in carbohydrate content. The conjugates prepared with FITC-dext,ran and with the dialyzable starch dextrin were separated from unattached carbohydrate by dialysis. The carbohydrate analysis of these and the lysozyme-T10-A and -B preparations was carried out by gas chromatography of the trimethyl silyl derivatives of the methyl glucosides after methanolysis.6
RESULTS AND DISCUSSION The results obtained previously by us7 and by Marshall and Ilabinowitzs are in agreement with the observations in the present report (Tables I1 and 111). Table I1 shows that a considerable resistance to proteolysis is imparted to lysozyme, chymotrypsin, casein, and serum albumin after conjugation with polysaccharide. The lysozyme-T10-A was more resistant than the lysozyme-T10-B preparation and this may be due to the higher content of carbohydrate. However even the low incorporation of dextrin into the complexes with casein and serum albumin yielded a significant reduction in proteolytic degradation. The thermal inactivation experiments in Table I11 revealed that the conjugates were more stable than the corresponding free enzymes. In the experiments with lysozyme a precipitation of protein could clearly be seen after heating, whereas the conjugates remained completely soluble. It is worth noting that the effect of protein concentration was different for the free lysozyme and for the conjugate. Nakamuri and Hayashi have observed that the carbohydrate constituents of glucose oxidase enhance the thermal stability in the presence of sodium dodecyl sulfate (Sl>S).g On heating the lysozyme and the conjugate with dextran T10 in the presence of 0.0170 SDS, we obtained a pronounced difference between the two preparations (Table 111). The control experiments with nonconjugated proteins were carried out in the presence of the appropriate concentration of free polysaccharide. However, the relatively low concentrations of carbohydrate did not appear to affect the results. The proteolytic activity in the various incubation mixtures were routinely assayed and only minor variations were found. Moreover, the chymotrypsin activity in the lysozyme mixtures remained very high. After five days incubation, it was about 60% of the initial activity. The specific activity of the enzymes was reduced on glycosylation. It is possible that the use of other polysaccharides and/or coupling conditions may yield products of higher activity. One reason for the reduction may be that carbohydrates are attached near or a t the active site of the enzyme molecule. The results obtained with different proteins and different polysaccharides attached by the cyanogen bromide method7.8 as well as the evidence from experiments on natural glycoproteinsg-'* indicate that the stabilization due to carbohydrate constituents may be a general phenomenon. Thus, carbohydrate attached to enzymes could possibly be advantageous for commercial uses. The glycosy-
COhTMUNICATIONS TO T H E EDITOR
1397
lated enzymes may, of course, be linked to an insoluble support and an attachment is made by links between the support and the carbohydrate constituent^.'^ In the case of relatively insoluble enzymes, glycosylation may be used to enhance the solubility when this is desirable. Another possible application is in the field of macromolecules applied in therapy. Thus, heparin coupled to Ficoll (a sucrose polymer produced by Pharmacia, Sweden) has been shown to have a distinctly longer half-life i n vivo than free heparin .I4 A possible explanation for the increased stability of protein-carbohydrate conjugates may be provided by the hypothesis of M a r k ~ s . ' ~According to this, binding of ligands to a polypeptide may reduce the tendency of oscillations between different conformational states. An effect of this nature might reduce the susceptibility to proteolysis as well as to thermal inactivation. References 1 . 1). N. Salter, J. E. Ford, and K. J. Scott, FEES Lett., 20, 302 (1972). 2. L. KHLgedal and S. Akerstrorn, Acta Chem. Scund., 25, 1855 (1971). 3. D. Shugar, Biochim. Biophys. Acta, 8, 302 (1952). 4. It. C. Hughes and R. W. Jeanloz, Biochemistry, 3, 1535 (1964). 5 . M. Kunitz, J . Gen. Physiol., 30, 291 (1947). 6. R. E. Chambers and J. R. Clamp, Biochem. J., 125, 1009 (1971). 7. T. B. Christensen and G. Vegarud, Int. Res. Commun. Syst. (Biochem.), 2, 1311 (1974). 8. J. J. Marshall and M. L. Kabinowitz, Arch. Riochem. Bzophys., 167, 777 (1975). 9. S. Nakamura and S. Hayashi, FEBS Lett., 41, 327 (1974). 10. T. S. A. Samy, Arch. Biochem. Biophys., 121, 703 (1967). 11. A. J. Birkeland and T. B. Christensen, J . Carbohydrates-NucleosidesNucleotides, 2,83(1975). 12. A. Goldstone and H. Koenig, Biochem. J., 141, 527 (1974). 13. 0. It. Zaborsky and J . Ogletree, Bzochem. Bzophys. Res. Commun., 61, 210 (1974). 14. A. Teien, 0. 11. QdegHLrd, and T. B. Christensen, Thromb. Res., in press. 15. G. Markus, Proc. Acad. Sci. U . S., 54, 253 (1965).
GERDVEGARUD TERJEB. CHRISTENSEN Dept. of Biochemistry University of Oslo Blindern, Norway Accepted for Publication June 5, 1976
BIOTECHNOLOGY AND BIOENGINEERING
Glycosylation of Proteins: A New Method of Ensyme Stabilization Conjugat.es between different polysaccharides and different proteins have been prepared by the cyanogen bromide method. .4lthough the enzymes which were modified in this way suffered a marked loss in specific activity, the stability towards thermal and proteolytic inactivation was significantly improved. I t is suggested that optimalization of the method with respect to the coupling procedure and the carbohydrate components used might lead to an economic method of enzyme stabilization.
EXPERIMENTAL PROCEDURE The conjugates prepared are listed in Table I . The lysozyme and chymotrypsin derivatives were made according to the method of Salter, Ford, and Scott,' whereby the carbohydrate is attached to free amino and carbox71 groups. The other conjugates were prepared by the method of Kdgedal and Akerstr@m*using free amino groups only. The polysaccharides were dextran T10 and FITC-dextran-3 with a molecular weight of 10,000 and 3,000 D, respectively. Both were obtained from Pharmacia, Sweden. Starch dextrin was obtained from Hopkin and Williams, England, and the fraction passing through a dialysis membrane (av mol wt 6,000 D ) was used for our experiments. Lysozyme was assayed by measuring the lysis of bacterial cell walls,3 P-glucosidase by the hydrolysis of nitrophenol glucoside,' the proteases by the caseinolytic method of Kunitz,6 and the extent of proteolysis of nonenzyme proteins was TABLE I Protein-Polysaccharide Preparations
Preparation
Relative specific enzyme activity ("/o)
Carbohydrate content (76, w h )
~~
Lysozyme-TI0 Lysozyme-T10-A Lysozyrne-T10-B Chymotrypsin-T10 8-glucosidase-FITC dextran 3 @-glucosidase-TlO Casein Aextrin Serum albumin-dextrin
40 16 24 .5.5 30 32
73 90-
2.5 3.5
*The carbohydrate content was obtained directly from the quantities used in the coupling procedure as no subsequent fractionation was carried out. 1391
@ 1975 by John Wiley & Sons, Inc.
25mM citratephosphate (pH 6.2) S5rnMCaCl2 25mM c i t r a t e phosphate (pH 6 . 2 ) +5mMCaC12 25mM citratephosphate (pH 6.2) +5mMCaC1, lOOmM phosphate (pH 7.5) lOOmM phosphate (pH 7.5)
0.05 0.20
0.05 0.20
0.50
0.70
Lysozyme-T10-A chymotrypsin
Lysozyme-T 10-B
Chymotrypsin
Chymotrypsin-T10
Lysozyme
+ chymotrypsin
+
+ chymotrypsin
lysozyme
chymotrypsin
lysozyme
lysozyme
lysozyme
Enzyme assay
100
100
100
100
100
0
72
52
40
52
13
1
60
41
32
45
0
50
29
28
42
0
40
17
27
40
0
Incubation time (days) 2 3 4
Buffer
Protein concn (mg/ml)
0.05 0.20
Contents
Remaining enzyme activity (%)
Incubation mixture8
TABLE I1 Proteolytic Inactivation of Protein-Carbohydrate Conjugates and of the Free Proteins
-
-
25
40
0
5
Casein 0.365
lOOmM phosphate (pH 7.5) lOOmM phosphate (pH 7.5) lOOmM phosphate (pH 7.5)
6.60 0.01
2.70 0.02 2.70 0.02 0.170
0.300
0.500
lOOmM phosphate (pH 7.5)
6.60 0.01
*All incubations were carried out a t 37°C except for the mixtures with serum albumin which were incubated a t 50°C.
+ trypsin Serum albumin + trypsin Serum albumin-dextrin + trypsin
Casein-dextrin
+ trypsin
Proteolytic degradation during 1 hr (absorbance a t 280 nm)
w
W
W
w
z
0
7
5
tr!
2M
c3 0
u)
Z
0
=!
Q P
5
c
kz2
Q
0.5
1.0
2.0
0.5
1. 0
2.0
Lysozyme
Lysosyme
Lysosyme-T 10
Lysozyme-T10
Lysozyme-T10
Protein concn (mg/ml)
Lysozyme
Contents
Incubation mixture
25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2) 25mM citratephosphate (PH 6.2)
Buffer
100
100
100
100
100
100
Temperature ("C)
100
100
100
100
100
100
1 -
99
80
65
20
36
38
Incubation time (min) 15 30
-
40
Remaining enzyme activity (yo)
TABLE I11 Thermal Inactivation of Enzyme-Carbohydrate Conjugates and of Free Enzymes
0.5
0.5
0.1
0.1
0.1
Lysozyme
Lysozy me-T10
@-glucosidase
8-glucosidase-T10
@-glucosidaseFITC dextran 3
lOOmM acetate (PH 5.5) lOOmM acetate (PH 5 . 5 ) l00mM acetate (pH ,5.5)
+
25mM citratephosphate (pH 6.2) 0.01% (w/w) SDS
+
25mM citratephosphate (pH 6.2) 0.01% (w/w) SDS
100 100 100
60 60
100
100
60
100
100
-
-
-
-
-
-
50
0
79
82
41
-
-
m
x
cl
0
cl
1396 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVII (1975) determined by measuring the absorbance a t 280 nm of the supernatant after precipitating undegraded protein with a solution of trichloroacetic acid.5 The percent relative specific enzyme activity was calculated on the basis of the protein content of the preparations and relates to the activity of the nonconjugated enzymes. Lysozyme-T10-A and -B were obtained by chromatography on Sephadex G-100 subsequent to the coupling procedure in order to obtain fractions varying in carbohydrate content. The conjugates prepared with FITC-dext,ran and with the dialyzable starch dextrin were separated from unattached carbohydrate by dialysis. The carbohydrate analysis of these and the lysozyme-T10-A and -B preparations was carried out by gas chromatography of the trimethyl silyl derivatives of the methyl glucosides after methanolysis.6
RESULTS AND DISCUSSION The results obtained previously by us7 and by Marshall and Ilabinowitzs are in agreement with the observations in the present report (Tables I1 and 111). Table I1 shows that a considerable resistance to proteolysis is imparted to lysozyme, chymotrypsin, casein, and serum albumin after conjugation with polysaccharide. The lysozyme-T10-A was more resistant than the lysozyme-T10-B preparation and this may be due to the higher content of carbohydrate. However even the low incorporation of dextrin into the complexes with casein and serum albumin yielded a significant reduction in proteolytic degradation. The thermal inactivation experiments in Table I11 revealed that the conjugates were more stable than the corresponding free enzymes. In the experiments with lysozyme a precipitation of protein could clearly be seen after heating, whereas the conjugates remained completely soluble. It is worth noting that the effect of protein concentration was different for the free lysozyme and for the conjugate. Nakamuri and Hayashi have observed that the carbohydrate constituents of glucose oxidase enhance the thermal stability in the presence of sodium dodecyl sulfate (Sl>S).g On heating the lysozyme and the conjugate with dextran T10 in the presence of 0.0170 SDS, we obtained a pronounced difference between the two preparations (Table 111). The control experiments with nonconjugated proteins were carried out in the presence of the appropriate concentration of free polysaccharide. However, the relatively low concentrations of carbohydrate did not appear to affect the results. The proteolytic activity in the various incubation mixtures were routinely assayed and only minor variations were found. Moreover, the chymotrypsin activity in the lysozyme mixtures remained very high. After five days incubation, it was about 60% of the initial activity. The specific activity of the enzymes was reduced on glycosylation. It is possible that the use of other polysaccharides and/or coupling conditions may yield products of higher activity. One reason for the reduction may be that carbohydrates are attached near or a t the active site of the enzyme molecule. The results obtained with different proteins and different polysaccharides attached by the cyanogen bromide method7.8 as well as the evidence from experiments on natural glycoproteinsg-'* indicate that the stabilization due to carbohydrate constituents may be a general phenomenon. Thus, carbohydrate attached to enzymes could possibly be advantageous for commercial uses. The glycosy-
COhTMUNICATIONS TO T H E EDITOR
1397
lated enzymes may, of course, be linked to an insoluble support and an attachment is made by links between the support and the carbohydrate constituent^.'^ In the case of relatively insoluble enzymes, glycosylation may be used to enhance the solubility when this is desirable. Another possible application is in the field of macromolecules applied in therapy. Thus, heparin coupled to Ficoll (a sucrose polymer produced by Pharmacia, Sweden) has been shown to have a distinctly longer half-life i n vivo than free heparin .I4 A possible explanation for the increased stability of protein-carbohydrate conjugates may be provided by the hypothesis of M a r k ~ s . ' ~According to this, binding of ligands to a polypeptide may reduce the tendency of oscillations between different conformational states. An effect of this nature might reduce the susceptibility to proteolysis as well as to thermal inactivation. References 1 . 1). N. Salter, J. E. Ford, and K. J. Scott, FEES Lett., 20, 302 (1972). 2. L. KHLgedal and S. Akerstrorn, Acta Chem. Scund., 25, 1855 (1971). 3. D. Shugar, Biochim. Biophys. Acta, 8, 302 (1952). 4. It. C. Hughes and R. W. Jeanloz, Biochemistry, 3, 1535 (1964). 5 . M. Kunitz, J . Gen. Physiol., 30, 291 (1947). 6. R. E. Chambers and J. R. Clamp, Biochem. J., 125, 1009 (1971). 7. T. B. Christensen and G. Vegarud, Int. Res. Commun. Syst. (Biochem.), 2, 1311 (1974). 8. J. J. Marshall and M. L. Kabinowitz, Arch. Riochem. Bzophys., 167, 777 (1975). 9. S. Nakamura and S. Hayashi, FEBS Lett., 41, 327 (1974). 10. T. S. A. Samy, Arch. Biochem. Biophys., 121, 703 (1967). 11. A. J. Birkeland and T. B. Christensen, J . Carbohydrates-NucleosidesNucleotides, 2,83(1975). 12. A. Goldstone and H. Koenig, Biochem. J., 141, 527 (1974). 13. 0. It. Zaborsky and J . Ogletree, Bzochem. Bzophys. Res. Commun., 61, 210 (1974). 14. A. Teien, 0. 11. QdegHLrd, and T. B. Christensen, Thromb. Res., in press. 15. G. Markus, Proc. Acad. Sci. U . S., 54, 253 (1965).
GERDVEGARUD TERJEB. CHRISTENSEN Dept. of Biochemistry University of Oslo Blindern, Norway Accepted for Publication June 5, 1976
