ARCHlVES OF BIOCHEMISTRY AND BIOPHYSICS 167, 777-779 (19%)

Enzyme Stabilization by Covalent Attachment of Carbohydrate

Soluble enzyme-carbohydrate conjugates have been prepared by coupling trypsin, a- amylase, and @amylase to cyanogen bromide activated dextran. All three conjugates are more stable to heat than the respective native enzymes. ,Loss of trypsin activity by autolytic digestion is also decreased by attachment of carbohydrate.

Glycoproteins often show unusual stability charac- teristics compared with carbohydrate-free proteins, the former being less sensitive to heat and other denaturing conditions, and more resistant to proteol- ysis (l-4). We have attempted to endow improved stability to a number of carbohydrate-free enzymes by covalent attachment of carbohydrate to them. We report, in this communication, the results of stability studies on three carbohydrate-enzyme conjugates, prepared by coupling of dextran to bovine pancreatic trypsin, Bacillus amyloliquefaciem o-amylase and sweet-potato @-amylase.

Feasible methods for attachment of carbohydrates to proteins through glycosidic linkages, such as occur in natural glycoproteins (1, 5) are not available. However, the syntheses of carbohydrate-protein con- jugates involving coupling by linkages of other types have been described. For the most part, these methods have been developed for the immobilization of enzymes on insoluble polysaccharides (6). We have selected one of the simplest of these methods for the studies reported here. After “activation” of carbohy- drate by treatment with cyanogen bromide, addition of protein results in coupling through the activated monosaccharide residues in the polysaccharide and the c-amino groups of lysine in the protein (7). When this procedure was applied directly for the synthesis of soluble polysaccharide-protein conjugates, immediate precipitation of the polysaccharide took place during the activation procedure. The same problem was experienced by Kagedal and Akerstrom (8) during their attempts to synthesize soluble dextran-insulin conjugates. I-Iowever soluble conjugates were success- fully prepared by using lower concentrations of cyano- gen bromide than that used earlier for the activation of insoluble polysaccharides.

Dextran solutions (clinical grade, 2.5 g dissolved in 250 ml water) were adjusted to pH 10.7 with sodium hydroxide and treated with two portions (0.625 g each) of cyanogen bromide, the second being added 30 min after the first. The pH was maintained at 10.7 during the activation procedure by addition of sodium hydroxide so:lution (0.5 M). Sixty minutes after the first addition of cyanogen bromide the pH was ad- justed to 9.0 ,with 0.1 N HCl, and the enzyme (0.25 g) added. The pH was readjusted to 9.0 by addition of

sodium carbonate solution (0.2 M) and coupling al- lowed to proceed during 12 h at 4°C. Glycine (2.0 g) was then added to saturate any unsubstituted reac- tive groupings in the polysaccharide, and the polysac- charide-protein conjugates were isolated by freeze- drying. In some cases, dialysis of the activated poly- saccharide was performed before coupling of the enzyme, to prevent excessive losses of activity. The conjugates were separated from small amounts of residual uncoupled enzyme by chromatography on Sephadex G-106 or G-200. In most cases the recovery of enzyme activity after the coupling and purification procedures was better than 50% of the original activ- ity.

Figures 1 and 2 show the loss of activity of the (Y- and /3-amylase conjugates during heat treatment, compared in each case with the corresponding native enzyme. In both cases, marked resistance to heat inactivation is observed. Thus the half-life of Bacillus amyloliquefaciens a-amylase at 65°C is increased from 2.5 min to 63 min (Fig. l), and that of sweet- potato fl-amylase at 60°C is increased from 5 min to 175 min (Fig. 2). In the case of trypsin heated at 60°C in 1 mM hydrochloric acid solution, an initial small (5-10s) loss of activity of the conjugated enzyme took place at the same rate as for the native enzyme, but thereafter the rate of inactivation decreased to 0.06 that of the native enzyme. Because the rate of activity loss was nonlinear and 50% inactivation was not reached in the duration of the experiment, half lives cannot be quoted for this enzyme and its dextran conjugate.

Figure 3 shows the loss of activity of trypsin and its dextran conjugate heated at 37°C under conditions favoring autolytic digestion (pH 8.1 in 100 mM borate buffer in the absence of calcium). After attachment of carbohydrate there is a marked reduction in the loss of activity by autolysis.

It is pertinent to consider the nature of the stabiliz- ing effect of the attached carbohydrate, and whether it is related to the role played by carbohydrate in natural glycoproteins. It seems likely that the im- proved resistance of trypsin to autolytic degradation after carbohydrate attachment is the result of steric interactions between carbohydrate and protease hin- dering attack on the polypeptide backbone of the

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778 COMMUNICATIONS

enzyme. Carbohydrate oligomers in ovine submaxil- lary gland glycoprotein function in the same way to prevent proteolysis (9). There is no evidence regarding how the carbohydrate in glycoproteins serves to confer heat stability, although a possible explanation (10) is that heat stability is associated with the degree of hydration, this being influenced, in turn, by the carbohydrate content. While the improved heat sta- bility of the enzyme-dextran conjugates could be explained in the same way, there is an alternative explanation. It seems more likely that the observed phenomenon results from stabilization of the tertiary structure and, in the case of sweet-potato fl-amylase which is a tetrameric protein (ll), the quaternary

o I.--.---

,0 20 30 40 50 60

DURATION OF HEATING tmd

FIG. 1. Loss of activity on heat treatment at 65°C of Bacillus amyloliquefaciens a-amylase (0) and its conjugate with dextran (0). Native and modified enzyme solutions (5.5 pg of protein/ml) in water were heated and samples removed for activity determina- tion at different times. cY-Amylase activity was deter- mined by the release of reducing sugars from soluble starch by using the dinitrosalicylic acid method (12).

DURATION OF HEATING h)

FIG. 2. Loss of activity on heat treatment at 60°C of sweet-potato @-amylase (0) and its conjugate with dextran (0). Samples (50 ~1) of native and modified enzyme (activity 0.5 U/ml) were heated for different lengths of time then the activity remaining was determined by release of reducing sugars from starch by using a copper-reducing power method (13).

01 ’ ’ I ’ ’ ’ 20 40 60 80 100 120

DURATION OF HEATING jm,nJ

FIG. 3. Loss of activity on heating trypsin (0) and its conjugate with dextran (0) at 37 “C. Samples (125 ~1) of native and modified enzyme (activity 2 U/ml measured towards p-toluene sulfonyl-r.-arginine methyl ester) were heated for different lengths of time in 80 mM borate buffer pH 8.1, then the activity remaining determined by the method of Hummel

(14).

structure, by cross-linking. This would result from two or more activated monosaccharide residues in the cyanogen bromide treated polysaccharide reacting with two or more e-amino groups in different parts of the polypeptide chain. The overall effect of such cross-linking on the tertiary and quaternary struc- tures of the enzymes is probably akin to the role played by disulfide bridges in maintaining protein conformation. This was shown most convincingly by incubation of native and conjugated trypsin in urea (8 M) and 2-mercaptoethanol (5 mM), conditions which completely inactivated the native enzyme within 7 min. The conjugated enzyme retained 81% of its activity under the same conditions in this time, indicating that the carbohydrate serves to prevent unfolding of the enzyme molecules.

Thus it is clear that attachment of enzymes to soluble carbohydrate polymers can result in the same sort of modifications in stability properties which are often observed after enzyme immobilization (6). Mod- ification in this way, particularly of enzymes which are very labile, may offer an attractive alternative to immobilization when improved stability properties, rather than a solid-phase product, are the primary requirement. Detailed characterization of the three enzyme conjugates described herein will be presented elsewhere.

ACKNOWLEDGMENTS

This work was supported by grants from the Heart Association of Greater Miami, National Institutes of Health (GM-21258) and NSF Institutional grant GU 4033. The technical assistance of Mrs. Hortensia Miyar is gratefully acknowledged.

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REFERENCES

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7. AXEN, R., AND ERNBACK, S. (1971) Eur. J. Bio- them. 18, 351-360.

8. KAGEDAL, L., AND AKERSTROM, S. (1971) Acta Chem. &and. 25, 1855-1859.

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Physiol. 37, 139331399.

J. J. MARSHALL’ M. L. RABINOWITZ

Laboratories for Biochemical Research, Howard Hughes Medical Institute, Miami, Florida and Department of Biochemistry, University of Miami School of Medicine, Miami, Florida 33152

Received November 14, 1974

’ Investigator of the Howard Hughes Medical Insti- tute.