Preparation and Characterization of a Dextran-Trypsin Conjugate*? t

(Received for publication, June 16, 1975)

J. JOHN MARSHALL$ AND MARK L. RABINOWITZ

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

Bovine pancreatic trypsin was coupled to dextran after activation of the polysaccharide by cyanogen bromide. The soluble dextran-trypsin conjugate was purified by molecular sieve chromatography. After coupling, ,53% of the esterase activity of trypsin remained, but the conjugate had only 7% of the caseinolytic activity of the native enzyme. The modified trypsin showed greater resistance than the native enzyme to inactivation by heat treatment, autodigestion, or denaturing agents, and was also more resistant to inhibition by trypsin inhibitors, particularly ovomucoid. Treatment with dextranase partly removed the improved stability properties and resistance to inhibition of the trypsin-dextran conjugate. The conjugated enzyme preparation consists of a heterogeneous mixture of macromolecular aggregates, each containing many trypsin and many dextran molecules linked together. Intramolecular cross-linking

of enzyme molecules by polysaccharide chains is considered to be responsible for stabilization of the tertiary structure of the enzyme molecules in the conjugate.

In recent years, much attention has been directed towards

the attachment of enzymes and nonenzymic proteins to insoluble supports such as Sepharose, cellulose, and polyacryl- amide (1, 2). There are, however, few reports of attachment of proteins to soluble polymers. Immobilization often results in increased stability of proteins towards, for example, heat inactivation, proteolytic degradation, and other unfavorable conditions. Conjugates prepared by coupling of enzymes to soluble polymers might also be expected to show the improved stability properties of the corresponding insoluble conjugates.

In the case where the attached polymer is carbohydrate, stabilization may also result from the factors, as yet unknown, which make naturally occurring glycoproteins more stable than their carbohydrate-free counterparts (3-5). Soluble enzyme-

polymer conjugates could be of value when stabilization is required but not when immobilization is unnecessary or even a disadvantage, for example in the enzyme therapy of metabolic disorders (6, 7). We have therefore prepared and characterized a series of such soluble conjugates, particularly those produced by attachment of enzymes to soluble polysaccharides. In this communication we report the coupling of bovine pancreatic trypsin to dextran, and a comparison of the properties of the native and conjugated enzymes.

A preliminary account of part of this work has been published (8).

*This work was supported, in part, by grants from the Heart Association of Greater Miami, Grant GM-21258 from the National Institutes of Health and National Science Foundation Institutional Grant GU 4033.

t Dedicated to Professor Karl H. Slotta on the occasion of his eight- ieth birthday, May 12th, 1975.

$ Investigator of Howard Hughes Medical Institute.

EXPERIMENTAL PROCEDURE

Materials

Tos-Arg-OMe’, Na dodecyl-SO,, dextran (average molecular weight, 40,000), dextranase (from a Penicillium sp., activity 32 units/mg), bovine pancreatic trypsin (activity approximately 160 units/mg), soy- bean trypsin inhibitor, ovomucoid, glycine, and tris(hydroxymethyl)- aminomethane (Tris) were purchased from Sigma Chemical Co. Bac- terial dextranase (Bacillus sp., activity 16 units/mg) was a gift from Beckman Microbics. Bovine pancreatic trypsin inhibitor was prepared as by Dlouha et al. (9), lima bean trypsin inhibitor was purchased from Worthington Biochemical Corp., and casein (Hammersten) was obtained from Schwartz/Mann. Urea was from Baker Chemical Co.; cyanogen bromide and 2.mercaptoethanol were from Eastman Organic Chemicals. Sephadex G-100 and Sepharose 4B were purchased from Pharmacia Fine Chemicals. All other chemicals were reagent grade.

Methods

Assay of Trypsin Actiuity-Trypsin activity was routinely measured spectrophotometrically by the method of Hummel (10). Enzyme (native or conjugated, 0.1 to 0.3 unit, usually in a volume of 0.1 ml) was added to a solution (2.9 ml) of buffered Tos-Arg-OMe. The final composition of the standard digests was Tos-Arg-OMe (1 mM), Tris buffer (40 mM, pH 8.1), calcium chloride (10 mM), and enzyme. In some cases, as indicated, borate buffer (87 mM, pH 8.1) was substituted for Tris. One unit of trypsin is the amount which causes the hydroly- sis of 1 pmol of Tos-Arg-OMe/min at 25” under these conditions. Caseinolytic activity of trypsin was determined by the method of Kunitz (ll), as described by Bergmeyer (12). Except where otherwise indicated, all dilutions of trypsin solutions were performed in plastic tubes to minimize losses caused by binding to glass.

Determination of Carbohydrate-Carbohydrate was measured by the phenol-sulfuric acid method (13), calibrated against glucose. Cyanogen bromide treatment of dextran results in a marked decrease in the color production in the phenol-sulfuric acid reaction; for this

‘The abbreviations used are: Tos-Arg-OMe, p-toluenesulfonyl-L- arginine methyl ester; Na dodecyl-SO,, sodium dodecyl sulfate.

1081

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1082 Dex tran- Trypsin Conjugate

reason carbohydrate contents of dextran-trypsin conjugates are appar- ent, rather than true, values.

Measurement of Protein-Protein contents of enzyme solutions were determined by using the method of Lowry (14). In column fractions protein was usually located qualitatively by measurement of the ultraviolet absorbance (A,,,).

Gel Filtration-Chromatography was performed at 4’ on columns (90 x 1.5 cm) of Sephadex G-100 or Sepharose 4B prepared according to the manufacturer’s recommendations (15). Elution was with 50 mM acetate buffer, pH 5.0, containing 10 rnM sodium chloride, and 3.0.ml fractions were collected automatically.

Polyacrylamide Gel Electrophoresis-Polyacrylamide gel electro- phoresis was carried out in the presence of Na dodecyl-SO, and 2mercaptoethano1, according to Weber and Osborn (16, 17). Protein samples were denatured by addition to a solution of Xa dodecyl-SO, and Zmercaptoethanol in phosphate buffer at 100”, and kept at this temperature for 5 min before loading on the gels.

RESULTS

Preparation of Dextran-Trypsin Conjugate-To a stirred solution of dextran (2.5 g) in water (250 ml), adjusted to pH

10.7 with 500 mM sodium hydroxide solution, cyanogen bro- mide (0.625 g) was added, followed by a second addition of cyanogen bromide (0.625 g), 30 min later. The pH was maintained at 10.7 during this process by addition of sodium hydroxide solution (500 mM). Thirty minutes after the second addition of cyanogen bromide, the pH was adjusted to 9.0 by addition of 100 mM hydrochloric acid solution. After dialysis at 4” for 2 hours, against 4 liters of sodium carbonate solution, pH 9.0 (prepared by addition of 1.0 M sodium carbonate solution to

distilled water until the pH reached 9.0), trypsin (0.25 g) was added. The pH was maintained at 9.0 during addition of trypsin, by addition of sodium carbonate solution (200 mM).

Coupling of trypsin to cyanogen bromide-activated dextran was then allowed to proceed during 12 hours at 4”. After this period, the solution was dialyzed for 2 hours at room tempera- ture against 4 liters of sodium carbonate solution (prepared as above), then 20 ml of glycine solution (100 mg/ml) were added. After standing for a further 12 hours at 4” the product (4.40 g) was isolated by lyophilization.

Purification of De&ran-Tvpsin Conjugate-A sample (200 mg) of the product was chromatographed on Sephadex G-100,

and the column fractions were analyzed for carbohydrate, protein, and trypsin activity. The majority of the enzyme activity was eluted at the void volume of the column, associ- ated with dextran (Fig. 1A). A control run, in which appropri- ate amounts of dextran and trypsin were mixed and subjected to chromatography on the same column, showed that the two components separated under such conditions (Fig. 1B). Thus, it was concluded that the trypsin had been covalently attached to the dextran. Fractions 12 to 16 from the column were

combined as purified conjugate. Analysis showed the purified conjugate to have a carbohydrate content of 89%. The specific activity of trypsin in the conjugate, determined with Tos-Arg- OMe as substrate, was 88 units/mg, this being 53% of the specific activity of the native enzyme. The caseinolytic activity of the conjugate was 0.24 Kunitz units/mg; this being 7% of that of native trypsin (3.3 Kunitz units/mg). The solution of purified conjugate obtained in this way was used for character- ization.

Polyacrylamide Gel Electrophoresis of Dextran-Trypsin Conjugate-Further evidence for covalent attachment of tryp- sin to dextran, with formation of a high molecular weight

product, came from a comparison of the behavior of free trypsin and the Sephadex G-loo-purified conjugate, on gel electrophoresis under denaturing conditions. The native en-

16r

50-

A

FRACTION NUMBER

FIG. 1. Sephadex G-100 column chromatography of (A) dextran- trypsin conjugate and (B) dextran-trypsin mixture. In each case the sample applied to the column contained approximately 115 mg of dextran and 11 mg of trypsin. Fractions were analyzed for protein by absorbance at 280 nm (- -), carbohydrate (---I and trypsin activity (-)

zyme migrated as a single band corresponding to a polypeptide of the expected molecular weight (23,300). The conjugated enzyme showed a band of protein at the top of the gel, indicating that it was of a molecular weight too large to permit migration through the gel. Material with mobility correspond- ing to that of unmodified trypsin could not be detected. The stained gels are shown in Fig. 2.

pH Optima for De&ran-Trypsin Conjugate and Native Trypsin-pH activity curves were determined by measurement of the rate of hydrolysis of Tos-Arg-OMe in digests of the standard composition, with Tris buffer (40 mM) of various pH values from 6.8 to 9.5, containing native enzyme (1 fig) or conjugated enzyme (2 fig of protein). The pH activity curves for both forms of the enzyme were closely similar, maximum activity being exhibited at pH 8.1 in both cases.

K, Values for Dentran-Trypsin Conjugate and Native Typsin-K, values for trypsin and its dextran conjugate were determined from Lineweaver-Burk plots, constructed by mea- surement of the rate of hydrolysis of Tos-Arg-OMe in standard digests in which the substrate was present at concentrations of 0.2 to 1 mM. The K, values for both forms of the enzyme were identical (0.078 mM).

Heat Stability of Dextran-Trypsin Conjugate and Native Tvpsin-Solutions of native trypsin (10 wg/ml) and its dex- tran-conjugate (20 rg/ml) in 1 mM hydrochloric acid solution were heated at 60”, and samples (0.1 ml) were removed at intervals from each for activity determination. The results are

shown in Fig. 3, for experiments performed in both glass test tubes and plastic test tubes.

When solutions of trypsin (10 wg/ml) and dextran-trypsin

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Dextran-Trypsin Conjugate 1083

FIG. 2. Polyacrylamide gel electrophoresis in the presence of Na dodecyl-SO, and 2-mercaptoethanol of native trypsin (left) and trypsin- dextran conjugate (r&t).

conjugate (20 pg/ml) in 1 mM hydrochloric acid solution were heated at 100” for 30 min, 36% loss of activity of the conjugate took place, as compared with 54% in the case of the native enzyme. In neither case was any significant amount of activity found to return after storage at 0” for 60 min.

Loss of Activity of Dextran-Trypsin Conjugate and Native Trypsin by Autodigestion-Solutions of native trypsin (14 &ml) and dextran-trypsin conjugate (26 pg/ml) in 80 mM borate buffer, pH 8.1, were incubated at 37”. At intervals samples (125 ~1) were assayed for residual activity in standard digests, buffered with 87 mM borate buffer, and containing 10 mM calcium chloride. The results are shown in Fig. 4.

Stability and Activity of Dextran-Trypsin Conjugate and Native Trypsin in Presence of Denuturants-Native trypsin

0’ I I I I I I 20 40 60 80 100 120

DURATION ’ OF HEATING (men)

FIG. 3. Heat inactivation of trypsin (0) and dextran-trypsin conju- gate (0) at 60” in 1 mM hydrochloric acid solution. Results are shown for experiments performed in glass tubes (- - -) and plastic tubes c-4.

01 I I I 1 I I 20 40 60 80 100 120

DURATION OF HEATING (mln)

FIG. 4. Autodigestion of trypsin (0) and dextran-trypsm conjugate (0) at pH 8.1 and 37”.

(10 pg/ml) and dextran-trypsin conjugate (20 pg of protein/ml) were incubated at 37” in Tris-HCl buffer (46 mM, pH 8.1) containing calcium chloride (11.5 mM) and 8 M urea. The same experiment was also performed with 5 mM 2-mercaptoethanol present. In both cases samples (0.1 ml) were removed at intervals and assayed for activity using standard digests. The results are shown in Fig. 5, a and b.

Native trypsin (10 pg/ml) and dextran-trypsin conjugate (20 fig of protein/ml) were incubated at 37” in borate buffer (100 mM, pH 8.1) containing 1% Na dodecylS0,. Samples (0.1 ml) were removed at intervals and assayed for activity using standard digests (borate buffer). The results are presented in Table I.

The effect of different urea concentrations on the inactiva- tion of native trypsin and the dextran-trypsin conjugate was also investigated. Native trypsin (10 fig/ml) and dextran-tryp- sin conjugate (20 fig of protein/ml) were incubated at 37” in borate buffer (100 mM, pH 8.1), containing calcium chloride (11.5 mM) and urea (0 to 8 M). Samples (0.1 ml) were removed after 60 min and assayed for activity using standard digests (borate buffer). The results are presented in Table II.

The esterase activity of both forms of trypsin in the presence of urea was measured by incorporating native enzyme (1 fig) or conjugated enzyme (2 fig) into standard digests (borate buffer, with and without calcium chloride (10 mM)), containing 8 M

urea. To compare the caseinolytic activity of native and conjugated trypsin in the presence of urea the method of Kunitz (11) was used. Since both forms of the enzyme were essentially inactive in the assay at 8 M urea concentration, the activities

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1084 De&ran-Trypsin Conjugate

a80- . ii?L

FIG. 5. Inactivation of trypsm (0) 2 \

and dextran-trypsin conjugate (0) at z 60- l \. 37” and pH 8.1 by 8 M urea in the 2 absence (a) and presence (5) of 5 mM s 2-mercaptoethanol. The broken line in z A’

l 1. (b) shows the rate of inactivation of 2 dextran-trypsm conjugate after treat- & ment with dextranase. Q: 20-

a 0

I I I I I ,

20 40 60 80 100 120

DURATION OF INCUBATION (mm)

Loss of actiuity of native trypsin and dextran-trypsin conjugate in Na dodecyl-SO, (1%)

TABLE I TABLE III

Actiuity of native trypsin and dextran-trypsin conjugate in urea

Native or conjugated enzyme was incorporated into digests contain- ing Tos-Arg-OMe in 8 M urea (esterase activity) or casein in 6 M urea (protease activity). The measured activity in each case is expressed as a percentage of the activity in the absence of urea.

Native trypsin (IO rg/ml) and dextran-trypsin conjugate (20 pg of protein/ml) Were incubated at 37” in borate buffer (100 m&t, pH 8.1) containing 1Yr Na dodecylS0,. Samples (0.1 ml) were removed at intervals and assayed for activity on Tos-Arg-OMe. Activities are expressed as percentages of the activity in the absence of Na dodecyl- SO,.

Activity

Duration of incubation

(min)

Aproximately 1 20 40 60

Activity remaining

Conjugate Native

9% 47 0 30 0 29 0 25 0

Enzyme Esterase (8 M urea) Protease (6 M urea)

+Ca2+ caz+ +Ca*+ -ca*+

% Native 30 0 26 21 Conjugated 73 50 38 21

TABLE II

Loss of actiuity of native trypsin and dextran-trypsin conjugate in different concentrution$ of urea

containing 11.5 mM calcium chloride (50 ~1) for 7 min at 25”. The amount of activity remaining after this period was determined in standard digests (borate buffer) and compared with the activity of the same amount of enzyme preincubated under identical conditions, in the absence of any inhibitor. The results are shown in Table IV.

Native trypsin (10 &ml) and dextran-trypsin conjugate (20 ).~g of protein/ml) were incubated in borate buffer (0.1 M, pH 8.1) containing calcium chloride (11.5 mM) and urea (0 to 8 M). Samples were removed after 60 min and assayed for activity on Tos-Arg-OMe. Activities are expressed as percentages of the activity in the absence of urea.

Activity remaining Urea concentration

Conjugate Native

CM) 70 0 100 100

2 100 92

4 100 81 6 100 43 8 91 78

The inhibition of trypsin and its dextran-conjugate by ovomucoid was examined in more detail -by preincubating native trypsin or dextran-trypsin conjugate (0.275 units in each case) with different amounts (0.25 to 20 pg) of ovomucoid as

above, and measuring the amount of residual activity. The results are illustrated in Fig. 6.

were compared at 6 M urea concentration. Digests were

buffered with borate (75 mM) and were performed with and without calcium (10 mM). The results are presented in Table III.

Inhibition of Dextran-Trypsin Conjugate and Native Tryp- sin by Trypsin Inhibitors-The effect of a number of trypsin inhibitors on both forms of trypsin was compared by prein- cubating native trypsin or dextran-trypsin conjugate (0.275 units in each case) with trypsin inhibitors (lima bean, soybean, ovomucoid, bovine pancreatic trypsin inhibitor, 5 fig) or normal human serum (25 ~1) in 100 mM borate buffer, pH 8.1,

Dextranase Treatment of De&ran-Trypsin Conjugate-Di- gests (0.1 ml) were prepared containing dextran-trypsin conju- gate (1 pg of protein), dextranase (bacterial or fungal, 16 units), and acetate buffer (50 mM, pH 5.5). After incubation at 37” for 3 hours, the activity towards Tos-Arg-OMe was determined by using standard digests. The activities in control digests containing dextran-trypsin conjugate incubated at 37” for 3 hours without dextranase and native trypsin incubated at 37’ for 3 hours with and without dextranase were also determined. Esterase activity of the dextran:trypsin conjugate was not increased by this treatment. Measurement of the activity of the dextran-trypsin conjugate towards casein, after dextranase treatment in a similar manner, showed there to be no effect on the caseinolytic activity of the conjugate.

To determine the effect of dextranase treatment on the stability properties of the dextran-trypsin conjugate, digests (2.0 ml) were prepared containing conjugate (11 rg of protein) and fungal dextranase (18 mg) in 10 mM acetate buffer, pH 5.5, and 20 mM calcium chloride. After incubation at 37” for 3 hours, the loss of activity of the dextranase-treated conjugate

b

20 40 60 80 100 120

DURATION OF HEATING (mm)

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Dextran-Trypsin Conjugate 1085

TABLE IV

Inhibition of natwe trypsm and de&ran-trypsin conjugate by trypsin inhibitors

Native and conjugated enzymes (0.275 unit) were Incubated with trypsin inhibitors (5 pg), or serum (25 ~1) for 7 mm. The activity remaining after this treatment is expressed as a percentage of the activity in controls in which the same amount of enzyme was preincubated in the absence of inhibitor.

Activity remaining

Enzvme BCW1ne pancreatic Lma bean Soybean

trypsm trypsm trypun Ovomucmd SeIULll

mhibenr mhlbltor inhIbItor

%

Native 6 6 6 9 43 Conjugated 13 24 29 70 71

100 r l - l -•

80 -

s 60-

/

p

./

/ .

2

-“i I’

0- 0-O 0

8 20

/ o If I I I I I I , I

100 200 300 400 500 600 700 800

OVOMUCOID (gg)

FIG. 6. Inhibition of trypsin (0) and dextran-trypsin conjugate (0) by various amounts of ovomucoid.

in 8 M urea/5 mM 2-mercaptoethanol was determined in the same way as for the native enzyme and the untreated conjugate (see above). The results are shown by the broken line in Fig. 5b. Dextranase treatment had no effect on the stability of native trypsin under the same conditions.

After treatment of dextran-trypsin conjugate (2.7 hg of protein) with fungal dextranase (1.0 mg) in 10 mM acetate buffer, pH 5.5, at 37” for 3 hours, the susceptibility to inhibition by ovomucoid (16 rg) was compared with that of the untreated conjugate. The extents of inhibition under the conditions described above were 30% (untreated conjugate) and 54% (dextranase-treated conjugate).

It was not possible to determine the amount of carbohydrate removed enzymically from the dextranase-trypsin conjugate because of interference arising from the presence of large amounts of carbohydrate in the dextranase preparation.

Fractionation of DextraniTrypsin Conjugate on Sepharose 4B-Unpurified dextran-trypsin conjugate (250 mg) was dis- solved in 1.5 ml of eluting buffer, then chromatographed on Sepharose 4B. For comparison purposes, appropriate amounts of dextran and trypsin were mixed and chromatographed in the same way. Fractions from both columns were analyzed for protein and carbohydrate and assayed for trypsin activity. The results are shown in Fig. 7, A and B. Fractions from the column were also tested for their susceptibility to inhibition by ovomucoid. Samples from selected fractions (containing 0.275 unit of trypsin activity) were treated with 20 pg of ovomucoid as before, and the extent of inhibition after preincubation for 7 min was determined. Tests on the stability of different fractions (for example, during treatment with Na dodecylS0,

Id-

12 -

P > lo- r p OS- <

g 06- z s 04- s

02-

O-

20 -

18 -

lb -

14 -

12 -

IO -

8-

b-

4-

2-

o-

250

8- 150- :^: B

7- : :

3 l- ,[ oi , , (c --“,‘--*‘: k ( /

15 20 25 30 35 40 45 50

FRACTION NUMBER

FIG. 7. Sepharose 4B chromatography of (A) dextran-trypsin conju- gate and (B) dextran-trypsin mixture. In each case the sample apphed to the column contained approximately 145 mg of dextran and 14 mg of trypsin.

and 2-mercaptoethanol as before) showed little difference in stability. Neither was the residual caseinolytic activity in the conjugate associated with any particular fractions.

The heterogeneity of the conjugate with respect to carbohy- drate content, specific activity, and susceptibility to inhibition by ovomucoid is illustrated in Table V.

DISCUSSION

Many methods have been developed for attaching proteins to insoluble polysaccharides (1). One of the simplest procedures (18) uses cyanogen bromide to introduce into polysaccharides imidocarbonate functional groupings, which can then react with t amino groups of lysine, forming covalent linkages between polysaccharide and protein. A modification of this method, employing low concentrations of cyanogen bromide to avoid irreversible precipitation of the polysaccharide during the activation reaction, has enabled us to synthesize a soluble dextran-trypsin conjugate. In contrast to the procedure for producing a Bacillus amyloliquefaciens a-amylase-dextran conjugate, where an active product was obtained simply by adding enzyme directly to the activated polysaccharide,2 it was necessary to incorporate a dialysis step, to remove by- products of the activation reaction, prior to coupling trypsin with activated dextran. After coupling, glycine was added to saturate unreacted imidocarbonate groupings and to prevent initially soluble conjugate from becoming insoluble during freeze-drying.

Evidence for covalent attachment of trypsin to dextran in the product came from gel chromatography on Sephadex G-100 and polyacrylamide gel electrophoresis. The former procedure showed the absence of enzymic activity at the normal position

* Marshall, J. J. (1976) Carbohyd. Res., in press.

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1086 Dextran-Trypsin Conjugate

TABLE V

Heterogeneity of dextran-tppsin conjugate

Unpurified dextran-trypsin conjugate was chromatographed on a column of’ Sepharose 1H (Fig. ;a). Selected fractions were analyzed for protein. carbohydrate, and trypsin activity. ‘The inhibition of 0.55 unit 01 acti\ ity from these tractions hy ‘0 ~g ot ovomucoid was also determined.

’ X.11.. not determined

of elution of trypsin: rather it emerged from the column at the void volume. associated with dextran (Fig. 1, A and B). On electrophoresis in the presence of sodium dodecyl sulfate and 2.mercaptoethanol (Fig. 2), protein stains showed that the

modified trypsin did not penetrate the polyacrylamide gel, indicating that it is of very high molecular weight. Native

trypsin migrated as was expected of a protein with molecular weight 23,300 (Fig. 2).

Conjugated trypsin, after purification, had a specific activity of 53% of that of the native enzyme, measured with Tos-Arg- OMe. The caseinolytic activity was, however, only 7% of that of native trypsin, indicating unfavorable steric interaction he- tween the conjugated enzyme and its macromolecular suh-

strate, a phenomenon also observed after attachment of

proteases to insoluble supports (1). Native and conjugated enzyme had the same K, for Tos-Arg-OMe, showing that attached carbohydrate does not interfere with action on the

ester substrate. Caseinolytic activity could not he increased by treating the conjugated enzyme with dextranase. In view of the poor activity of conjugated trypsin towards macromolecular substrate, most experiments were performed with the ester substrate.

The effect of pH on activity was closely similar for.the native and conjugated forms of trypsin. This finding is not surprising

in view of the uncharged nature of the carbohydrate attached to the enzyme.

Heat treatment of trypsin and its dextran conjugate at 60” in 1 rnM hydrochloric acid in glass tubes showed a much faster loss of activity of the native enzyme than of the dextran conjugate (Fig. 3, broken lines). In plastic tubes, there was still a significantly faster rate of inactivation of the native than of the conjugated enzyme (Fig. 3, solid lines), but the IOSS of activity of the former was much slower than in the glass tubes. In view of the possibility that the difference in the results

obtained using glass and plastic tubes resulted from spurious binding to glass, all further experiments were performed in plastic tubes. When heated at 100” the conjugated enzyme lost

36% and the native enzyme 54% activity in 30 min. However, although carbohydrate attachment stabilizes trypsin against heat inactivation, it was not possible to demonstrate that it assists renaturation after heat inactivation at 100”.

Autodigestion, at pH 8.1 and 60” in the absence of calcium resulted in loss of 80% of the activity of native trypsin in 96 min; the conjugated enzyme lost less than 10% activity under

these conditions (Fig. 4). The small amount of activity lost by the conjugated enzyme may even he due to heat inactivation

(cf. Fig. 3), rather than to proteolysis. Attachment of carhohy- drate to trypsin, therefore, renders the enzyme essentially completely resistant to autodigestion. It remains to he deter- mined whether it is possible to prepare a conjugated form of trypsin which is resistant to autodigestion hut still has significant protease activity, e.g. towards casein.

Denaturing agents (urea, Na dodecylS0,) inactivate tryp- sin rapidly, particularly in the presence of a reducing agent such as 2-mercaptoethanol. However. the conjugated enzyme is denatured at a much slower rate by these agents (Tables I and II; Fig. 5, A and B). As well as increasing the stability OI

trypsin in denaturants, conjugation also causes a significant increase in the activity of the enzyme in urea solution, this being most apparent in the case of esterase activity where the activity of the conjugate is more than double that of the native enzyme (Table III). Treatment of the conjugate with dextran- ase destabilizes the conjugate (Fig. 5B). Attached carhohy- drate clearly stabilizes the tertiary structure of the enzyme, an effect which is considered in greater detail below.

Conjugated trypsin shows resistance to inhibition by pro- tease inhibitors, the difference between the extents of inhihi- tion of the native and modified enzyme increasing as the mo- lecular weight of the inhibitor increases (Table IV). Serum, which contains trypsin inhibitors, also causes a lower extent of

inhibition of the conjugated enzyme than of the native enzyme. Dextranase treatment of conjugated trypsin results in a signifi- cant increase in the extent of inhibition by ovomucoid, showing that removal of carbohydrate increases the accessibility of inhibitor to the active site of the conjugated enzyme molecules.

It is pertinent to consider the molecular nature of the dextran-trypsin conjugate. Fractionation on Sepharose 4B (Fig. 7) indicates that the product consists of a heterogenous

population of macromolecular aggregates, the largest of which must contain many trypsin and many dextran molecules. Such aggregates will he produced by intermolecular reaction he- tween activated dextran molecules (each of which contains many activated residues) and trypsin molecules (each of which has more than one c amino group with which imidocarbonate groupings can react).

Fig. 6 indicates that a large proportion of the conjugated enzyme is completely resistant to inhibition by ovomucoid, rather than all molecules being inhibited at a slower rate than in the case of the native enzyme. The extent of inhibition of conjugated trypsin decreases as the molecular size increases (Table V). Thus, the larger dextran-trypsin aggregates contain

higher proportions of buried enzyme molecules which are inaccessible to trypsin inhibition. Smaller trypsin inhibitors can, however, reach the buried trypsin molecules more easily than can larger inhibitors (Table IV). Dextranase treatment causes partial breakdown of the conjugate, resulting in a smaller proportion of buried trypsin molecules and increased susceptibility to inhibition.

Protease activity of conjugated trypsin does not vary with

the molecular size of the conjugated molecules. nor does it increase after treatment with dextranase. It can, therefore, be concluded that carbohydrate attachment per se is responsible for the drastic reduction in protease activity, rather than concomitant burying of enzyme molecules resulting from

cross-linking. In addition to intermolecular reactions between dextran and

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Dextran-Trypsin Conjugate 1087

trypsin, which cause aggregation, a second type of dextran- trypsin interaction can occur. This is the reaction of two or more activated monosaccharide residues in a single polysac- charide chain with two or more t amino groups of trypsin in a :tqgle enzyme molecule, introducing cross-links into trypsin molecules. That such interactions occur, and play an important part in the stabilization of trypsin, is apparent from the de- naturation experiments in 8 M urea and 2-mercaptoethanol (Fig. 5B). The disulfide bridges of native trypsin are reduced and the protein unfolds rapidly, complete loss of activity taking place within about 5 min. On the other hand, the dextran-trypsin conjugate is only slowly inactivated, retaining over 50% of its activity after 2 hours. Dextranase treatment of the conjugate substantially removes the stabilization, as a result of enzymic cleavage of some cross-links. It can be con- cluded that stabilization of trypsin by carbohydrate attach- ment results, to a large extent, from cross-linking. The car- bohydrate bridges which are introduced have an effect on the conformation of the enzyme similar to that of disulfide bridges.

The rationale for attempting to stabilize enzymes by attach-

ment of carbohydrate was that glycoproteins often show unusual stability properties compared with carbohydrate-free enzymes, the former being much less sensitive to heat and other denaturing conditions and more resistant to proteolysis (3-5). Attachment of carbohydrate results in the predicted stabilization, but the role of carbohydrate in stabilizing the

enzyme is largely different from the stabilizing role of carbohy- drate in natural glycoproteins. In the latter there is no contribution to the stability from cross-linking; each carbohy-

drate chain is attached to the polypeptide backbone through a single linkage (19).

1.

2.

:3.

4.

5.

6.

7.

8.

9.

10. 11. 12.

13.

14.

15.

16. 17.

18. 19.

REFERENCES

Zaborsky, 0. R. (1973) Immobilized Enzymes, C.R.C. Press, Cleveland

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J J Marshall and M L RabinowitzPreparation and characterization of a dextran-trypsin conjugate.

1976, 251:1081-1087.J. Biol. Chem. 

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