stability characteristics of chemically-modified soluble trypsin

ELSEVIER Journal of Biotechnology 49 (1996) 163-171

Stability characteristics of chemically-modified soluble trypsin

Ann Murphy, Ciarkn 6 FAgBin*

School of Biological Sciences, Dublin City University, Dublin 9, Ireland

Received 5 October 1995; revised 22 April 1996; accepted 22 April 1996

Abstract

Bovine pancreatic trypsin was modified with acetic acid N-hydroxy-succinimide ester (AANHS) which neutralizes the positive charge on lysine residues. Approximately eight out of 14 lysine residues per trypsin molecule were modified. The AANHS-treated trypsin showed enhanced thermostability compared with the native between 30 and 70°C. Tm values for native and AANHS trypsin were 46°C and 51”C, respectively. At 55°C the modified trypsin’s half-life is doubled to 8.7 min versus 4.3 min for the native enzyme. AANHS trypsin exhibited a decreased rate of autolysis and also showed enhanced stability at 65°C in aqueous mixtures of the following organic solvents: 1,4-dioxan, dimethylformamide, dimethylsulphoxide and acetonitrile.

Keywords: Enzyme stability; Organic solvents; Succinimide; Trypsin

1. Introduction

There is much interest in the use of proteases in

peptide synthesis (Kasche and Haufler, 1984;

Morihara, 1987; Andersen et al., 1991). They offer numerous advantages over conventional

Abbretiiations: AANHS, acetic acid N-hydroxy succinimide

ester; BAPNA, benzoyl-Dr-arginine p-nitroanilide; DC, denat-

uration capacity; DMF, dimethylformamide; DMSO, dimethyl

sulphoxide; NPGB, p-nitrophenyl-p’-guanidinobenzoate;

PAGE, polyacrylamide gel electrophoresis; SDS, sodium dode- cyl sulphate; THF, tetrahydrofuran; TNBS, 2,4,6_trinitroben-

zene sulphonic acid.

*Corresponding author. Fax: + 353 1 7045412; e-mail:

[email protected]

chemical methods including reduction of racem- ization and side reactions. However there are drawbacks associated with their use, including destabilization and loss of activity both at high temperatures and at high concentrations of organic solvents. Another major problem is autolysis which can occur when proteases are in solution. It is, therefore, often necessary to mod- ify the enzyme so as to improve its stability. Conventional modifications include immobilization and crosslinkine. which have been shown to enhance protease stability (Means and Feeney, 1990; Gianfreda and Scarfi, 1991). Recently protein engineering has been used to stabilize proteases (Arnold, 1990; Wong, 1992).

0168.1656/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

PII SO168-1656(96)01539-S

Bis-N-hydroxysuccinimide esters are crosslinking agents which have been shown to successfully stabilize enzymes against autolysis and thermal denaturation (Tafertshofer and Talsky, 1989; Tal- sky et al., 1990; Gleich et al., 1992). When N-hydroxysuccinimide esters are reacted with an amine, an amide is formed; thus the positive charge of the original amino group is lost (Ji, 1983). One can neutralize the positive charge on the lysine residue without crosslinking the enzyme using the monofunctional acetic acid N-hydroxy- succinimide ester (AANHS). It is therefore possible to look at the effect of changing the charge on the surface of the enzyme.

In this paper the effects of modifying trypsin lysines with AANHS are examined. Trypsin is a serine protease with a narrow specificity. It hy- drolyzes L-lysyl and L-argininyl bonds of polypep- tides. AANHS trypsin, with modified lysine residues, should be more stable towards autolysis. Thermostability and organotolerance properties of the modified trypsin are also described.

2. Materials and methods

2.1. Muleriuls

Bovine pancreatic trypsin (EC 3.4.21.4) type 111, benzoyl Dt_-arginine p-nitroanilide (BAPNA), acetic acid N-hydroxy-succinimide ester (AANHS), glycine, Tris, calcium chloride, 2,4,6- trinitrobenzenesulfonic acid (TNBS), urea, guanidine hydrochloride, sodium dodecylsulfate (SDS) and benzamidine were all purchased from Sigma. Acetone, dimethylformamide (DMF), dimethylsulphoxide (DMSO), 1,4-dioxan, methanol and tetrahydrofuran (THF) were purchased from Labscan Ltd., Dublin, Ireland. Acetic acid, acetonitrile, sodium acetate and hy- drochloric acid were purchased from BDH. Coomassie blue protein assay was obtained from Bio-Rad. All chemicals were analytical grade.

2.2. Reaction of trypsin with AANHS

Reaction of trypsin with AANHS was carried out in aqueous solution (3 mM KH,PO,/

K,HPO,, 0.1 M KCl, pH 8.2 containing 3 mM benzamidine) at room temperature for 20 min with a working concentration of 2 mg ml _ ’ of both enzyme and AANHS. Reaction mixture con- tained 20 mg of trypsin in 10 ml of buffer and 0.5 ml of DMSO containing 20 mg AANHS. The reaction was terminated with 10.5 ml of 0.1 M Tris-HCl pH 7. The sample was dialyzed in 3 mM KH,PO,, 0.1 M KCl, pH 8.2, for 20 h at 4°C.

2.3. Trypsin activity assay

Amidase activity of trypsin was measured using a scaled down version of the method described by Erlanger et al. (1961) using BAPNA as a substrate; 200 pl of 1.67 mM BAPNA (29 mg dis- solved in 1 ml DMSO and added to 39 ml of 0.1 M Tris-HCl, pH 8.2 containing 20 mM CaCl,) was equilibrated in a waterbath at 30°C; 50 pl of sample was added and incubated in the waterbath at 30°C for 15 min. The reaction was then terminated with 50 ,~l of 30”/0 acetic acid. The absorbance was read at 405 nm on a Titertek Mk II reader.

2.4. Active site titration

Determination of the active site was carried out usingp-nitrophenyl-p’-guanidinobenzoate (NPGB) as described by Walsh (1970) but modified slightly; 30 ,~l of 0.01 M NPGB (3.37 mg in 1 ml DMF) was added to 3 ml of sample in the aqueous buffer (1 mg ml ‘) and absorbance read at 410 nm after 15 min.

2.5. Amino group estimation

The method of Rajput and Gupta (1987) using TNBS for the estimation of free amino groups in trypsin was followed.

2.6. Trypsin uutolysis

The rate of autolysis was measured using Coomassie blue dye as described by Bickerstaff and Zhou (1993). Approximately 0.2 mg ml _- ’ enzyme solution in 0.1 M Tris-HCl pH 8.2 was

A. Murphy, C. b Fcigbin / Journal of Biotechnology 49 (1996) 163- 171 165

incubated in a waterbath at 50°C. At regular

intervals, aliquots of 2.6 ml were taken, cooled quickly to room temperature on ice and 0.4 ml of Coomassie blue added. The sample was mixed and after 5 min the absorbance was read at 595 nm.

and glycine-NaOH buffer respectively. Enzyme solution of approximately 0.05 mg ml - ’ was di- luted in each pH. Substrate solutions were pre- pared using buffer of each pH and samples’ activities were measured at each pH as described above. The activity was calculated (%) relative to the maximum activity of each sample.

2.7. Thermoinactivation 2.11. Enzyme stability in denaturing agents

Approximately 0.05 mg ml - ’ trypsin in aqueous buffer (3 mM KH,P0,/K2HP04, 0.1 M KC1 pH 8.2) was incubated in a waterbath at 55°C for both native and modified trypsin. At intervals aliquots were taken, cooled quickly to room temperature in ice and the residual activity of trypsin assayed. Autolysis under these conditions was negligible.

Approximately 0.05 mg ml - ’ trypsin in aqueous buffer containing guanidine HCl (in the range O-l M) or urea (in the range O-12.5 M) were incubated at 30°C for 1 h, after which time the residual activity of the trypsin was assayed. Controls containing aqueous buffer only were used for comparisons with the samples.

2.8. Temperature proji le 2.12. SDS-gel electrophoresis

Approximately 0.05 mg ml-’ of trypsin in aqueous buffer was incubated for 10 min over a range of temperatures between 30°C and 75°C. The samples were cooled on ice for 1 min, residual activity was assayed and compared with the activity of a sample incubated at 30°C.

SDS-polyacrylamide gel electrophoresis (10% and 15% acrylamides) was performed according to Laemmli (1970).

3. Results

2.9. Enzyme stability in organic solvents 3.1. AANHS modiJied trypsin

Approximately 0.05 mg ml- ’ trypsin in an aqueous/organic mixture in the range O-90% (v/v) solvent was incubated at 30°C for 1 h. The residual activity of the trypsin was assayed and compared with controls containing no organic solvent. To show the combined effects of organic solvents and elevated temperature, the above procedure was followed but the samples were incubated at 65°C. Controls (containing no solvent and incubated at 30°C for 1 h) were used for comparisons with the samples.

2.10. pH profile

Buffer solutions (0.1 M), containing 20 mM CaCl,, of pH 4.5-6, 7-9, and 8-10 were made with sodium acetate buffer, Tris-HCl buffer,

The modified trypsin showed a 30% enhance- ment of amidase activity compared with native trypsin. Benzamidine had no adverse effect when included in the reaction mixture. From the free amino group determination using TNBS, it was found that approximately 40% of the lysine residues remained unaltered, suggesting that eight lysine residues per trypsin molecule were modified. Bovine trypsin has 14 lysine residues (Walsh, 1970). This result was identical for the reaction carried out in the presence and absence of 3 mM benzamidine. The active site titration of trypsin with NPGB showed that the modified trypsin had slightly lower active site binding than that of the native trypsin, retaining approximately 80% compared to that of the native. Again the presence of benzamidine had little effect on the active site titration values.

3.2. SDS-polyucrylmnide gel rkctrophorrsis

The stained gel showed bands in similar posi- tions for all the samples (Fig. 1). This indicates that no agglutination or artefacts resulted from the modification procedure and that the enzyme mixture was homogeneous.

3.3. Auto1.ysi.s of’ trypsin

The rate of autolysis of the native and AANHS modified trypsin was studied. The assay is based on the finding that peptides of molecular weight 3000 and lower do not support the Coomassie dye reaction and hence the absorbance at 595 nm decreases. As autolysis of trypsin occurred there was a decrease in the absorbance at 595 nm (Bickerstaff and Zhou, 1993). The modified trypsin showed enhanced stability towards autolysis compared with the native (Fig. 2). The absorbance of the native trypsin had dropped to 60% of the initial value after 2 h while the modified trypsin remained stable. The modified trypsin showed a slight increase in absorbance.

Fig. I. SDS-PAGE of AANHS-modified and native trypsin. Modified and native trypsins (20 ~lg per well in both cases)

each migrated as a single band. The molecular weight markers

on the right were (from top) bovine serum albumin (66 kDa).

egg albumin (45 kDa), glyceraldehyde-J-phosphate dehydroge- nase (36 kDa), carbonic anhydrase (29 kDa), bovine trypsino-

gen (24 kDa), soybean trypsin inhibitor (20.1 kDa). Empty wells were run between the trypsin samples and between the

samples and molecular weight markers.

0 50 100 150 200 250 300

TIME (min)

Fig. 2. Autolqsis of native and modified trypsin at 50°C. The

absorbance is shown in percentages relative to initial ab-

sorbancc of samples.

3.4. Efkcts of’ tenpruture

Thermostability of AANHS trypsin was compared with native trypsin. Modified trypsin was more thermostable than the native (Fig. 3A,B). The residual activity of modified trypsin compared with native trypsin over the temperature range 30--75°C was higher (Fig. 3A). The modified trypsin retained 100% activity up to 50°C while the native trypsin lost some of its residual activity at 40°C. Tm values for native and AANHS trypsin were 46°C and 51°C respectively. The half-life of the modified at 55°C was double that of the native: 8.7 min (k = 0.08 min ~ ‘) for the AANHS trypsin compared with 4.3 min (k = 0.16 min ~ ‘) for the native trypsin. A number of different buffering systems were tested to ascertain conditions of minimal autolysis. Under the conditions described for thermoinactivation, autolysis was negligible.

3.5. StuhilitJ, in organic solwnts

The stability of both native and AANHS trypsin were compared in the following solvents: acetone, DMF, DMSO, 1,4-dioxan, methanol and THF. After incubation for 1 h at 30°C. the

A. Murpl~y, C. b F&&n i Journal of Biotechnology 49 (1996) 163- 171 167

modified enzyme showed no enhanced stability

towards any of the solvents tested. However,

when the samples were incubated at 65°C the

following organic solvents showed protecting ef-

fects against thermoinactivation: acetone, DMF,

DMSO, acetonitrile, 1,bdioxan and methanol.

The modified trypsin in these solvents showed enhanced stability. (Fig. 4 shows plots for DMF (A), acetonitrile (B) and methanol (C) as exam- ples.) For methanol, the AANHS trypsin showed enhanced stability at lower solvent concentrations while the native trypsin was more thermostable at higher concentrations (Fig. 4C). THF afforded little protection against thermoinactivation. Little or no activity was retained over the concentration range 0&90”/0 THF by either the native or AANHS trypsin.

3.4. EJiicts oj’ pH

The activities of native and AANHS trypsin were compared over a range of pH values. The modified trypsin retained slightly higher activity in alkaline conditions compared with native trypsin (Fig. 5). Both native and modified forms of trypsin exhibited similar activities in acid and neutral pH ranges.

0 30 40 50 60 70 60

A Temperature

3.7. Stability in guanidine hydrochloride and uwc(

The denaturing effects of urea and guanidine hydrochloride on both the native and modified trypsin were investigated. Modified trypsin showed no increase in stability compared to native trypsin in either denaturant.

I I I I I I

100 0 NATIVE TRYPSIN vM-NHsTRYP6m -

0 10 20 30 40 50 60 70

B TIME (min)

Fig. 3. (A) Effect of IO-min incubations at increasing tempera-

ture on native and modified trypsin. Activities were measured at various temperatures and are ‘74 values relative to activity at

30°C; (B) Thermoinactivation of native and modified trypsin

at 55°C. The activity is shown as ‘%I values relative to initial

activity of samples.

4. Discussion

There have been previous reports of trypsin stabilization by crosslinking agents (Rajput and Gupta, 1987; Rajput and Gupta, 1988) including bis-succinimide esters (Gleich et al., 1992). We undertook a non-crosslinking covalent modification of trypsin’s lysine residues to distinguish between the effects of charge-retaining imidates and charge-neutralizing succinimides (Ji, 1983). Bi- functional imidates and succinimides differed in their ability to stabilize horseradish peroxidase even though both are amino-specific (Ryan et al., 1994). Among proteolytic enzymes, notable stability gains resulted for chymotrypsin (Melik- Nubarov et al., 1987) and trypsin (Mozhaev et al., 1988) following non-crosslinking chemical modification of surface groups.

168 A. Murphy. C. b F&&n / Journal of Biotechnology 49 (1996) 163- 171

80

e = 60

a

3 Sr s 40

a s

20

20 40 80 80 100

% DMF

.2? 70

.z 2 60

7 50 % cz 40

s 30

20

10

0

1

,1 0 20 40 60 80 100

6 % Acetonitrile

Fig. 4. Effects of various concentrations of (A) DMF, (B)

acetonitrile and (C) methanol on native and modified trypsin

at 65°C. The activity is shown as ‘/o values relative to activity

of solvent-free samples.

Trypsin cleaves proteins and peptides at the carboxylic side of the basic amino acids arginine and lysine. Modification of lysine residues pro- vides some protection against digestion. As ex- pected, the AANHS trypsin was more resistant to autolysis than native trypsin. The absorbance of AANHS trypsin increased slightly initially but then remained constant throughout the experi- ment, showing that autolysis was not occurring.

> ._ 2 60-

7 50- a

0

C

Fig. 4(C).

20 40 80

% Methanol

80 100

The initial increase could be due to the formation of artifacts or to slight aggregation.

Tm value for the AANHS derivative was 5°C higher than that of the native (51°C and 46°C respectively). Our twofold thermostabilization at 55°C is modest compared with that achieved by Mozhaev et al. (1988) who gained a > lOO-fold increase in stability at 56.5”C by hydrophilization of a hydrophobic patch on the surface of the trypsin molecule. Gaertner and Puigserver (1992) conjugated polyethylene glycols (PEGS) of vary- ing molecular masses to trypsin amino groups. Although the inactivation at 60°C was polyphasic (so no comparison of half lives could be at- tempted), PEG,.,,,-modified trypsin retained approx. 70% initial activity after 2 h while native trypsin lost 50% activity within approx. 6 min. Our twofold stabilization at 55°C is nonetheless significant since it has resulted from a very small and simple chemical change. Approximately eight lysine residues per trypsin molecule were modified. All of trypsin’s lysines are located on the enzyme surface (T. Higgins, personal commu- nication) and hence should be easily accessible to AANHS, although their relative reactivities may differ. The AANHS reaction leads to an apparent increase in amidase (BAPNA) activity to 1.3 times that of the native trypsin.

A. Murphy, C. b Fcigciin 1 Journal of Biotechnology 49 (1996) 163-171 169

Tolerance towards organic solvents (acetone, DMF, DMSO, 1,4-dioxan, THF and methanol) was not enhanced in AANHS trypsin. However, when the AANHS trypsin was subjected to the combined effects of elevated temperature (65°C) and organic solvents, it showed enhanced stability for DMF, DMSO, acetone, acetonitrile, 1,4-dioxan and methanol compared with the native. In the absence of these solvents (i.e. in aqueous buffer) both the native and AANHS trypsin inactivated very rapidly at 65°C. Possibly, the reduced water content in the cosolvent mixtures decreases autolysis. Note, however, that activity was compared with a control incubated at 30°C to observe the combined effect of temperature and organic solvent. The control was incubated under conditions of negligible autolysis. Autolysis was likewise negligible throughout the l-h thermoinactivation at 55°C in buffer (Fig. 3B). Thus, we believe that the persistence of activity at 65°C in the presence of solvents is due to increased solvent tolerancebyAANHStrypsinandnotjusttodecreased autolysis. All of these solvents showed a protecting effect against thermoinactivation. Methanol was the least protecting solvent. Methanol is protic, i.e. it

Table 1

Threshold concentrations of organic solvents for trypsin

Solvent log P” DC”

Tetrahydrofuran 0.46 100

Acetone -0.24 78.2

1,4-Dioxane -0.27 92.1

Acetonitrile - 0.34 64.3

Methanol -0.74 30.5

Dimethylformamide -1.01 63.3

Dimethylsulphoxide -1.35 60.3

P is the solvent’s partition coefficient in a water/octanol bipha-

sic system, while DC is the solvent’s Denaturation Capacity, a

quantitative relationship between a solvent’s physicochemical

and enzyme-inactivation properties.

a Values taken from Khmelnitsky et al. (1991).

solvates cations through unshared electron pairs and solvates anions through hydrogen bonding. Like water, methanol has an - OH group but is less polar. The other solvents tested are aprotic. These do not hydrogen-bond to anions and they dissolve ionic compounds chiefly through solvation of cations (Morrison and Boyd, 1987). Of this group, THF afforded little or no protection against thermoinactivation. At lower concentrations, THF probably inactivates trypsin due to its high denaturation capacity (DC) (Table 1) (Khmelnitsky et al., 1991). The other solvents, which protect against thermoinactivation at lower concentrations, have lower DC values.Volkinetal.(199l)showedthatsomeenzymes are extremely thermostable in anhydrous organic solvents. Enzymes have been shown to lose their bound water when suspended in organic solvents, with polar solvents resulting in the highest degree of desorption (Gorman and Dordick, 1992). Polar cosolventscansolvateapartiallydehydratedprotein, replacing the displaced bound water. Only above a critical polar solvent concentration does this process lead to denaturation [unfolding] (Khmelnitsky et al., 1991).Belowtheircriticaldenaturingconcentrations, they protect the enzyme against thermoinactivation by removing some of the water. However, at higher concentrations the solvent itself causes inactivation.

I I I I ,

4 5 6 7 8 9 10 11

PH

Fig. 5. Effects of pH on the amidase activity of native and

AANHS modified trypsin. The buffer solutions, (containing 20 mM CaCl,) of pH 4.556, 7-9, 8-10 were made with sodium acetate, Tris-HC1 and glycine-NaOH buffer (I = 0.1 M),

respectively. The activity is shown as percentage of the maxi-

mum activity of each sample.

The pH optimum for amidase activity of modified trypsin is shifted slightly towards alkaline conditions (Fig. 5).ThiscorrelateswiththefindingsofLabouesse and Gervais (1967), who found that pH for esterase activityofacetylatedtrypsinshiftedtoamorealkaline pH as compared with trypsin.

No differences were observed between the effects of denaturants urea and guanidine HCl on native and modified trypsin. Since the AANHS modification affects only the surface of trypsin without forming any crosslinks, one would not expect any increased resistance to these conformation-disrup- tive denaturants.

Labouesse and Gervais ( 1967) described an acetylated trypsin (‘acetyl trypsin’). Use of acetic anhydride at pH 6.7 led to complete acetylation of lysine epilson-amino groups and to acetylation of exposed tyrosines of trypsin. A comparison of native and acetyl trypsin (Sigma) by Gleich et al. (1992) showed no enhanced stability at 50°C for acetyl trypsin. In contrast, our AANHS trypsin shows decreased autolysis at 50°C and increased thermal stability at 55°C. Unlike acetic anhydride, AANHS is specific for lysine residues. Only 60% of these are modified, leaving some lysines free for other purposes such as immobilization.

We have shown that the treatment of trypsin with a non-crosslinking succinimide (AANHS) ester leads to improved thermostability in aqueous buffer and in the presence of the solvents acetone, acetonitrile, DMF, DMSO, 1,6dioxane and methanol. Autolysis is much reduced but resistance to urea and guanidine HCl is unaltered. These findings are relevant to the use of trypsin in organic milieux (Sakurai et al., 1990) and for protein engineering of enzymes for non-aqueous systems (Arnold, 1990). We are exploring this AANHS trypsin derivative further in terms of its esterase activity and possible usefulness for peptide synthesis.

Acknowledgements

We thank Dr Tim Higgins (University College, Galway, Ireland) and Dr J.M. Guisan (CSIC, Madrid. Spain) for helpful correspondence. AM received financial support from Forbairt and from Dublin City University.

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stability characteristics of chemically-modified soluble trypsin

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