358
Bloconlugate Chem. 1993, 4, 358-361
Trypsin Modification by Vinyl Polymers with Variable Solubilities in Response to External Signals Yoshihiro Ito,' Masaaki Kotoura, Dong-June Chung, and Yukio Imanishi Department of Polymer Chemistry, Faculty of Engineering, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto, Japan 606-01. Received April 12, 1993"
Trypsin was modified with various vinyl polymers by the graft polymerization of vinyl monomers using a trypsin derivative containing aliphatic azo groups as the initiator. The graft polymers were chosen to be sensitive to external signals, e.g., a redox-sensitive poly[3-carbamoyl-l-@-vinylbenzyl)pyridinium chloride] and a pH-sensitive poly(methacry1ic acid-co-methyl methacrylate). The trypsin modified with the redox-sensitive polymer became insoluble in water in the presence of Na2S204,and redissolved when H202 was added. However, the enzymatic activity of the homogeneous solution was not proportional to the concentration of the modified trypsin remaining in the solution. The trypsin modified with the pH-sensitive polymer was insoluble and soluble in acidic and neutral solutions, respectively. The enzymatic activity of the homogeneous solution changed reversibly with pH in proportion to the concentration of the modified trypsin remaining in the solution.
INTRODUCTION Immobilizationis useful in the stabilization and repeated use of enzymes. On the other hand, soluble state is necessary when the enzymes are used to digest insoluble substrates such as cellulose, and when the diffusiondetermining reaction speed in the homogeneous state is higher than that in the heterogeneous state. Therefore, an enzyme modification that enables an efficient catalysis in solution during the reaction and easy recovery by subsequent insolubilization has been desired. Toward this goal, several attempts have been made including modifying enzymes with polyelectrolytes carrying carboxylic acid groups to control the solubility according to pH or the ionic strength of the solution (1-3) and magnetic recovery of an enzyme hybrid (4). In this study, we considered a redox reaction as well as the variation of pH or ionic strength as external signals. Poly[3-carbamoyl-l-@-vinylbenzyl)pyridinium chloride] (PCVPC)l(5)and poly(methacrylicacid-co-methyl methacrylate) [P(MA-MMA)] (1-3) were chosen a redoxresponsive and pH-responsive polymers, respectively. We reported a novel method for grafting a wide range of vinyl polymers to enzymes (6-8). According to this, aliphatic azo groups were connected to trypsin. The azo-containing trypsin was used as an initiator for graft polymerization of monomers for signal-responsive graft chains. The solubility change of the modified enzyme and the consequent catalytic activity of the homogeneous solution in response to external signals was investigated. EXPERIMENTAL PROCEDURES Reagents. Trypsin was of bovine pancreas origin (type 1,T-8003, Mw 23 000) and purchased from Sigma Chemical Co. (St.Louis, MO). 4,4'-Azobis(4-~yanovalericacid) was purchased from Aldrich Chemical Co. (Milwaukee, WI). 4-Morpholineethanesulfonic acid was purchased from Dojin Chemical Institute (Kumamoto, Japan). 2,4,6e Abstract published
in Advance ACS Abstracts, August 15,
1993. l Abbreviations used: PCVPC, poly[3-carbamoyl-l-@-vinylbenzy1)pyridiniumchloride]; [P(MA-MMA)],poly(methacry1ic acid-co-methyl methacrylate).
1043-1802/93/2904-0350$04.00/0
Trinitrobenzenesulfonicacid and l-ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochloride,which is watersoluble, were purchased from Wako Pure Chemical Ind. Co. (Osaka, Japan). p(Chloromethy1)styrenewas a product of Tokyo Kasei Co. (Tokyo, Japan). Nicotinamide, methacrylic acid, and methyl methacrylate were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Nicotinamide was recrystallized from acetone. Monomers were used after purification as described below. Sodium hydrosulfite and N*-benzoyl-DL-argininep-nitroanilide hydrochloride were purchased from Nacalai Tesque Inc. and used without further purification. Synthesis and Purification of Monomers. 3-Carbamoyl-1-@-vinylbenzy1)pyridinium chloride was synthesized as reported previously (9). p(Chloromethy1)styrene (11.9 g, 78.2 mmol) and nicotinamide (15 g, 123 mmol) were mixed in NJV-dimethylformamide (250 mL) and the solution was stirred for 24 h at 4 "C. A white precipitate was recovered by centrifugation and crystallization from ethanol. Anal. Calcd of 3-carbamoyl-1-@vinylbenzy1)pyridinium chloride (C15HlsN20Cl): calculated to be C, 65.58; H, 5.50; N, 10.20, Cl, 12.90. Found: C, 65.65; H, 5.49; N, 10.23; C1, 12.97. Mp 228-229 "C [lit. (9) mp 228 OC1. Methacrylic acid was purified by distillation under reduced pressure (63 OC, 12mmHg). Methyl methacrylate was washed with 5% NaOH and double-distilled water, dehydrated with anhydrous sodium sulfate overnight, and then distilled under reduced pressure (50 OC, 160mmHg). Trypsin Modification with Vinyl Polymers. The synthetic scheme of trypsin modification with vinyl polymers is shown in Figure 1. First, aliphatic azo groups were connected to trypsin by the following method. 4,4'Azobis(4-cyanovaleric acid) (73 mg) and water-soluble carbodiimide (50 mg) were added to an aqueous solution (20 mL) buffered to a specific pH value with 0.05 M 4-morpholineethanesulfonate buffer (pH 5.51, 0.05 M phosphate buffer (pH 6.7),0.05 M phosphate buffer (pH 7.2), or 0.05 M borate buffer (pH 8.7). The solution was incubated for 2 h at 4 "C, and trypsin (10 mg) was added. The mixture was further incubated for 24 h at 4 OC. Unreacted chemicals were removed by dialysis for 2 days, 0 1993 American Chemical Society
Trypsln Modification with
Bloconjugate Chem., Vol. 4, No. 5, 1993 359
Solubility-VariablePolymers CH3
CH,
HOOCCH2CH2-r-N=N-{-CH2CH2-C-OH II CN
CN
wsc -
0
ACV
c=o I N-H
I
Graft polymerization UV light, 4 T , 24h
-
ACV-TRP Figure 1. Synthetic scheme of azo-modified trypsin and vinyl polymer-modified trypsin. WSC, water-soluble carbodiimide; TRP, trypsin.
and the trypsin attached to 4,4’-azobis(4-cyanovalericacid) was freeze-dried. The graft polymerization of vinyl monomers to trypsin was carried out as follows. Azo-containing trypsin (5 mg) was added to aqueous 3-carbamoyl-l-@-vinylbenzyl)pyridinium chloride (20 w t %, 2.5 mL) or a mixture of methacrylic acid (3.18 mL) and methyl methacrylate (1.34 mL) (3/1 mol/mol), and the mixtures were irradiated with a 120-W mercury lamp for 24 h under a nitrogen atmosphere. The polymerization solution was poured into acetone to precipitate the graft-polymerized 3-carbamoyl-1-@vinylbenzy1)pyridinium chloride. The precipitate was redissolved in 0.05 M phosphate-buffered saline (pH 7.0), and the grafted trypsin was purified by fractionation through a column packed with Sephadex G-200. Graft copolymerization of methacrylic acid and methyl methacrylate was achieved by pouring the polymerization solution into methanol containing hydrochloric acid to precipitate the product. The precipitate was redissolved in a 0.05 M phosphate buffer (pH 7.01,and the solution was fractionated through a column packed with Sephadex G-200 to separate trypsin grafted with poly(methacry1ic acid-co-methyl methacrylate) [P(MA-MMA)]. The molecular weight of polymer-modified trypsin was calculated from the retention volume in a Sephadex G-200 column fractionation by using a calibration curve, which was obtained by using proteins of known molecular weight, namely, ribonuclease A, chymotrypsinogen A, ovalbumin, and albumin. Determination of the Azo Content in Trypsin Containing Azo Groups. The level was determined as follows (10). Three solutions were prepared: (A) an aqueous trypsin (0.5 mg/mL), (B) aqueous NaHC03 (4 % , pH 8.51, (C) aqueous 2,4,6-trinitrobenzenesulfonicacid (0.1%). Portions of each solution (1 mL of each) were mixed and incubated in the dark for 2 h at 37 “C. The mixtures were 10-fold diluted with solution B, and the absorption intensity at 350 nm due to amine-substituted 2,4,6-trinitrobenzenesulfonicacid was measured. The amount of unreacted amino groups in the azo-containing trypsin is proportional to 350-nm absorption; thus, the content of the azo group in the azo-modified trypsin was determined. Measurement of Enzymatic Activity. Nu-benzoylDL-arginine p-nitroanilide hydrochloride was used as the substrate for trypsin. A 1.2 mM substrate solution in a 0.05 M phosphate buffer (pH 7.0)was prepared. The
1 100 t
8ol
60
0
5
6
7
8
9
PH Figure 2. Content of azo groups coupled with trypsin at various
pH values.
enzyme activity was estimated by measuring the intensity of absorption at 410 nm due to p-nitroaniline, which is a hydrolysis product of the substrate at 35 “C. The enzyme activity was represented as the amount of p-nitroaniline produced per unit time per unit weight of trypsin at pH 7.0 (11). The solubility of the polymer-modified trypsin was determined by measuring the dry weight of the precipitate produced on adding a reducing reagent or on lowering the pH of the solution.
RESULTS AND DISCUSSION Synthesis of Azo-Containing Trypsin. I t is shown in Figure 2 that the number of azo groups in the azomodified trypsin is higher under more acidic conditions. This should have been caused by the faster activation of carboxy groups in the 4,4’-azobis(4-~yanovaleric acid) with water-soluble carbodiimide under more acidic solutions. A similar observation has been reported elsewhere (12). In this investigation, azo-containing trypsin at pH 5.5 was used to initiate graft polymerization. About 6-7 azo groups were connected to amino groups in trypsin, which possesses 15 amino groups. Graft Polymerization of 3-Carbamoyl-l-(pvinylbenzy1)pyridiniumChloride. The elution patterns of the native and grafted trypsins through a Sephadex G-200 column are shown in Figure 3. The crude product of graft polymerization, which was obtained by pouring the reaction solution into acetone, should contain a homopolymer PCVPC (curve b), and therefore the enzymatic
Bioconlugate Chem., Vol. 4, No. 5, 1993
Ito et el.
a)
_j/
+ +
60
-
40
-
20
-
0.0 500
250
0.2
0.4
0.6
0.8
1.0
Molar ratio of NazSz04/CVPC
750
Elution volume (ml) Figure 3. Elution patterns of native and modified trypsin: (a) native trypsin, (b)PCVPC-trypsin as polymerized, (c) enzymatic activity of fractionated PCVPC-trypsin [separation column, Sephadex G-200 (diameter, 3.3 cm; length, 87 cm); eluent, 0.05 M phosphate buffer (pH 7.0); detection, UV absorption at 276 nm in a and b and 410 nm @-nitroanilineof substrate) in c].
h
E
loo+
I
Table I. Effect on Enzymatic Activity of Trypsin by Polymer Modification and by Addition of Redox Reagents amount of p-nitroaniline Relative produced by the reaction enzymatic of trypsin and BANA" at activity SamDle 35 "C (umol/min me of trwsin) (% ) 100 trypsin 4.80 azo-modified trypsin 2.51 52 PCVPC-trypsin 0.81 17 trypsin + NazS20db 4.36 91 trypsin + H20zb 1.50 31
~~
~
~
~~
.2
~
a Na-Benzoyl-DL-argininep-nitroanilide hydrochloride. Reaction conditions are the same as those for Figure 5.
activity of each fraction was measured (curve c). Consequently, the high molecular weight component, which appeared after the graft polymerization, was considered to be the PCVPC-trypsin. The molecular weight of the PCVPC-trypsin was calculated to be 143 000. This value indicates that the molecular weight of the graft chain is 18 OOO, on the assumption that all azo groups in the azomodified trypsin were used in the initiation of graft polymerization. The effects of the introduction of azo groups to trypsin and the graft polymerization on the enzymatic activity of trypsin were estimated by measuring the catalytic activities of the modified trypsin during hydrolysis. As is shown in Table I, the catalytic activity of azo-containing trypsin decreased to about half that of the original and that of PCVPC-trypsin to about 16% of the native. The drastic decrease in the activity of the graft polymerized enzyme should be due to a conformational change induced by the graft polymerization or to coverage of the catalytic site of trypsin with graft chains. Figure 4a showsthat the water solubility of the modified trypsin (PCVPC-trypsin) as well as the graft chain (PCVPC) decreases with increasing concentration of reducing reagent. The solubility decrease should be due to the loss of electric charges by the reduction of PCVPC chains. The solubilitydecrease of PCVPC was faster than that of PCVPC-trypsin. This could be explained by taking into consideration the heightened water solubility of PCVPC-trypsin due to the presence of trypsin. I t was anticipated that the enzyme activity of the homogeneous solution would be lowered on adding re-
7 d
20 0
0.0
0.2
0.4
0.6
0.8
1.0
Molar ratio of Na2&04/CVPC
Figure 4. The change of the water solubilityon PCVPC-trypsin (a) and the enzymatic activity remaining in a homogeneous solution (b) at various molar ratios of Na2S20I/CVPC: ( 0 ) PCVPC-trypsin (1.25 mg/mL phosphate buffer; concentration of CVPC, 3.6 mM), (A)PCVPC (1.0 mg/mL phosphate buffer;3.6 mM). ducing reagent because of the decreased enzyme concentration resulting from precipitation of reduced PCVPCtrypsin. As shown in Figure4b, a small amount of Nafi204 decreased the enzyme activity of the homogeneous solution. However, the extent of the activity decrease was more than that of solubility decrease. This may be due to the obstruction of substrate access to the partially reduced PCVPC-trypsin, which might form microaggregatea in aqueous solution or might be blocked by adsorption of hydrophobic reduced graft chains. Table I shows that the reducingreagent NazS204 does not seriouslydeteriorate the enzymatic activity of trypsin. The solubility of PCVPC-trypsin and the enzyme activity remaining in the homogeneous solution by alternate reductions and oxidations were investigated, and the results are shown in Figure 5. The water solubility of PCVPC-trypsin in an midized state,which carries positive charges and is highly soluble in water, decreased on adding Na2S204, and was precipitated. Finally the enzyme activity of the homogeneous solution totally disappeared. After adding H202, PCVPC-trypsin recovered total water solubility. However,the enzyme activity of the homogeneous solution was not fully restored by the oxidation. As shown in Table I, under the same conditions, the catalytic activity of the native trypsin decreased to one-third of the original activity upon addition of HzO2. These results show that the absence of activity reversibility after redox reactions are due to the damage sustained by trypsin during oxidation.
Biomnjtgate Chem., Vol. 4, No. 5, l9g3
Trypsln Modification with Solubility-Variable Polymers
961
~~
A
A
A
PH7
B
B
Figure 5. The change of water solubility of PCVPC-trypsin
(1.25mg/mL phosphate buffer; concentration of CVPC,3.6 mM) and the enzymatic activity remaining in a homogeneoussolution in response to repeated redox reactions by adding HzOz (0.02 mM, A) and NazS204 (0.2 mM, B): ( 0 )solubility, ( 0 )activity.
PH7
PH3
PH3
Figure 7. Reversible changeof the solubility in water of P(MA-
MMA)-trypsin (1 mg/mL phosphate buffer; concentration of trypsin, 17 pM) and the enzymatic activity remaining in a homogeneous solution in response to repeated pH changes: ( 0 ) solubility, ( 0 )activity. In summary, modified trypsins were synthesized, the solubilities of which varied in response to external signals such as redox reactions and pH variations. More useful devices can be synthesized by choosing suitable external signabensoring polymer combinations, in which possible damage to the enzyme can be minimized. LITERATURE CITED (1) Fuijimura, M., Mori, T., and Tosa, T. (1987) Preparation
and properties of soluble-insoluble immobilized proteases. Biotechnol. Bioeng. 29, 747-752. (2) Taniguchi,M., Tanahashi, S., and Fujii, M.(1990)Properties PH Figure 6. The solubility of P(MA-MMA)-trypsin (1 mg/mL phosphate buffer; concentration of trypsin, 17 rM) and the enzymaticactivity remainingin a homogeneoussolutionat various pH values: ( 0 )solubility, ( 0 )activity.
pH-Dependent Solubility and Activity of P(MAMMA)-Trypsin. The water solubility of P(MA-MMA), which was prepared from a methacrylic acid/methyl methacrylate mixture at a molar ratio of 2/1 [rmethcrylic acid = 0.63 and rmethyl methacrylate = 1.18 as previously reported (1411,changed drastically at pH 5. P(MA-MMA) was not soluble in water below pH 4, but soluble above pH 6. The molecular weight of P(MA-MMA)-trypsin was 80000, corresponding to the molecular weight of the graft chain being 8500. The enzyme activity of P(MA-MU)-trypsin was about 14% of native trypsin, which is nearly the same as PCVPC-trypsin. As shown in Figure 6, the water solubility of P(MA-MMA)-trypsin was low under acidic conditions. The enzyme activity remaining in the homogeneous solution also decreased with decreasing pH values. A slight decrease of the solubility in water was accompanied by a sudden decrease of the enzyme activity remaining in the homogeneous solution. This phenomenon occurred with PCWC-trypsin, too. The changes of the solubility of P(MA-MMA)-trypsin in water and the enzyme activity remaining in the homogeneous solution at various pH values ranging from 3 to 7 were investigated,and the results are shown in Figure 7. Either solubility or enzyme activity changed almost reversibly with the pH variation. These results show the absence of an irreversible trypsin transformation in the range of pH values studied.
and repeated use of a reversibly soluble-insolubleyeast lytic enzyme. Appl. Microbiol. Biotechnol. 33, 629-635. (3) Fujii, M., and Taniguchi, M. (1991)Applicationof reversibly soluble polymers in bioprocessing. TIBTECH. 9, 191-196. (4) Y&moto,T., Mihama,T.,Takahashi,K., Saito, Y., Tamura, Y., and Inada, Y. (1987) Chemical modification of enzymes with activated magnet modifier. Biochim. Biophys. Res. Commun. 145,908-914. (5) Eismer, U., and Kuthan, J. (1972) The chemistry of dihydropyridines. Chem. Rev. 72, 1-42. (6) Ito, Y., Fujii, H., and Imanishi, Y. (1992) Enzyme hybridization with synthetic polymers for use in organic media. Makromol. Chem. Rapid Commun. 13,315-319. (7) Ito, Y., Fujii, H., and Imanishi, Y. (1992)Lipase modification by various synthetic polymersfor use in chloroform. Biotech. Lett. 14, 1149-1152. (8) Ito, Y., Fujii, H., and Imanishi, Y. (1993) Catalytic peptide synthesisby trypsin hybridizedwith polystyrenein chloroform. Biotech. h o g . 9, 128-130. (9) Endo, T., and Okawara, M. (1979) Preparation of polymers containing 1,4-dihydronicotinamide structure and their use in the reduction of vicinal tricarbonyl compounds. J.Polym. Sei., Polym. Chem. Ed. 17, 3667-3674. (10) Habeeb, A. F. S. A. (1966) Determination of free amino groups in proteins by trinitrobenzene-sulfonicacid. Anal. Biochem. 14,328-383. (11) Mole, J. E., and Horton, H. R. (1973) Kinetics of papaincatalyzed hydrolysis of a-N-benzyl-L-arginine-p-nitroanilide. Biochemistry 12, 816-825. (12) Papisov, M. I., Maksimenko, A. V., and Torchilin, V. P. (1985) Optimization of reaction conditions during enzyme immobilization on soluble carboxyl-containingcarriers. Enzyme Microb. Technol. 7, 11-20. (13) Ryabov, A. V., Smimova, L. A., and Panova, G.D., and Tsareva, L. V. (1970) Effect of water on the copolymerization on methyl methacrylate with methacrylic acid. Tr. Khim. Khim. Tekhno. 2,221-229.
