Method to maintain the activity in polyethylene glycol-modified proteolytic enzymes

A method for preparing protease-polyethylene glycol adducts is presented wherein the immobilized reversible inhibitor, benzamidine, prevents reaction of activated polyethylene glycol with the active site of the protease. Improved activity against macromolecular substrates is obtained compared to when the benzamidine is in solution during the conjugation reaction.

The present invention relates to a method to avoid binding of polyethylene 
glycol (PEG) at the active site and its surroundings in proteolytic 
enzymes. 
The method, based on the modification reaction of an 
enzyme-macromolecularized inhibitor complex in the heterogeneous state, 
allows to obtain enzyme-PEG adducts in which the proteolytic activity 
toward macromolecular substrates is preserved. 
BACKGROUND OF THE INVENTION 
The modification of enzymes with polyethylene glycol (PEG) is a technology 
that has markedly been developed in recent years to obtain adducts having 
valuable properties for the use both in the biomedical field and as novel 
biocatalysts, due to the presence of polyethylene glycol chains linked at 
the surface. 
In fact, the enzyme-PEG adducts lose the major part of the typical 
properties of naturally occurring enzymes, such as immunogenicity and 
antigenicity, rapid clearance from circulation, easy degradability by 
proteases and instability in diluted solutions A. Abuchowski et al., J. 
B. C., 252 3582, 1977!, that often prevent their use in therapy. 
In the use of enzymes in biocatalysis, the PEG-enzyme adduct acquires a 
quite different characteristic, i.e. the solubility in organic solvents, 
thus allowing a better use of the enzymes in converting liposoluble 
substrates Y. Inada et al., Thrends Biotech., 4 190, 1986!. 
The properties of such novel biotechnologic products are due to the fact 
that PEG binds to the enzyme surface, thus protruding with its hydraration 
cloud toward the outer protein solvent, preventing the access of large 
molecules, such as proteolytic enzymes, as well as the recognition by the 
immune system. On the other hand, as PEG also has amphyphilic properties, 
the PEG-enzyme adduct can acquire solubility in organic solvents. 
However, the polymeric cloud surrounding the PEG-enzyme adduct also limits 
the general use of said derivatives: in fact, the enzymatic activity is 
maintained toward small substrates, that can have access to the active 
site diffusing among the PEG polymer chains, but it is prevented toward 
large substrates, that cannot reach the active site due to steric 
hindrance. 
There fore, convenient PEG-enzyme adducts are obtained with enzymes such as 
superoxide dismutase, catalase, asparaginase, arginase, urease, adenosine 
deaminase, phenylalanine ammonium liase etc., which are nowadays under 
pharmacological and clinical tests, but not with enzymes acting on large 
substrates such as proteins, nucleic acids and polysaccharides. In fact, 
substantial activity losses are de scribed following a PEG-modification of 
-trypsin, chymotrypsin, urokinase, ribonuclease, lysozyme and the like. 
A proposed solution consists in preparing adducts having only a few polymer 
chains linked to the enzyme, thus decreasing the loss in enzymatic 
activity. However, this result, which can be attained carrying out the 
reaction in a PEG molar defect, suffers from drawbacks such as attainment 
of very heterogeneous products and poor reproducibility. 
SUMMARY OF THE INVENTION 
To allow access of macromolecular substrates, large polypeptides or 
proteins, in case of proteolytic enzymes, to the active site, the PEG 
binding to the enzyme is carried out in heterogeneous phase, in which the 
enzyme is linked to an inhibitor thereof that is, in its turn, immobilized 
on a highly hydrated insoluble polysaccharide (Sepharose). In such a way, 
the PEG polymer will bind to enzyme areas far form the active site and its 
proximity, thus allowing the approach of the substrate macromolecules. 
The method was investigated with two serine-dependent proteolytic enzymes, 
trypsin and urokinase, the first being used in medicine, for instance in 
the removal of necrotic tissues, in digestive disorders or in 
ophthalmology in the elimination of protein deposits from contact lenses; 
the latter, i.e. urokinase, being of specific therapeutical interest as a 
plasminogen activator. 
Benzamidine, an inhibitor of serine enzymes, was used as a linker to keep 
Sepharose in the surrounding of the active site (example 2). The method 
could also be used for the site-protection of other serine enzymes, such 
as tissue plasminogen activator (tPA), plasmin, chymotrypsin, elastase, 
kallikrein and the like.

DESCRIPTION OF PREFERRED EMBODIMENTS 
The following examples report the comparison of the activities of the 
starting enzyme A), PEG-modified either with no protection or in the 
presence of free benzamidine in solution B) and obtained carrying out the 
modification in heterogeneous phase with the complexed enzyme with the 
benzamidine-Sepharose macromolecularized inhibitor C) (examples 1, 3, 5, 
7). 
The obtained results will be described with reference to the figures and 
tables reported in the following. 
In the various modifications, monomethoxypolyethylene glycol of MW 5000 
(PEG) was used, bearing norleucine as a spacer between polymer and 
protein, activated at the carboxy group as the succinimidyl ester. PEG 
with norleucine was used as it allows a precise evaluation of the linked 
polymer chains, by means of amino acids analysis L. Sartore et al., Appl. 
Biochem. Biotechnol., 27 45, 1991!. Trypsin and urokinase used in the 
tests were previously purified by affinity chromatography. In the reported 
examples, the activities of the enzyme various forms (A, B, C) were 
compared for equimolecular amounts of enzyme, i.e. without taking into 
account the weigh of the bond polymer. 
Table 1 reports the esterase activity toward the small substrate 
tosyl-arginyl-methyl ester (TAME) of: 1) native trypsin A), trypsin 
randomly modified with PEG B) and trypsin linked to benzamidine-Sepharose 
C). In these cases, the catalytic activity is not lost, on the contrary, a 
small increase in activity occurs in the PEG-modified adducts, which 
proves that PEG chains do not prevent the access to the active site of a 
small substrate (example 4). Similar increases in activity have already 
been observed in proteolytic enzymes, following different chemical 
modifications. 
The same Table also reports the hydrolyric activity toward casein 2a), that 
is a protein of MW 23,600 turning out to be strongly lowered to about 30%, 
following a random modification with PEG B) , whereas it is totally 
maintained when the modification is carried out in heterogeneous phase 
with trypsin linked to benzamidine-Sepharose C) (example 5). 
Finally, Table 1 shows that the hydrolytic activity toward bovine serum 
albumin (BSA) 2b), that is a protein of higher molecular weight, namely 
64000, is completely lost in the sample that was PEG-modified in the 
absence of site-protectors B), whereas a high activity degree is still 
retained (55% compared with native trypsin) when the modification is 
performed with trypsin linked to benzamidine-Sepharose C) (example 5). 
FIG. 1 shows the autolysis of the various trypsin sample, evaluated by 
means of the determination of the esterase activity. Trypsin in aqueous 
solution undergoes degradation very rapidly; when it is randomly modified 
with PEG B) it does not undergoes self-digestion, whereas the one obtained 
carrying out the modification in the presence of benzamidine-Sepharose C) 
shows a slow starting loss of activity, that however becomes stable at 
about 70%. This behaviour is in agreement with the above results, namely 
the incapability of randomly modified trypsin B) to digest a large 
molecule (in the case represented by trypsin itself). The decreased 
autolysis of the sample modified as a benzamidine-Sepharose complex C) is 
consistent, on the one hand, with the persistence of the proteolytic 
activity of this species toward macromolecular substrates, on the other 
with a more difficult access for proteases, due to steric hindrance of the 
PEG chains masking the enzyme surface, with the exception of the active 
site (example 6). 
Table 2 reports the properties of native and modified urokinases, measured 
by means of the esterolytic activity on a small substratum, namely 
carbobenzoxy lysine-p-nitrophenyl ester 1 ), the thrombolytic activity on 
the synthetic thrombus be means of the resistance to penetration of a 
glass bead 2) and the affinity to the synthetic thrombus by means of 
colorimetric measurements of thrombus degradation products 3 ), eventually 
by the evaluation of the capability to hydrolyse free plasminogen in 
solution 4). 
In this case also samples of native urokinase A), randomly PEG-modified 
urokinase B) and urokinase modified by site-protection linking urokinase 
to benzamidine-Sepharose C) were compared. 
Table 2 shows that, analogously to trypsin, the activity toward a small 
substrate increases (example 9). 
On the contrary, thrombolytic activity on the synthetic thrombus, measured 
by means of the resistance to the penetration of a glass bead, disappears 
in the randomly modified urokinase sample, whereas the samples obtained by 
site-protection keep a high thrombolytic activity (example 10). 
The affinity to a synthetic thrombus decreases of 10 orders of magnitude 
for sample C (modified in the presence of benzamidine-Sepharose, and of 
about 500 orders of magnitude for sample B) (obtained by random 
modification ) (example 11). 
The activity toward plasminogen in solution decreases by one order of 
magnitude in sample C), and by 2 orders in the randomly modified sample B) 
(example 12). 
Example 1) Preparation of Trypsin-PEG in Conditions of Random Modification 
10 mg di trypsin are dissolved in 2 ml di borate buffer 0.2 M, pH 8.0 and 
added under stirring to 160 mg of activated PEG, to obtain a molar ratio 
of the amino groups present in the protein to PEG of 1:5, while pH is kept 
to 8.0 in a pHstat with 0.2 M NaOH. After 30' the solution is diluted with 
8 ml of HCl 10 nM and it is ultrafiltered with a membrane of cut-off 
10,000 to reduce the volume to 2 ml. The dilution and concentration 
process is repeated for 5 times. Finally, the solution is purified by gel 
filtration chromatography and the trypsin-PEG peak is concentrated by 
ultrafiltration to 2 ml, added again with 8 ml of 10 mM HCl and 
ultrafiltered repeating the procedure for 5 times. (The modification was 
carried out also in the presence of benzamidine for an enzyme/benzamidine 
1:100 molar ratio and the obtained sample was purified as reported above). 
The trypsin-PEG adduct has about 13 PEG chains linked per enzyme molecule. 
Example 2 ) Preparation of benzamidine-Sepharose 
Sepharose 6B (50 ml) activated by CNBr P. Cuartecasas, J. B. C., 245 3059, 
1970 ! is reacted in borate buffer 0.1 M pH 9.5 with 200 mg of 
p-aminobenzamidine dissolved in 25% dimethyl formamide 25%. The resin was 
washed with 25% dimethyl formamide in borate buffer and finally with 0.5 M 
NaCl, in which it was subsequently preserved. The resin has 5-20 .mu.M of 
benzamidine per ml. A commercially available benzamidine-Sepharose resin 
can also be used. 
Example 3) Preparation of trypsin-PEG in Conditions of Protection of the 
Active Site and the Surroundings Thereof 
15 mg of trypsin are dissolved in 5 ml of 0.2 M borate buffer, pH 8.0 and 
added to 5 ml of benzamidine-Sepharose resin, previously washed with 100 
ml of 1.5 M NaCl and equilibrated with 0.2 M borate buffer, pH 8.0. After 
addition of trypsin, the resin is filtered, washed 3 times with 10 ml of 
0.2 M borate buffer, pH 8.0 and further filtered. 
The resin is added with 5 ml of borate buffer and, under stirring, with 360 
mg of activated PEG, to reach a 1:7.5 protein amino groups to PEG molar 
ratio. The suspension is stirred for 1 hour, filtered and washed 3 times 
with 10 ml of borate buffer. The trypsin-PEG adduct is removed from the 
resin by repeated washings (10 times) with 10 ml of 10 nM HCl. The 
solution is concentrated by ultrafiltration on a membrane and purified by 
gel filtration chromatography, as in example 1. The trypsin-PEG peak is 
collected and finally concentrated to 2 ml and diluted with 8 ml of 10 mM 
HCl and concentrated to 2 ml. This procedure is repeated for 5 times. The 
modified protein contains about 12 polymer chains per enzyme molecule. 
Example 4) Enzymatic Activity of Trypsin and PEG-Trypsin Toward a Low 
Molecular Weight Substrate 
This activity is measured using N.alpha.-p-tosyl-arginyl-methyl ester 
(TAME) as the substrate. The increase in optical density of a solution of 
800 .mu.l of 0.046 M Tris HCl, 0.015 M CaCl.sub.2 at pH 8.0, 100 .mu.l of 
1 mM HCl, 100 .mu.l of a 0.01 M substrate aqueous solution and 0.5 to 6 
.mu.g of enzyme. 
The activity of the modified products is expressed as a percentage compared 
to the activity of the native enzyme (Table 1). 
Example 5) Activity of Trypsin and PEG-trypsin Toward a High Molecular 
Weight Substrate 
To evaluate the protease activity toward high molecular substrates, casein 
(MW 23000) or serum albumin (MW 64000) were used as standard substrates. A 
solution of 0.4 ml of 0.1 M Tris HCl, pH 8.0, 0.4 ml of a 1% substrate 
solution in 0.1 M Tris HCl, pH 8.0 and an enzyme amount from 0.25 to 3 pg 
is incubated at 30.degree. C. for 20 minutes. This solution is added with 
1.2 ml of 5% trichloroacetic acid and the optical density at 280 nm is 
evaluated on the supernatant after centrifugation and removing of the 
precipitate. The residual activity of the PEG-trypsin products is 
evaluated as a percent activity of the native form (Table 1). 
Example 6) PEG Autolysis of Trypsin and its Derivatives 
0.25 mg of enzyme or PEG-trypsin samples in 1 ml of 0.1 M Tris buffer at pH 
8.0 are incubated at 37.degree. C. The esterase activity test is carried 
out at preset times to evaluate the percentage of still active enzyme 
Example 7) Preparation of PEG-urokinase in Random Modification Conditions 
2.5 mg of urokinase in 2 ml of 0.2 M borate buffer, pH 8.0, in a 
polyethylene container, are added under stirring with 30 mg of activated 
PEG at a protein amino groups to PEG 1:5 molar ratio. After 60 minutes the 
solution is diluted with 8 ml of 10 mM HCl and concentrated by 
ultrafiltration. The PEG-urokinase adduct is purified following the 
procedure of example 1, always working with polyethylene containers. (The 
modification was carried out also in the presence of benzamidine for a 
1:100 urokinase/benzamidine molar ratio and the obtained sample was 
purified as reported above ). The resulting urokinase has about 14 polymer 
chains per protein molecule. 
Example 8) Preparation of PEG-urokinase in Conditions of Protection of the 
Active Site and of the Surroundings Thereof 
3 mg of urokinase in 3 ml of 0.2 M borate buffer, pH 8.0 are added with 1.5 
ml of benzamidine-Sepharose resin, previously washed with 30 ml of 0.5 M 
NaCl and equilibrated with the same borate buffer. The obtained suspension 
is added with 72 mg of activated PEG at a protein amino groups to PEG 1:10 
molar ratio. After 60 minutes the resin is washed 3 times with 5 ml of 0.2 
M borate buffer, pH 8.0 and then the obtained adduct is removed from the 
resin by washing with 50 ml of 10 mM HCl and purified as in example 3. The 
obtained adduct has 13 PEG chains covalently linked per enzyme molecule. 
Example 9) Urokinase and PEG-urokinase Enzymatic Activities Toward a Low 
Molecular Weight Substrate 
This activity is spectrophotometrically evaluated using carbobenzoxy L-Lys 
-p-nitrophenyl ester hydrochloride (Z-Lys-OpNO.sub.2) as the substrate. 65 
.mu.l of substrate (2.5 mg/ml of water) are added to 935 .mu.l of 0.1 M 
K.sub.2 HPO.sub.4 /KH.sub.2 PO.sub.4 buffer, pH 6.8 containing 0 to 5 
.mu.g of urokinase. The increase in the optical density at 360 nm per 
minute is reported against the enzyme amounts. The esterase activity of 
the adducts is expressed as a percentage compared with that of the native 
enzyme and it is reported in Table 2. 
Example 10 ) Urokinase and PEG-urokinase Fibrinolytic Activity Inside a 
Synthetic Cloth 
500 .mu.l of a fibrinogen solution (30 .mu.g/ml ) in 0.1 M Na.sub.2 
HPO.sub.4 /K.sub.2 HPO.sub.4, 0.5% of BSA, pH 7.2, containing 25 to 100 
urokinase enzyme Units and finally 100.mu.l of thrombin (500 UI/ml) in 0.1 
M KH.sub.2 PO.sub.4 /Na.sub.2 HPO.sub.4 buffer pH 7.2 containing 1 mg of 
BSA are subsequently placed into a glass test tube (9 mm.times.100 ram). 
The test tube is turned upside down 2 times and placed into a 
thermostatized bath at 37.degree. C. After 1 hour, a glass bead of 0.3 g 
weight is placed onto the formed cloth and the time necessary for the bead 
to reach the test tube bottom is evaluated The fall time of the bead is 
reported on a logarithm scale against the corresponding enzyme Units. The 
fibrinolytic activity, expressed as the percentage compared with standard 
urokinase of known activity, is reported in Table 2. 
Example 11!Urokinase and PEG-urokinase Affinity to a Synthetic Cloth 
200 .mu.l of a fibrinogen solution (4.2 mg/ml ) in 0.05 M Tris HCl, 0.15 M 
NaCl and 0.01 M CaCl.sub.2 buffer pH 8.0 and 20 .mu.l of a thrombin 
solution (500 UI/ml) in 0.1 M Na.sub.2 HPO.sub.4 /KH.sub.2 PO.sub.4 
buffer, 1% BSA%, pH 7.2, are placed into a test tube. The tubes are 
centrifuged at 2500 rpm for 20 minutes, to squeeze the cloth that is 
subsequently extruded, washed with 5 ml of 1.5 M NaCl and dried. The cloth 
is placed into a cuvette containing urokinase amounts varying from 0.1 to 
10 .mu.g 0.05 M Tris HCl, 0.015 M NaCl buffer pH 8.0. Optical Density 
values, recorded at 3 minutes intervals, are plotted against the 
time.sup.2. The ratio of the concentration of the used enzyme to the 
obtained slopes is plotted against the urokinase concentration to obtain 
lines from which the affinity value of the enzyme to the cloth can be 
evaluated G.A. Homandberg and T. Wai, Thrombosis Res., 55 493, 1989!. The 
results are reported in Table 2. 
Example 12) Urokinase and PEG-urokinase Activity Toward Plasminogen in 
Solution 
In a cuvette containing 900 .mu.l of 0.05 M Tris HCl buffer, 0.15 M NaCl pH 
7.0, 50 .mu.l of a solution of 4.41 mg/ml in the same buffer 
Val-Leu-Lys-pNO.sub.2 anilide, 40 .mu.l of a plasminogen 0.125-12.5 mg/ml 
solution and 10 .mu.l of an urokinase solution or a modified urokinase 
solution in 0.05 M Tris HCl buffer, 0.15 M NaCl, bovine serum albumin 5 
mg/ml pH 7.0. The change in the optical density recorded at 405 nm is 
plotted against the time.sup.2 expressed in minutes, to obtain the plasmin 
formation rate V. Eli is et al., J. B. C., 262 14998, 1987!. In Table 2 
the enzymatic activities of the various samples are reported. 
TABLE 1 
______________________________________ 
Native trypsin and PEG-modified trypsin characteristics 
Protease 
Esterolytic activity 
activity versus: 
versus TAME casein BSA 
(1) % (2a) % (2b) % 
______________________________________ 
Native trypsin A) 
100 100 100 
PEG-trypsin modified 
120 30 0 
randomly or with an in- 
hibitor in solution B) 
PEG-trypsin modified by 
115 100 55 
site-protection in 
heterogeneous phase C) 
______________________________________ 
(1) Values obtained in Example 4, (2) Values obtained in Example 5. 
TABLE 2 
__________________________________________________________________________ 
Native urokinase (UK) and PEG-modified UK characteristics 
Fibrinolytic activity 
Affinity to 
Activity versus 
with UK inside the 
the synthetic 
Activity toward 
Z-Lys-.phi.pNO.sub.2 
synthetic thrombus 
thrombus 
plasminogen 
(1) % (2) % (3) nM (4) Kcat sec.sup.-1 
__________________________________________________________________________ 
Native UK A) 
100 100 2.1 25.5 
PEG-UK modified 
115 0 1079 0.5 
randomly or with 
an inhibitor in 
solution B) 
PEG-UK modified 
115 21 24.3 2.7 
by site-protection 
in heterogeneous 
phase C) 
__________________________________________________________________________ 
(1) Values obtained in Example 9, (2) Values obtained in Example 10. 
(3) Values obtained in Example 11, (4) Values obtained in Example 12.