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Description  |
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BACKGROUND OF THE INVENTION
The present invention relates to chemical modification of polypeptides by
means of covalent attachment of strands of polyalkylene oxide to a
polypeptide molecule such as is disclosed in U.S. Pat. No. 4,179,337, to
Davis, et al. It is disclosed in Abuchowski & Davis "Enzymes as Drugs",
Holcenberg & Roberts, eds., pp. 367-383, John Wiley & Sons, N.Y. (1981)
that such preparations of polypeptides have reduced immunogenicity and
antigenicity and also have a longer lifetime in the bloodstream as
compared to the parent polypeptides. These beneficial properties of the
modified polypeptides make them very useful in a variety of therapeutic
applications, such as enzyme therapy.
The active groups that are introduced onto polyalkylene oxides for the
purpose of subsequent attachment of these polymers to proteins must
satisfy the following requirements:
1. The active groups have to be reactive enough to afford fast reaction
with a protein under mild conditions;
2. The residues released from the active groups during the process of
modification have to be non-toxic and/or readily separable from the
protein-polymer adduct.
To effect covalent attachment of polyethylene glycol (PEG) to a protein,
the hydroxyl end-groups of the polymer must first be converted into
reactive functional groups. This process is frequently referred to as
"activation" and the product is called "activated PEG".
Methoxypolyethylene glycol (mPEG) derivatives, capped on one end with a
functional group, reactive towards amines on a protein molecule, are used
in most cases.
The most common form of activated PEG heretofore used for preparation of
therapeutic enzymes is poly(ethylene glycol)
succinoyl-N-hydroxysuccinimide ester (SS-PEG) [Abuchowski, et al. Cancer
Biochem. Biophys. 7, 175-186 (1984), Scheme 1]. Use of this activated
polymer satisfies both of the requirements listed above. However, it has
one major drawback. The ester linkage between the polymer and succinic
acid residue has limited stability in aqueous media [U.S. Pat. No.
4,670,417, to Iwasaki, et al. (1987); Ulbrich, et al., Makromol. Chem.
187, 1131-1144 (1986)]
##STR1##
Various functionalized polyethylene glycols (PEG) have been effectively
used in such fields as protein modification (Abuchowski & Davis, 1981,
supra), peptide chemistry [Zalipsky, et al., Int, J. Peptide Protein Res.,
30, 740-783 (1987)] and preparation of conjugates with biologically active
materials [Zalipsky, et al., Eur. Polym. J. 19, 1177-1183 (1983) and
Zalipsky and Barany, Polymer Preprints, Am. Chem. Soc. Div. Polym. Chem.
27(1), 1-2 (1986)]. PEG protein conjugates useful in medical applications
have shown promise, particularly with regard to their stability to
proteolytic digestion, reduced immunological response and longer half-life
times in the bloodstream.
To accomplish this, the prior art has activated the hydroxy group of PEG
with cyanuric chloride and the resulting compound then coupled with
proteins (Abuchowski, et al. (1977) J. Biol. Chem. 252, 3578; Abuchowski
and Davis, 1981, supra). However, various disadvantages of using this
method exist, such as the toxicity of cyanuric chloride and the
non-specific reactivity for proteins having functional groups other than
amines, such as free essential cysteine or tyrosine residues.
In order to overcome these and other disadvantages, alternative procedures,
such as succinimidyl succinate derivatives of PEG (SS-PEG) have been
introduced (Abuchowski, et al. 1984, supra, see Scheme 1, above). It
reacts quickly with proteins (30 min) under mild conditions yielding
active yet extensively modified conjugates use of this activated polymer
has one major disadvantage. The ester linkage between the polymer and the
succinic acid residue has limited stability in aqueous media [U.S. Pat.
No. 4,670,417 to Iwasaki, et al. and Ulbrich, et al. Makromol Chem., 187,
1131-1144 (1986)].
Formation of urethane linkages between amino groups of a protein and PEG
overcomes the problem of hydrolytic loss of the polymer chains [Veronese,
et al., Appl. Biochem. Biotechnol. 11, 141-152 (1985)]. In fact, it was
demonstrated on radioactively labeled PEG-derivatives that urethane links
are completely stable under a variety of physiological conditions [Larwood
& Szoka J., Labeled Compounds Radiopharm., 21, 603-614 (1984)]. The
attachment of PEG to a protein via a carbamate derivative was accomplished
[Beauchamp, et al. Analyt. Biochem. 131, 25-33 (1983)] using
carbonyldiimidazole-activated PEG. However, the polymer activated in this
manner is not very reactive and therefore long reaction times (48-72 hrs
at pH 8.5) were required to achieve sufficient modifications. Therefore,
the carbonyldiimidazole-activated agent clearly does not satisfy the first
requirement noted above. An additional disadvantage of this approach is in
the relatively high cost of carbonyldiimidazole.
Use of PEG-phenylcarbonate derivatives for preparation of urethane-linked
PEG-proteins was reported [see Veronese, et al. (1985), supra]. The main
drawback of this approach lies in the toxicity of the hydrophobic phenol
residues (p-nitrophenol or 2, 4, 5-trichlorophenol) and their affinity for
proteins. Clearly this method does not satisfy the second requirement
noted above.
Each of the activated forms of the polymer has properties which can be
considered advantageous or disadvantageous, depending on the system of
use. In light of the many applications of PEG-polypeptides, it is
desirable to broaden the arsenal of protein modifying PEG-reagents made
for a specific end use.
SUMMARY OF THE INVENTION
The present invention provides a compound having the structure
##STR2##
wherein R.sub.1 is H--, H.sub.3 C--, an oxycarbonyl N-dicarboximide group,
or any other functional group;
wherein each R.sub.2, R.sub.3, and R.sub.4 is an alkyl group which may be
straight, branched, disubstituted, or unsubstituted, and wherein each
R.sub.2, R.sub.3, and R.sub.4 may be independently the same as, or
different from, the others of R.sub.2, R.sub.3, and R.sub.4 ;
wherein R.sub.5 is an N-dicarboximide group; and
wherein a is an integer between 1 and 1000 and each of b and c is an
integer between 0 and 1000, and the sum of a, b, and c is between 10 and
1000.
The present invention also provides a process for preparing the compound
which comprises reacting a compound having the structure
##STR3##
wherein R.sub.1 is H--, H.sub.3 C--or any other functional group; wherein
each R.sub.2, R.sub.3, and R.sub.4 is an alkyl group which may be
straight, branched, disubstituted, or unsubstituted, and wherein each
R.sub.2, R.sub.3, and R.sub.4 may be independently the same as, or
different from, the others of R.sub.2, R.sub.3, and R.sub.4 ;
wherein X is a halogen;
wherein a is an integer between 1 and 1000 and each of b and c is an
integer between 0 and 1000, and the sum of a, b, and c is between 10 and
1000.
with an N-hydroxydicarboximide in the presence of a base.
The present invention further provides a modified polypeptide comprising a
polypeptide having bound thereto at least one polymer having the structure
##STR4##
wherein R.sub.1 is H--, H.sub.3 C--, an oxycarbonyl-N-carboximide or any
other functional group;
wherein each R.sub.2, R.sub.3, and R.sub.4 is an alkyl group which may be
straight, branched, substituted, or unsubstituted, and wherein each
R.sub.2, R.sub.3, and R.sub.4 may be independently the same as, or
different from, the others of R.sub.2, R.sub.3, and R.sub.4 ;
wherein a is an integer between 1 and 1000 and each of b and c is an
integer between 0 and 1000, and the sum of a, b, and c is between 10 and
1000.
wherein each polymer is covalently bound to an amine group of the
polypeptide by a urethane linkage.
The invention also provides a process for preparing a modified polypeptide
comprising reacting a polypeptide with the compound at a pH in the range
of about 5.8-11.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Release of mPEG from PEG-BSA conjugates. Experimental conditions:
Solutions of both types of PEG-BSA conjugates (-61% modification) at
concentration 4 mg/ml (by Bluret assay) were incubated in the appropriate
buffer. At given time intervals aliquots of these solutions were injected
into an HPLC equipped with Zorbax GF-450 column and RI-detector to
quantitate mPEG-5000.
FIG. 2. Stability of SS-PEG and SC-PEG at 22.degree. C. Experimental
conditions: The activated PEG's were stored in the form of fine powder in
tightly closed polypropylene containers. At given time intervals samples
of each polymer were assayed for active acyl content.
FIG. 3. Reactivity of activated PEG's as a function of pH. Experimental
conditions: To triethanolamine-borate buffer (0.3M, 1 ml) at the
appropriate pH, stock solution of NAL in water (50 mM, 0.1 ml) was added
followed by stock solution of the appropriate activated PEG in CH.sub.3 CN
(50 mM active acyl, 0.1 ml). The resultant solution was vortexed and
incubated at 28.degree. C. for 1 hr. A mixture of the same components but
leaving out SX-PEG was used as a control. The TNBS - assay version of
Snyder, et al. [(1975) Anal. Biochem. 64, 284] was used to determine the
unreacted NAL.
DESCRIPTION OF THE INVENTION
The present invention describes activated polyalkylene oxides having the
general structure:
##STR5##
wherein R.sub.1 is H--, H.sub.3 C--, an oxycarbonyl N-dicarboximide group,
or any other functional group; wherein each R.sub.2, R.sub.3, and R.sub.4
is an alkyl group which may be straight, branched, disubstituted, or
unsubstituted, and wherein each R.sub.2, R.sub.3, and R.sub.4 may be
independently the same as, or different from, the others of R.sub.2,
R.sub.3, and R.sub.4 ; wherein R.sub.5 is an N-dicarboximide group; and
wherein a is an integer between 1 and 1000 and each of b and c is an
integer between 0 and 1000, and the sum of a, b, and c is between 10 and
1000.
More specifically, the invention relates to preparation and use of a new,
activated PEG, namely, poly(ethylene glycol)-succinidyl carbonate (SC-PEG)
and the bifunctional derivative of PEG, namely, polyethylene
glycol-bis-succinidyl carbonate (BSC-PEG). Furthermore, heterobifunctional
derivatives of PEG are possible (Zalipsky and Barany, supra); one of the
end groups is succinidyl carbonate and the other end group (R.sup.1, see
Scheme 2, infra) contains a different reactive functional group, such as a
free carboxyl group or an amine acid. These materials react attachment
through stable urethane linkages (Scheme 2, below). The reactivity of the
new agents, SC-PEG and BSC-PEG, are comparable to the conventionally used
SS-PEG. Thus, high degrees of modification are achievable in mild
conditions (aqueous buffers, pH 5.8-11, preferably pH 7.0-9.5) within
about 30-60 min. and moderate temperatures (4.degree.-40.degree. C.).
Additionally, the agents are soluble in a variety of organic solvents,
thus being useful and important in the coupling of low molecular weight,
partially protected peptides and other biologically useful ligands.
The PEG does not have to be of a particular molecular weight, but it is
preferred that the molecular weight be between 500 and 40,000; more
preferably between 2,000 and 20,000. The choice of molecular weight of PEG
is made based on the nature of the particular protein employed, for
example, the number of amino groups available for modification.
The N-hydroxysuccinidyl released during protein modification is non-toxic
material that is often used in protein chemistry, especially for
preparation of biologically active protein-adducts. As in the case of
above mentioned carbonyldiimidazole activated PEG and
PEG-phenylcarbonates, the product of protein modification using SC-PEG or
BSC-PEG has PEG-chains grafted onto the polypeptide backbone through
carbamate (urethane) linkages. However, due to the higher reactivity of
the new agents, higher degrees of modification are achievable in shorter
periods of time. An additional advantage of succinidyl carbonate activated
PEG is that those active functional groups that do not react with amino
groups of a protein undergo fast aqueous hydrolysis producing
N-hydroxysuccinimide, carbon dioxide and hydroxy-terminated PEG. This is
of particular importance in the case of bifunctional PEG derivatives
(BSC-PEG). These materials can serve a dual purpose: PEGylation and
crosslinking at the same time. The BSC-PEG, like any homobifunctional
material can be used to crosslink two different proteins. When a BSC-PEG
molecule reacts with a protein via only one end group, the other SC-group
of the polymer, which does not react with the amine, is hydrolyxed and
therefore no extraneous (potentially antigenic) residues are introduced
onto the PEG-protein conjugate.
Biological activities of proteins modified with SC-PEG and BSC-PEG are
preserved to a large extent as shown by the examples below. There is one
literature precedent in which protein (tissue plasminogen activator)
convalently conjugated with PEG via urethane links had a higher specific
activity than the same protein modified with SS-PEG to approximately the
same extent [Berger & Pizzo, Blood, 71, 1641-1647 (1988)].
##STR6##
Naturally, the utility of SC-activated PEG-derivatives extends to
preparation of PEG-conjugates of low molecular weight peptides and other
materials that contain free amino groups.
A one-pot procedure for introduction of SC-groups onto PEG was developed
(Scheme 3 below). First, polyethylene glycol chloroformate was generated
in situ by treatment of the polymer (PEG) with phosgene. The resulting
chloroformate was then reacted with N-hydroxysuccinimide (HOSu) followed
by triethylamine (TEA) to yield the desired activated derivatives of PEG.
The activated polymer preparations were purified from low molecular weight
materials and determined to contain the theoretical amounts of active
groups.
##STR7##
EXAMPLE 1
Preparation of SC-PEG: Methoxypolyethylene glycol of molecular weight 5000
(Union Carbide, 60 g, 12 mmol) was dissolved in toluene/dichloromethane
(3:1, 200 ml) and treated with a toluene solution of phosgene (30 ml, 57
mmol) overnight. The solution was evaporated to dryness and the remainder
of phosgene was removed under vacuum. The residue was redissolved in
toluene/dechloromethane (2:1, 150 ml) and treated with solid
N-hydroxysuccinidyl (2.1 g, 18 mmol) followed by triethylamine (1.7 ml, 12
mmol). After 3 hours, the solution was filtered and evaporated to dryness.
The residue was dissolved in warm (50.degree. C.) ethyl acetate (600 ml),
filtered from trace insolubles and cooled to facilitate precipitation of
the polymer. The product was collected by filtration and then
recrystallized once more from ethylacetate. The product was dried in vacuo
over P.sub.2 O.sub.5. The yield was 52.5 g (85% of theory).
To determine the active carbonate content of the product, samples of the
polymer were reacted with a measured amount of benzylamine in
dichloromethane and the excess of amine was titrated with perchloric acid
in dioxane. These titrations indicated that 1 g of the product contained
1.97.times.10.sup.-4 mole of active carbonate (101% of theoretical
content). I.R. (film on NaCl, cm.sup.-1) characteristic bands at : 1812
and 1789 (both C.dbd.O, succinimide); 1742 (C.dbd.O, carbonate); 1114
(CH.sub.2 OCH.sub.2). .sup.13 C-NMR (CDCl.sub.3):.delta. 168.5 (CH.sub.2
C=O); 151.3 (0--CO.sub.2); 71.9 (CH.sub.3 OCH.sub.2) 70.2 (PEG); 68.7
(CH.sub.2 CH.sub.2 OCO.sub.2); 68.0 (CH.sub.2 CH.sub.2 OCO.sub.2); 58.9
(CH.sub.3 O); 25.2 (CH.sub.2 C.dbd.O) ppm.
EXAMPLE 2
Preparation of BSC-PEG: Polyethylene glycol of molecular weight 4600 (Union
Carbide, 50g, 21.7 mequiv. OH) was converted to the corresponding
bis-N-hydroxysuccinidyl carbonate using a toluene solution of phosgene (50
ml, 96.5 mmol) and then N-hydroxysuccinidyl (3.8 g, 23 mmol) and
triethylamine (3.2 ml, 23 mmol) following the procedure described in
Example 1. After purification the product was obtained as a white powder
(51 g, 96%). Active carbonate content was 4.0.times.10.sup.-4 mole/g (98%
of theoretical) as determined by titrations with benzylamine-perchloric
acid. I.R. (film on NaCl, cm.sup.-1) characteristic bands at : 1812 and
1789 (both C.dbd.O, succinimide); 1742 (C.dbd.O, carbonate); 1114
(CH.sub.2 OCH.sub.2). .sup.13 C--NMR(CDCl.sub.3) .delta. 168.5 (CH.sub.2
C=O); 151.3 (O--CO.sub.2); 70.2 (PEG); 68.7 (CH.sub.2 CH.sub.2 OCO.sub.2);
68.0 (CH.sub.2 CH .sub.2 OCO.sub.2); 25.2 (CH.sub.2 C.dbd. O) ppm. H-NMR
(CDCl.sub.3):.delta. 4.35 (m, 4H, CH.sub.2 OCO.sub.2); 3.55
(s,.about.400H, PEG); 2.74 (s, 8H, CH.sub.2 C.dbd.O) ppm.
EXAMPLE 3
Preparation of polyethylene glycol-Bovine Serum Albumin conjugates
(PEG-BSA):
A. SC-PEG (1 g) was added to a stirred solution of Bovine Serum Albumin
(BSA) (100 mg) in 0.1M sodium phosphate, pH 7.8 (20 ml). Sodium hydroxide
(0.5N) was used to maintain pH 7.8 for 30 min. The excess of free PEG was
removed by diafiltration using 50 mM phosphate buffered saline. The extent
of modification of BSA was approximately 50% (30 amino groups of BSA out
of total 60 reactive with SC-PEG.) as determined by
trinitrobenzenesulfonate (TNBS) titration of amino groups [Habeeb, Analyt.
Biochem. 14, 328-336 (1966)].
The same degree of modification was obtained when the experiment was
repeated under identical conditions using SS-PEG instead of SC-PEG.
B. SC-PEG (1 g) was added to a stirred solution of BSA (100 mg) in 0.1M
sodium borate, pH 9.2. Sodium hydroxide (0.5N) was used to maintain pH 9.2
for 30 min. The excess of free PEG was removed by diafiltration and the
product assayed for the number of free amino groups. Approximately 68%
(41) of the amino groups of the native BSA were modified.
C. BSC-PEG (1 g) was added to a stirred solution of BSA (100 mg) in 0.1M
sodium borate, pH 9.2. Sodium hydroxide (0.5N) was used to maintain pH 9.2
for 30 min. The excess of free PEG was removed by diafiltration and the
product assayed for the number of free amino groups. Approximately 80%
(48) of the amino groups of the native BSA were modified. Analysis of the
product by HPLC (Gel Filtration) indicated that over 65% of PEG-BSA was in
intermolecularly crosslinked form and about 35% of the product had the
same molecular weight as PEG-BSA from Example 3B.
EXAMPLE 4
Preparation of PEG-glutaminase: A solution of glutaminase Pseudomonas 7A
(200 mg) in 0.1M sodium phosphate, pH 7.8 was treated with SC-PEG (4.7)
g). The solution was stirred and pH 7.8 was maintained for 30 minutes. The
excess free PEG was removed by diafiltration using 50 mM PBS. The extent
of modification of glutaminase was 74% as determined by
trinitrobenzenesulfonate titration of amino groups (see Habeeb, 1966
above). The PEG-glutaminase product preserved 81% of the enzymatic
activity of the parent glutaminase.
EXAMPLE 5
Preparation of PEG-Trypsin: A solution of bovine pancreatic trypsin
(Boeringer-Mannheim, 120 mg in 20 ml), that was dialyzed overnight at
4.degree. C. against 20 mM CaCl.sub.2 in 1 mM HCl, was brought to
25.degree. C. and treated with SC-PEG (600 mg) for 30 min. During this
time pH 7.8 was maintained in the reaction vessel by automatic titration
with 0.5N NaOH. The solution was acidified to pH 3 and extensively
diafiltered to remove excess of free polymer using 20 mM CaCl.sub.2 in 1
mM HCl as a replacement fluid. The modified trypsin had approximately half
of the free amino groups of the parent enzyme (7 PEG chains per trypsin
molecule) as determined by TNBS titration of amino groups (Habeeb 1966).
The PEG-trypsin product preserved 96% of enzymatic activity of the parent
enzyme towards N.sup..alpha. -benzoyl-L-arginine ethyl ester.
EXAMPLE 6
Preparation of PEG-Arginase: A solution of bovine liver arginase (Sigma, 90
mg) in 0.1M NaCl (20 ml) was treated with SC-PEG (1.3 g) at 27.degree. C.
while pH 8.0 was maintained by automatic titration with 0.5N NaOH. After
30 min. the reaction mixture was diafiltered using 50 mM PBS as a
replacement fluid. Approximately 64% of the amino groups of the native
arginase were modified (56 PEG chains per arginase molecule). The
PEG-arginase product retained 70% of specific activity of the native
enzyme when assayed with 2,3-butanedione (BUN-urea reagent) at pH 9.5.
EXAMPLE 7
Other polypeptides, including chymotrypsin, asparaginase, and adenosine
deaminase, have been modified with SC-PEG using the procedures set forth
herein.
The methods of using SC- and/or BSC-functionalized polyalkylene oxides,
such as PEG and its copolymers are generally applicable to the preparation
of other modified polypeptides and other biologically active components
having amino groups.
While the present invention has been described by reference to
N-hydroxysuccinidyl derivatives, it will be obvious to those skilled in
the art that other N-hydroxydicarboximides may be substituted therefor.
Typical of such derivatives are N-hydroxyphthalimide,
N-hydroxyglutarimide, N-hydroxytetrahydrophthalimide,
N-hydroxy-5-norbornene-2,3-dicarboximide or other N-disubstituted
derivatives of hydroxylamine.
EXAMPLE 8
Comparison of SC-PEG To SS-PEG
A. As seen in Scheme 2, the product of protein modification using SC-PEG
has PEG-chains grafted onto the polypeptide backbone through carbamate
(urethane) linkages. Greater stability of the urethane linkage relative to
the ester bond produced upon use of SS-PEG (see Scheme 1) was expected to
prove a key difference between the two activated PEG's and the
corresponding PEG-protein conjugates. Our studies indeed confirmed this
expectation. FIG. 1 shows the results of GF-HPLC measurements of the
amounts of free mPEG produced as a result of incubation of PEG-BSA
conjugates derived from each of the activated PEG's. Considerably higher
stability of the SC-PEG-derived conjugate is apparent
B. To estimate the reactivities of SC-PEG and SS-PEG, kinetic measurements
of hydrolysis of the activated polymers in phosphate buffer and their
aminolysis by Na-acetyl-lysine (NAL) were performed. The results of these
experiments are summarized in Table 1. It is clear from these data that
SS-PEG is a more reactive reagent than SC-PEG. The difference in
hydrolysis rates was larger than the difference in aminolysis rates;
consequently, SC-PEG showed more favorable K.sub.am /K.sub.n ratios. The
slower hydrolysis of SC-PEG was also manifested in superior storage
stability of the reagent (FIG. 2).
C. Reactivity of the activated PEG's as a function of pH was determined
using NAL as a model for the .epsilon.-amino group of a protein. Each of
the activated PEG S was reacted with an equimolar amount of NAL at
different pH's, and measured the unreacted NAL using the TNBS-assay (FIG.
3). The optimal pH for use of SC-PEG was found to be about 9.3. It is not
advisable to use SS-PEG at pH>8.0, due to the limited stability of
PEG-succinate ester. However, even at pH values less than 8.0 this
activated PEG was found to be very reactive.
D. Both reagents showed high reactivity towards Trypsin yielding comparably
modified enzyme derivatives in mild conditions (pH 7.5-8.5) within 30 min.
The products were purified by diafiltration, and the degrees of
modification were determined by fluorometric assay, according to Stocks,
et al. [(1986) Anal. Biochem. 154, 232]. All PEG-modified trypsin
derivatives were essentially lacking (<1% of native) proteolytic activity
as determined by the Azocoll assay [Chavira, et al. (1984) Anal. Biochem.
136, 446]. The specific activities of representative SS-and SC-PEG
modified trypsins towards low molecular weight substrates are summarized
in Table 2. The modifications produced hardly any changes in esterolytic
activities towards benzoyl-L-arginine ethyl ester, but did enhance the
activities towards p-nitroanilides. Michaelis-Menten kinetic constants for
several SC- and SS-PEG modified trypsins were measured using ZAPA as the
substrate. These results, summarized in Table 3, indicate that, while
V.sub.max, K.sub.cat and K.sub.cat /K.sub.m were increasing gradually with
the extent of modifications, K.sub.m values were decreasing.
As compared to SS-PEG, SC-PEG is a less reactive yet more selective
reagent. This is evidenced by its higher K.sub.am /K.sub.h ratios and
better storage stability. SC-PEG is a sufficiently reactive reagent to
produce PEG-protein conjugates under mild conditions within 30 min. SC-PEG
can be used in a broader pH range than SS-PEG, showing the highest
reactivity at pH=9.3. PEG-protein conjugates obtaining through use of
SC-PEG are chemically more stable than SS-PEG derived conjugates. The
PEG-Trypsin conjugates produced by both activated PEG's have very similar
properties: They show no proteolytic activity, well preserved esterolytic
activity, and dramatically increased activity towards p-nitroanilide
substrates. Michaelis-Menten constants of the modified enzymes indicate
that the attachment of PEG to trypsin causes an increase in both the rate
of turnover of ZAPA and its affinity towards the modified enzymes.
TABLE 1
__________________________________________________________________________
Comparison of first order rate constants for hydrolysis (K.sub.h)
and aminolysis (K.sub.am) of SC-PEG and SS-PEG.sup.a
Hydrolysis: K.sub.h .sup.b (min.sup.-1) .times. 10.sup.3
Aminolysis: K.sub.am .sup.c (min.sup.-1) .times.
10.sup.3
Temp.
and [h.sub.1/2 (min)]
and [K.sub.am /K.sub.h ]
pH (.degree.C.)
SC-PEG SS-PEG SC-PEG SS-PEG
__________________________________________________________________________
7.0
4 0.87 [793]
1.84 [376]
2.64 [3.0]
3.74 [2.0]
27 6.05 [115]
10.4 [67]
26.4 [4.4]
41.4 [4.0]
37 14.2 [49]
25.9 [27]
81.7 [5.8]
104 [4.0]
7.4
22 5.37 [129]
10.7 [65]
29.1 [5.4]
42.7 [4.0]
27 9.0 [77]
16.0 [43]
48.6 [5.4]
73.6 [4.6]
37 19.3 [36]
37.6 [18]
145 [7.5]
193 [5.1]
7.8
4 1.37 [505]
2.58 [268]
12.4 [9.1]
15.0 [5.8]
27 10.3 [67]
21.6 [32]
130 [12.6]
152 [7.0]
37 21.8 [32]
48.8 [14]
226 [10.6]
267 [5.5]
__________________________________________________________________________
.sup.a All the measurements were performed by following the appearance of
Nhydroxysuccinimide anion (OSu) at 260 nm in 0.008 M sodium phosphate;
concentration of PEGbound succinimidyl active acyl at time zero
[SXPEG].sub.0 was 0.1 mM; in aminolysis experiments concentration of
N.sup..alpha.acetyl-lysine at time zero [NAL].sub.0 was 3 mM.
.sup.b K.sub.h = Rate.sub.h /[SXPEG].sub.0, where Rate.sub.h = dA.sub.260
/dt .times. 1 E.sub.260 .times. 1F; .epsilon..sub.260 = 8500 M.sup.-1
cm.sup.-1 is an extintion coefficient of OSu; and F = OSu]/([HOSu] + OSu)
= (1 + 10.sup.6.0 pH ).sup.-1.
.sup.c K.sub.am = Total Rate/[SXPEG].sub.0 - K.sub.h. The Total Rate in
aminolysis experiments was calculated the same way as Rate.sub.h in
hydrolysis experiments.
TABLE 2
__________________________________________________________________________
SUMMARY OF MODIFICATION, ESTEROLYTIC ACTIVITY,
AND AMIDOLYTIC ACTIVITY DATA FOR TYPSIN
AND ITS mPEG DERIVATIVES
Trypsin Modif.sup.b
BAEE.sup.c
% BAPA.sup.d
% ZAPA.sup.d
%
Derivatives.sup.a
(%) (u/mg)
Native
(u/mg)
Native
(u/mg)
Native
__________________________________________________________________________
Native Trypsin
0 92.4 100 1.26 100 7.81 100
SC-PEG.sup.M -Trypsin
N = 6 42.3
103 112 2.26 179 15.3 196
N = 7 45.8
87.9 95.1
2.38 188 17.5 224
N = 9 58.8
90.1 97.5
2.67 212 18.9 242
N = 12 77.9
85.1 92.2
3.83 304 25.5 326
SS-PEG.sub.h -Trypsin
N = 7 44.8
102 110 3.25 258 18.8 241
N = 12 770 94.3 102 4.34 344 24.7 316
__________________________________________________________________________
.sup.a For SXPEG.sub.hTypsin, N = 15 .times. (% Modif)/100 and is rounded
to the nearest integer.
.sup.b The percent of amino groups modified was determined by the
fluorescamine assay [Stocks, et al. (1986) Anal. Biochem. 154,
.sup.c The BAEE (Nabenzoyl-L-arginine ethyl ester) trypsin assay was done
at pH 7.8, 37.degree. C. w/ a substrate conc'n of 0.5 mM. The extinction
coefficient was .epsilon..sub.285 = 808 M.sup.-1 cm.sup.-1 [Kezdy, et al.
(1965) Biochemistry 4, 99].
.sup.d The BAPA (Nabenzoyl-DL-arginine-p-nitroanilide) and ZAPA
(NaCBZ-L-arginine-p-nitroanilide) amidolytic assays were done w/ a
substrate conc'n of 1 mM in 50 mM TrisHCl pH 8.1, 10 mM CaCl.sub.2, at
37.degree. C. The extinction coefficient for pnitroaniline,
.epsilon..sub.410 = 8800 M.sup.-1 cm.sup.-1, was used in both assays.
TABLE 3
______________________________________
MICHAELIS-MENTEN CONSTANTS FOR THE
AMIDOLYTIC ACTIVITY OF NATIVE
TRYPSIN AND ITS mPEG DERIVATIVES.sup.a
Km V.sub.max K.sub.cat
K.sup.cat /K.sup.m
Trypsin Derivatives
(mM) (.mu.M/min)
(min.sup.-1)
(.sup.mM min.sup.-1)
______________________________________
Native Trypsin
1.08 15.7 378 349
SC-PEG.sub.H -Trypsin
N = 7 0.29 19.6 470 1626
N = 9 0.21 20.2 484 2290
N = 12 0.11 22.9 549 4973
SS-PEG.sub.h -Trypsin
N = 7 0.21 18.6 447 2172
N = 12 0.13 22.5 539 4159
______________________________________
.sup.a The measurements took place at 37.degree. C. with a constant
trypsin protein concentration of 1.0 .mu.g/ml (via Bluret assay).
Nacarbobenzoxy-L-arginine-p-nitroanilide (ZAPA was used as a substrate in
concentrations varying from 0.02 to 1.71 mM in 50 mM TrisHCl pH 7.8, 10 m
calcium chloride. The constants were calculated from LineweaverBurk plots
of the initial rates of the appearance of pnitroaniline (.epsilon..sub.41
= 8800 M.sup.-1 cm.sup.-1).
______________________________________
EFFECT OF N.alpha.-Ac-L-Tyr (NAT) ON THE EXTENT
OF REACTION OF SS-PEG AND SC-PEG
WITH N.alpha.-Ac-L-Lys (NAL)
% of NAL Reacted with.sup.a
NAT/NAL SS-PEG SC-PEG
______________________________________
0 78.55 (100).sup.b
53.75 (100)
1.0 77.15 (98.2) 55.25 (103)
2.5 74.50 (94.8) 52.55 (97.8)
5.0 68.50 (87.2) 48.60 (90.4)
______________________________________
.sup.a To triethanolamineborate buffer (0.3 M, pH 8.1) the following was
added: a solution of NAL (50 mM, 0.1 ml) and a solution of NAT (100 mM,
volume corresponding to the ratios given in the table and bringing the
combined volume to 1.1 ml) both in the same buffer, and lastly the
appropriate activated PEG in CH.sub.3 CN (50 mM active acyl, 0.1 ml). The
resultant solution was vortexed and incubated at 28.degree. C. for 1 hr.
mixture of the same components but leaving out SXPEG
was used as a control. The TNBS assay version of Snyder, et al. [(1975)
Anal. Biochem. 64, 284] was used to determine the unreacted NAL.
.sup.b The numbers given in the parentheses represent the values of
percent of NAL reaction divided by the percent of NAL reaction when
NAT/NAL = 0.
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