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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a novel method for covalently binding biologically
active compounds to carriers and to the complex formed by the method. More
particularly, the invention relates to a novel method for covalently
binding enzymes to carriers and to the complex formed by the method.
2. Background of the Prior Art
It is known that proteins such as enzymes may be bound onto or into
water-insoluble carriers to form bound or "immobilized" enzymes. These
immobilized enzymes may be used in various reactions especially in
commercial applications, e.g. food processing. Most prior art methods for
immobilizing enzymes to carriers require the presence of functional groups
in the carrier, usually nucleophilic groups. The present invention does
not have such a requirement. U.S. Pat. No. 3,824,150 describes enzymes
bound to polymeric sheets with a triazine bridging group.
SUMMARY OF THE INVENTION
The invention relates to a method for binding biologically active compounds
to carriers comprising reacting an asymmetric bifunctional linking
compound having at least one phenyl azide moiety and at least one
s-triazine moiety with a carrier in the presence of light to form a
reaction product comprising the carrier covalently bound to the linking
compound through the phenyl azide moiety and further reacting said
reaction product with a biologically active compound to form a final
product comprising the reaction product having the biologically active
compound covalently bound to the linking compound through the s-triazine
moiety.
The invention further relates to a complex comprising an asymmetric
bifunctional linking compound having at least one phenyl azide moiety and
at least one s-triazine moiety, said phenyl azide moiety being covalently
bound to a carrier.
The invention also relates to a complex comprising an asymmetric
bifunctional linking compound having at least one phenyl azide moiety and
at least one s-triazine moiety, said phenyl azide moiety being covalently
bound to a polymer.
The invention also relates to a complex comprising an asymmetric
bifunctional linking compound having at least one phenyl azide moiety
linked to a carrier and at least one s-triazine moiety linked to a
protein.
DETAILED DESCRIPTION OF THE INVENTION
The asymmetric bifunctional linking compound which may be used in the
present invention must have at least one phenyl azide moiety and at least
one s-triazine moiety. These linking compounds per se do not form a part
of this invention but form the subject matter of co-pending U.S. patent
application Ser. No. 573,364 filed Apr. 30, 1975.
Linking compounds which satisfy the foregoing criteria include those
compounds having the structural formula
##STR1##
where B is NR, O, S,
##STR2##
or CH.sub.2 ; (M).sub.n is a saturated or unsaturated hydrocarbon chain; X
is NR, O or S; A is SO.sub.2 R, CN, NO.sub.2 or H; Y is NR.sub.2, N.sub.3,
halogen or SH; R is H and/or a lower alkyl group; D is N.sub.3 or halogen
and n is 1-12.
A preferred linking compound has the following structural formula
##STR3##
The linking compounds described herein are asymmetric bifunctional
compounds. One functional moiety is a phenyl azide. This functionality may
be used because of its photochemical reactivity to form a nitrene which
inserts into covalent bonds e.g. --C--H, C=C and C--O, of many polymeric
organic molecules. The second functional moiety is an s-triazine group
which reacts with biologically active compounds such as proteins, as
discussed in more detail below.
Carriers which may be used in this invention include polymeric and
non-polymeric carriers. Polymers which may be used as carriers are water
insoluble organic polymers of synthetic or natural origin. These polymers
all react with the phenyl azide moiety of the linking compound. Examples
of suitable carriers include polyethylene (conventional and linear),
polypropylene, polymethylpentene, ethylene propylene copolymer,
polystyrenes, polycarbonate, polyvinyl chloride, etc.; organic polymers
from biological origin, such as, for example, cellulose, starch, pectin,
etc. and proteins such as enzymes, lipoproteins, mucopolysaccharides,
antibodies, etc. Other suitable polymers include aminoethylated cellulose,
diazobenzyl cellulose, diazotized p-aminobenzyl cellulose,
amino-s-triazine cellulose, acid chlorides of carboxylic or sulfonic acid
ion-exchange resins, carboxymethyl cellulose azide bromoacetyl cellulose,
methacrylic acid-methacrylic acid-3-fluoro-4,6-dinitroanilide copolymers,
the diazotized-m-aminobenzyloxy-methyl ether of cellulose, diazotized
poly-p-aminostyrene, the diazotized copolymer of p-aminophenylalanine and
leucine, phosgenized poly-p-aminostyrene, ethylene-maleic anhydride
copolymers, polyisothiocyanate derivatives of poly-p-aminostyrene,
polystyrylmercuric acetate, acrylamide-methylene-bis acrylamide copolymer
gels, polyacrylamide, poly-4-hydroxy-3-nitrostyrene and the like.
Examples of carbonyl polymeric carriers which may be used herein include
those produced according to any known procedure from such aldehyde
monomers as acrolein; .alpha.-alkyl acroleins, e.g. methacrolein,
.alpha.-propylacrylein; crotonaldehyde; 2-methyl-2-butenal;
2,3-dimethyl-2-butenal; 2-ethyl-2-hexenal; 2-decenal; 2-dodecenal;
2-methyl-2-pentenal; 2-tetradecenal and the like, alone or in admixture
with up to 95 percent, by weight, based on the total weight of the
copolymer, of each other and/or such other copolymerizable monomers known
to react therewith such as unsaturated alcohol esters, e.g., the allyl,
crotyl, vinyl, butenyl, etc., esters of saturated and unsaturated
aliphatic and aromatic monobasic and poly-basic acids such as acetic,
propionic, butyric, valeric, adipic, maleic, fumaric, benzoic, phthalic,
terephthalic, etc., acids; vinyl cyclic compounds (including monvinyl
aromatic hydrocarbons) e.g., styrenes, o-, m-, and p-chlorostyrenes,
-bromostyrenes, -fluorostyrenes, -methylstyrenes, -ethylstyrenes, various
polysubstituted styrenes, e.g., di-, tri-, and tetrachlorostyrenes,
-bromostyrenes, etc.; vinyl naphthalene, vinyl chloride, divinyl benzene,
allyl benzene, vinyl pyridine, diallyl benzene, various
.alpha.-substituted and .alpha.-substituted, ring-substituted styrenes,
e.g., .alpha.-methyl styrene, .alpha.-methyl styrene,
.alpha.-methyl-p-methyl styrene, etc., unsaturated ethers, e.g.
ethylvinylether, etc., unsaturated amides, e.g., acrylamide,
methacrylamide, etc.; N-substituted acrylamides, e.g.,
N-methylolacrylamide, N-allyl acrylamide, N-methyl acrylamide, etc.;
acrylates such as the methyl, ethyl, propyl, butyl, etc., acrylates and
methacrylates; nitriles such as acrylonitrile and other comonomers shown,
for example, in U.S. Pat. No. 2,657,192.
Examples of other carbonyl polymers which may be utilized as carriers
herein include those produced according to any known procedure and in
amounts similar to those indicated above in regard to the aldehyde
polymers from such ketone monomers as methyl vinyl ketone, methyl allyl
ketone, ethyl vinyl ketone, methyl isopropenyl ketone, ethyl allyl ketone,
etc., phenyl vinyl ketone, p-tolyvinyl ketone. Also, such polymers as
poly(vinylpyridinium ketones) and haloketones; copolymers of the
above-mentioned aldehyde monomers and ketone monomers with or without the
above-disclosed copolymerizable comonomers; polyacetal and the like. The
molecular weights of the polymers used is not critical and those as low as
1,000 can be used.
Similarly, such polymers as the copolymers of ethylene and carbon monoxide
and various glyoxal adducts, all well known in the art, can be utilized
herein.
The compounds defined as biologically active compounds include a broad
variety of compounds. Suitable biologically active compounds include
proteins broadly, that is, compounds which consist of or contain protein.
One major class of proteins are compounds which can be broken down from
proteins, and enzymes, such as, for example those conventionally used
commercially and industrially, e.g. in the fields of tanning of leather,
beer-making, sugar processing, etc. Thousands of enzymes are known to
exist.
Exemplary of the enzymes which may be utilized herein include proteolytic
enzymes, hydrolases, amylases, dehydrogenases, kinases, oxidases,
deaminases, amidases, enzyme anticoagulants, etc., including lactic
dehydrogenase, creatine, phosphokinase, trypsin, papain, alk. phosphatase,
amyloglucosidase, dextranase, glucose oxidase, glucose isomerase, amidase,
penicillin amidase, chymotrypsin, .beta.-galactosidase, pyruvate kinase,
ficin, pepsin, carboxypeptidase, streptokinase, plasminogen, urease,
invertase, alcohol dehydrogenase diastase, .beta.-glycosidase, maltase,
aldolase, lactase, amygdalase, lipase, steapsin, erepsin, zymase,
catalase, melibiase, pectolase, protease, bovine erythrocyte and/or horse
serum chlolinestrerase, tyrosinase, L-asparaginase, glucose isomerase,
cytase, adenase, guanidase, carboxylase, inulase, vinegar oxidase
aldehydase, rhamnase, myrosinase, phytase, tannase, carbamase, nuclease,
guanase, adenase, thrombase, chymase, cozymase and the like.
Other proteins which may be used in the invention include antigens,
antibodies, peptides, amino acids, lipoproteins, co-enzymes, etc.
Other biologically active compounds which may be used in this invention
include carbohydrates, enzyme inhibitors, biologic markers e.g.
radioactively labeled compounds and dyes, and drugs.
Generally, the reaction is carried out in two separate steps. In step one,
the phenyl azide moiety of the bifunctional linking compound is linked to
a polymeric carrier by photochemical reaction. To carry out the reaction,
a bifunctional linking compound, as described above, is reacted with a
polymeric carrier in a suitable solvent and exposed to light at a
wavelength of about 350 to about 800 nm and preferably about 420 to about
500 nm for about 0.1 to about 60 minutes at temperatures ranging from
about -20.degree. to about 50.degree. C and preferably for about 0.5 to
about 2 minutes at temperatures between about 0.degree. and 25.degree. C.
Upon completion of the reaction, unbound linking agent is removed from the
reaction mixture, e.g., by washing with a suitable solvent. In step two,
the polymeric carrier with covalently bound linking reagent is incubated
with a biologically active compound, e.g., an enzyme, for about 0.5 to
about 2 hours at a temperature of about 0.degree. to about 30.degree. C
and at a pH of about 4.5 to about 6.5. During this reaction, the
s-triazine moiety reacts with the biologically active compound. The phenyl
azide-polymer bond is not affected by this reaction. Unbound biologically
active compound is then removed from the reaction mixture, e.g., by
washing with a suitable solvent. The resultant complex consists of a
biologically active compound such as an enzyme bound through the
bifunctional linking compound to a polymeric carrier. The complex formed
by step 1 is also useful in that the biologically active compound may be
added to the complex at a later time.
An alternative method for covalently binding biologically active compounds
to a polymeric carriers is to activate the carrier with the s-triazinyl
azide derivative of the preferred linking reagent. The activated carrier
is then incubated with the biologically active compound at 0.degree. to
about 5.degree. C for about 1 to about 20 minutes while illuminating the
ultraviolet light of 180 to about 350 nm, preferably 200 to about 300 nm.
The excess non-bound biologically active compound is removed as described
previously under the general reaction. This alternative method is the
preferred method when the biologically active compound contains no
nucleophilic groups or when the linking reaction (the nucleophilic
replacement reaction) may be detrimental to the biological activity of the
biologically active compound. This alternative method would not be the
method of choice when the biologically active compound is a protein for
the reason that proteins may be damaged by ultraviolet irradiation.
The insolubilized enzymes of the invention can be used in a wide variety of
enzymatically catalyzed reactions, and are often suitable for use in
processes in which soluble enzymes have previously been used. Thus they
may, for example, be used in the preparation of penicillins, beer
clarification, the preparation of glucose using amyloglucosidase, the
preparation of optically active amino acids, and the formation of
L-alanine by transamination. Other potential uses include enzymatic
hydrolysis of carbohydrates and proteins, the processing of waste
materials, the specific manipulation of large natural molecules such as
steroids, alkaloids, chloramphenicol and riboflavine, alcoholic and other
kinds of fermentation, the fixing of nitrogen, a luciferase system for
A.T.P. estimation, biochemical fuel-cells, and the specific oxidation and
reduction of organic materials, including carbon dioxide fixation.
The insolubilized enzymes may also be used in enzymatic analysis,
particularly in the sequential analysis of proteins, RNA and DNA. In this
case the substrate can be, for example, forced through a permeable sheet
by means of a syringe. Where chromatography follows the reaction, it may
be possible to chromatograph the substrate across a permeable sheet
containing the enzyme, for example in urea analysis.
The following is an example of the foregoing method.
EXAMPLE I
Method of immobilizing glucose-6-phosphate dehydrogenase to polycarbonate
Two mg of the linking compound
1-N-(2-nitro-4-azidophenyl)-6-N-(4,6-dichloro-sym-triazinyl)-diaminohexane
having the structural formula
##STR4##
was dissolved in 100 ml ethyl alcohol and 2 ml of this solution was placed
in a polycarbonate test tube and exposed to sunlight at ambient
temperature for about 20 minutes. The test tube was washed with ethanol to
remove unbound linking compound and an enzyme solution, made by adding 10
.mu.g glucose-6-phosphate dehydrogenase to 2 ml of 0.05M Na acetate buffer
having a pH of 5.5, was added to the test tube and incubated for 2 hours
at ambient temperature. The enzyme solution was then removed and the test
tube was twice rinsed with 0.1M Tris buffer to remove any unbound enzyme.
Subsequent enzyme assay of the test tube confirmed the presence of bound
enzymes. The enzyme-polycarbonate preparation showed no significant
decrease in enzyme activity over a period of 60 days after forming the
complex.
EXAMPLE II
Method of immobilizing glucose-6-phosphate dehydrogenase to Tygon tubing
3 ml of the linking compound in ethyl alcohol as in Example I was allowed
to flow through Tygon tubing, Formula R3603, 1/8 in. I.D. 3/16 O.D., 2 ft
long, for 3 minutes under an ultraviolet lamp. The tubing was rinsed 3
times with 5 ml of ethyl alcohol and 2 times with water. One ml of enzyme
solution 20 .mu.g/ml in pH 5.5 acetate buffer was flowed through the
tubing for 1 hour at room temperature. The enzyme-tubing complex was
washed as in Example I. Subsequent enzyme assay of the tubing confirmed
the presence of bound enzyme. The loss of enzyme activity bound in the
tubing was negligible over a period of 30 days.
EXAMPLE III
Method of immobilizing hexokinase to polycarbonate
Example I is repeated, except hexokinase is used in the place of
glucose-6-phosphate dehydrogenase. Comparable results are obtained.
EXAMPLE IV
Method of immobilizing glucose-6-phosphate dehydrogenase to polystyrene
The method of Example I is used, except polystyrene discs are used in place
of polycarbonate and an artificial light source is used as in Example II.
Comparable results are obtained.
EXAMPLE V
Method for immobilizing alcohol dehydrogenase to cellulose
Cellulose particles were suspended in 5 ml of the linking reagent (shown in
Example I) (2 mg/100 ml in ethyl alcohol), and irradiated with sunlight at
ambient temperature. The activated cellulose was washed with 25 ml of
ethyl alcohol. 50 mg of the activated cellulose was incubated with 0.1 ml
of alcohol dehydrogenase solution (100 mg/ml pH 5.5 acetate) for 95
minutes at 25.degree. C. The enzyme-cellulose complex was washed 3 times
in 5 ml 0.1M Tris pH 8.1. An enzyme assay of the cellulose confirmed the
presence of the enzyme.
EXAMPLE VI
Method of simultaneously binding glucose-6-phosphate dehydrogenase and
alcohol dehydrogenase to cellulose
The activated cellulose was prepared as in Example V. 50 ml of the
activated cellulose was incubated with a mixture of glucose-6-phosphate
dehydrogenase and alcohol dehydrogenase (each 50 mg/ml in acetate pH 5.5)
for 1 hr. The cellulose carrier was washed as in Example V. Subsequent
enzyme assays confirmed the presence of both enzymes and that the enzymes
were bound in direct proportion to the concentration of each enzyme in the
solution surrounding the activated carrier. The enzymes were confirmed to
be active both individually and when carrying out simultaneous reactions.
EXAMPLE VII
Method of simultaneously binding hexokinase and glucose-6-phosphate
dehydrogenase to polycarbonate tubes
The method of Example VI is used, except hexokinase and glucose-6-phosphate
dehydrogenase are the enzymes used and polycarbonate tubes (as in Example
I) are used in place of cellulose.
EXAMPLE VIII
Method of immobilizing bovine serum albumin to cellulose for use as an
immunoabsorbent
The method of Example V is used except the protein used is bovine serum
albumin. The protein-carrier complex thus formed may be used as an
immunoabsorbent to remove anti-bovine serum albumin (antibody) from rabbit
serum.
EXAMPLE IX
Method of immobilization of antibody to cellulose for use as
immunoabsorbent
Example VIII is repeated, except anti-bovine serum albumin antiserum is
bound to cellulose by the method shown in Example V. The bound antibody
has affinity for bovine serum albumin.
EXAMPLE X
Method of covalently binding glucose-6-phosphate dehydrogenase to bovine
serum albumin
In this example, the serum protein bovine serum albumin can be considered
to be the carrier polymer. The linking reagent was bound to the albumin by
illumination (with Hanovia lamp) of a 2 ml solution of albumin (5 mg/ml)
and 0.02 ml linking reagent (1 mg/100 ml ethyl alcohol) at 0.degree. C pH
5 acetate for 2 minutes. The excess linking reagent was removed by passing
the albumin solution over a Sephadex G-25 column eluted with pH 5.5 sodium
acetate at 0.degree. C. The enzyme was bound to the albumin by incubation
of 5 mg of enzyme with the albumin at room temperature pH 5.5 for 2 hours.
Subsequent analysis confirmed that the two proteins, i.e., albumin and
enzyme, were bound to each other.
EXAMPLE XI
Method of binding glucose-6-phosphate dehydrogenase to regenerated
cellulose
The method of Example II is used, except cellulose tubing is used instead
of Tygon tubing. Comparable results are obtained.
EXAMPLE XII
Method of binding glucose-6-phosphate dehydrogenase to polyethylene
The method of Example II is used, except polyethylene tubing is used
instead of Tygon tubing. Comparable results are obtained.
EXAMPLE XIII
Example I is repeated, except the glucose-6-phosphate dehydrogenase is
replaced with each of the following enzymes: lactate dehydrogenase
6-phosphogluconate dehydrogenase, trypsin, peonase and amylase. Comparable
results are obtained.
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