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Description  |
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The invention is concerned with the production of a vehicle substance which
is capable of covalent bonding with biologically active matter, and is
formed from at least a vehicle preferably consisting of several --NH.sub.2
or --OH groups and at least one bonding agent having at least two reactive
groups. The invention moreover relates to a vehicle substance comprising
at least one reactive group capable of immobilising biologically active
matter, especially enzymes, and an agent which contains a biologically
active substance which is immobilised by a vehicle substance.
The following terms are defined as follows for better understanding of the
invention:
A "vehicle" is a starting material, for example cellulose, agarose,
dextranes or the like, the essential feature being that the vehicle
comprises NH.sub.2 or OH-groups. In case a vehicle does not contain these
groups they have to be made available in a preliminary reaction.
A "bonding agent" is a substance capable of adding biological matter or of
condensing with the same. On the other hand, the bonding agent establishes
the link with the vehicle, either directly or indirectly.
A "coupling agent" is a substance which always establishes a direct link
with the vehicle. It may therefore be a "bonding agent". It is common to
bonding agents and coupling agents that either may comprise two reactive
groups.
A "spacer" is a chemical compound which generally contains two or several
groups capable of reaction so that it can form a link on the one hand with
a coupling agent, and on the other hand with a bonding agent. It is the
function of a spacer to increase the distance between vehicle and bonding
agent and to counteract thereby steric interference.
The term "vehicle substance" denotes a chain which consists at least of one
vehicle and one bonding agent; however, the chain may be composed of a
vehicle, a coupling agent, a spacer, and a bonding agent. In a chain
formed in this fashion in the vehicle substance, each of the groups
capable of reaction will react with a reactive group of the substances,
thus forming chains. If, for example, the vehicle includes a group capable
of reaction, the coupling agent for this compound must comprise a reactive
group.
A "group capable of reaction" is a nucleophilic reagent, especially
hydroxy- or amino-groups. A "reactive" group is a reagent group of a
substance which readily enters into an addition- or condensation reaction
with a group capable of reaction.
A "biologically active" substance may be a biological substance as such or
a substance which is always capable of interacting with a biological
substance. For example, it may be a protein, an enzyme or the like, or it
may be any organic or inorganic matter constituting the substrate for an
enzyme.
An "agent" is a vehicle substance containing a biologically active
substance.
It is known that vehicles containing hydroxyl groups especially
polysaccharides such as cellulose or dextrane and agarose, may be
activated so as to enable them to bond biologically active matter
containing amino groups or hydroxyl groups. For example, agarose gels may
be activated with cyanogen bromide in the presence of alkali to enable
them to bond proteins and other biological substances. However, the
bonding effect is not stable enough. A number of bonding systems evolve,
whose stability is dissimilar. The vehicle substance has to be prepared
immediately prior to use, that is to say it is not storable. Several
theories are held with regard to accurate conversion. No exact explanation
has been offered up to now. (Methods in Enzymology, vol. 34, pp 13 to 30)
According to another, known, method, the coupling between a vehicle and a
biologically active substance may be effected by means of reagents
containing two or more reactive groups. For example, cellulose is
activated by cyanuric chloride, the resultant vehicle substance thereby
retaining chemically active chlorine groups which are capable of reacting
with protein. The drawback of this method is that cyanuric chloride
entails the denaturation of sensitive biologically active matter. (Nature,
vol. 216, 1967, pp. 514 to 515).
It is also known that agarose can be coupled with divinyl sulfone. The
resulting vehicle substance adds biologically active matter, for example,
protein preserving, and the yield is good. However, bondage is very weak
and the agent consequently sheds the bonded protein very quickly. (Methods
in Enzymology, as quoted).
Also known is the manufacture of a vehicle substance in a reaction between
agarose and Bisoxiranes. The resultant vehicle substance bonds protein
under the formation of stable linkages. However, in order to effect this
linkage, the pH conditions have to be extreme, and there is therefore the
danger of sensitive substances such as biologically active matter being
destroyed. (Methods in Enzymology, as quoted).
To bond macro molecules for the purpose of creating interaction with other
macro molecules, the distance from the vehicle surface should be great
enough to allow for the space required by the interaction. It is known
that this distance may be bridged as required by means of suitable
intermediate compounds defined as spacers. For example, it is known that
agarose is activated by means of cyanogen bromide and to bond with the
product a diamine of extended chain-length, which acts as a spacer. The
free group is succinylated, and the carboxyl groups is activated through
N-hydroxy-succinimide and dicyclohexyl carbodiimide. However, considerable
chemical expenditure is necessary. Manipulation during reactions is
difficult and in the case of large scale reactions it can hardly be
carried out. (Biochemistry, vol. 11; 1972, 2291-99).
It is an object of the invention to synthetise a vehicle substance which
permits biologically active matter to be bonded to an insoluble vehicle in
such a manner that the capability of the biologically active substance of
interacting with another biological substance is not affected. In
particular, it is an object of the invention to present an agent which
contains covalently bonded enzymes in a high yield, and which has the
following properties:
(a) has a temperature resistance up to 100.degree. C.
(b) is mechanically stable and resistant to compressive load
(c) has a low flow resistance
(d) is free from charge carriers
(e) is hydrophilic
The linkage of the biologically active matter, for example, enzyme, with
the vehicle, should
(f) be very stable against hydrolysis in the pH range 3 to 11,
(g) affect the vehicle characteristics as little as possible,
(h) be affected under very gentle conditions (aqueous solution, neutral pH
range),
(i) be carried out specifically,
(k) not interfere with enzymetic effect when coupled with the vehicle and
(l) not sterically hinder the reaction of enzymes with high molecular
substances.
It is emphasized in this connection that there are certain applications
where it is not necessary for all of the above listed characteristics to
be reached. However, with practically all sensitive enzymes, and with
enzymes having high molecular co-reactants it is important that the
characteristics listed under (a) to (l) should be simultaneously present.
The method according to the invention, relating to the production of a
vehicle substance, distinguishes itself in that one or preferably both
reactive groups of the bonding agent are isocyanates. The method is
founded on the sequence of an interrupted process, i.e., a vehicle
substance being produced and if required isolated, in a first stage of the
process. Even this first stage is carried out step by step, and the
intermediate product obtained from each step is isolated, say by
filtration. When the process is completed in one single stage, only one
bonding agent is involved however, as soon as a coupling agent and a
spacer are introduced, the process is divided into several stages. The
numerous possibilities given when a bonding agent, a coupling agent, and a
spacer are used, and their various feasible combinations enable the
product to be accurately adjusted so that it has the properties required
for any particular application, the vehicle substance being built up
gradually and in conformity with these requirements.
The vehicle itself for example, is responsible for properties (b) to (c).
The bonding agent and the coupling agent essentially determine the
properties (f), (g), (h), (i), and (k). The spacer determines mainly the
properties of (l). The other characteristics, which are not specifically
defined, are affected by the combination of the vehicle, the bonding
agent, the coupling agent, etc. Thus it is possible for the biologically
active agent to be directly attached to the vehicle through the bonding
agent alone. Alternatively, the bonding agent can be linked with the
vehicle through spacer and coupling agent, so as to counteract steric
interference and to increase the efficiency of biologically active
substances which are bonded with a vehicle substance.
A vehicle may consist of natural organic substances such as cellulose,
agarose, dextrane or the like. Also synthetic polymers have been tried
out, for example the derivatives of acryl amide. Alternatively, the
vehicle material may be an inorganic substance such as glass. When
deciding on which substance to chose it is necessary to take a number of
parameters into consideration. the macroscopic form of the vehicle is
important. The material may for example be globular or in pearl form, or
in fibre form for example be cotton wool or the like, or it may be
granular. When pearl shaped particles are used, two additional parameters
namely grain size and porosity are to be taken into account. Depending on
the above parameters, it is feasible to make the bondage on the surface or
in the inner regions of the material predominant. With coarse grain sizes,
the occupation density is usually moderate. The occupation density is
generally considered as a standard for the assessment of the quantity of
biologically active matter which is bonded per unit volume of the vehicle
substance. The porosity affects essentially the mechanical stability and
resistance to compression. In cases where these properties are
particularly important it is recommended to use as a vehicle substance a
small-pored or non-porous material. The flow resistance of the vehicle
substance depends on the macroscopic form of the vehicle substance and on
the particle size.
The choice of the bonding agent and also of the coupling agent will be
influenced by the desire to maintain the vehicle properties as constant as
possible. Therefore the chosen material will be such that the number of
secondary reactions which could adversely change the vehicle substance are
as small as possible. When using isocyanate it is therefore understood
that the vehicle should be anhydrous and that also the process uses no
water. This means that one has to operate with organic solvents. Aprotic,
polar, solvents such as cyclic ethers, for example THF (tetrahydrofurane)
or dioxan have given excellent experimental results. Also aceto nitrile,
dimethyl formamide, and formamide are highly suitable aprotic solvents.
Since the method uses an interrupted process and the solvent has to be
removed after completion of each stage it is an advantage if the solvent
has a relatively low boiling point. It should be approximately below
100.degree. C.
The coupling agent is preferably an aromatic or aliphatic di-isocyanate.
The coupling agents should be not more than slightyly hydrophobic. When
operating with aliphatic di-isocyanates a short-chain di-isocyanate should
be used which may be saturated or non-saturated. Aromatic di-isocyanates
should have the smallest possible number of hydrophobic groups. The
following di-isocyanates are especially suitable:
toluene-2,4-di-isocyanate, toluene-2,6-di-isocyanate, commercial mixtures
of toluene-2,4 and 2,6 di-isocyanate, ethylene di-isocyanate, ethylidene
diisocyanate, propylene-1,2-di-isocyanate, cyclohexylene
1,2-di-isocyanate, cyclohexylene-1,4-di-isocyanate, m-phenylene
di-isocyanate.
Since the groups capable of reaction of numerous vehicles are slow to
react, it is necessary to use a catalyst. These may be a strong bases,
especially sodium imidazolide. Similarly, metallorganic catalysts of the
polyurethane chemistry could be used. The charge density may be controlled
by varying the quantity of the catalyst employed. The coupling- and
bonding agents should always be present in excess. Frequently five to ten
times the necessary quantity are present. The reaction temperature is
between room temperature and the boiling temperature of the solvent used
with the process. For example when the vehicle consists of microporous,
pearl-shaped particles having a granular size between approximately 10 and
50 m, result will be satisfactory when the reaction is controlled in such
a manner that the the charge density amounts to 25 to 200 mols per ml of
the bulk volume. The reaction is completed by scrubbing the excess
reagents, using an anhydrous solvent, preferably the solvent which will be
used with the consequent reaction.
The spacer should be a substance containing at least two groups capable of
reaction. When two groups are used, a linear chain is formed. When
substances containing more than two groups capable of reaction are
involved, the chain will branch out. Substances which are especially
suitable for this purpose are aromatic or aliphatic saturated or
unsaturated diamines, amino-alcohols, or diols. Obviously a substance used
as a spacer should not contain hydrophobic groups. Saturated aliphatics
having a short chain length and containing two groups which are capable of
reacting, will yield good results.
If it is desirable to increase the chain length, it is advisable to use
polyethylene glycols having a low molecular weight. By this method it is
possible to avoid the hydrophobic effect of long aliphatic chains.
Unsaturated aliphatic or aromatic diols or diamines offer the special
advantage of yielding a spacer which is essentially rigid or rather
moderately flexible, the advantage being that the biologically active
substance which is subsequently attached to it is freely supported and
thus prevented from clinging to the vehicle substance. Thus the substance
cannot fold back. Analoguously, ramification is a means by which to
increase the occupation density.
Numerous substances have been tested and found to provide excellent
spacers. Only several preferred examples are named hereinbelow:
Diamines:
Ethylendiamine, tetramethylendiamine, 1,5-diamine-3-azapentane,
m-phenylendiamine etc.
Amino-alcohols:
Ethanolamine, 1-aminobutene-2,3-ol-4,3-aminocyclohenxanol, p-aminophenol
etc.
Diols:
Ethyenglycol, glycerol, butanediol-1,4: 2-2-butene-2,3-diol-1,4,
1,4,-hydroquinone, resorcin, phloroglucinol etc.
Polyethylenglycol fractions having a low molecular weight.
In order to ensure that only one of the groups capable of reacting which
are contained reacts with the coupling agent, the spacer substance is
present in a high excess, preferably 10 times. It is normally possible to
operate in the absence of a catalyst. However, a weak base such as
tri-ethylamine may be used. The solvents, temperature and scrubbing of the
reaction products specified above apply analogously to the spacer
reactions. When using a spacer it is necessary to insert a bonding agent
between the spacer and a biologically active substance. Again, the bonding
agent should contain two reactive groups. The same substances which were
specified above as suitable coupling agents may advantageously be used as
bonding agents. Regarding the choice of the coupling agent, analogous
criteria are to be considered. However, since the groups capable of
reacting which were used with the spacer are less inert than the groups
capable of reacting which are contained in the vehicle, it is possible for
a catalyst to be chosen which causes the bonding agent to react
exclusively with the spacer. Catalysts suitable for these reactions are
aprotic, weak bases such as triethylamine. Again, the reaction takes place
place in the presence of a high excess. The information given above in
respect of solvents, temperature and isolation apply analogously. The
scrubbing of excess substances is followed by the evaporation of the
solvent so that the vehicle substance is obtained for example in powder
form in which it may safely be stored. The vehicle substance includes at
its end an isocyanate group. This is not, however, an absolutely necessary
condition. The end might equally well be a deactivation agent which
enables the end to dissolve again when a biologically active substance is
attached. Compounds having these characteristics have been defined as
"isocyanate in disguise". The deactivation agent is introduced in solution
in an organic solvent. Again the product may be isolated and stored.
When coupled with biologically active substances the deactivation agent is
exchanged against the biologically active substance. Suitable
desactivation agents are, theophenols, phenol derivatives, for example
2,4dinitrophenol, hydroxyl amine derivatives such as N-hydroxysuccinimide,
cyclic amines for example benzimidazol or similar substances.
The reactive mechanism demonstrated in the example of isocyanate and
di-isocyanate may of course be extended over other coupling agents and
bonding agents. Further reactive groups which could be used as bonding
agents or coupling agents, are acyl halogenides, activated esters,
iso-thiocyanates, sulphonic chlorides, chloroformates, and similar
substances.
The coupling of biologically active substances is extremely simple. The
vehicle substance is stirred into the solution of the biologically active
substance in a suitable solvent. The charge density can be enhanced to an
especially high level by using biologically active substances which are
soluble in aprotic organic solvents. There are no secondary reactions.
This applies especially to certain classes of material such as
antibiotics. Enzymes and other proteins are normally used in aqueous
solution. The secondary reaction which occurs in this case is the
hydrolysis of the isocyanate. However, the amino groups in the
biologically active substance react with isocyanate much more rapidly than
the water which means that even with these reactions a high occupation
density may be produced. The solution of enzymes and other proteins in
water must be buffered. Any buffer substance which is free from groups
capable of reacting is suitable. Good results have been obtained with
phosphate buffers imidazol buffers, and Good's buffer solutions. The pH
range should be neutral or slightly alkaline. Good results have been
reported from solutions with pH range 6 and 10. The bonding reaction is
carried out at room temperature or in the cold. Once established, the bond
is very firm. All enzymes examined were found to be free from leakage. The
resulting vehicle substance including the biologically active material is
free from additional ionic groups.
Approximately 20 enzymes were bonded with a vehicle material. Because of
their considerable sensitivity some of these have previously resisted all
attempts of immobilisation.
All enzymes examined in this connection were bonded in their active form
with the vehicle substance. This seemed to indicate a definite probability
of the method being generally applicable.
Again, the coupling agent may be a bonding agent with two reactive groups,
and isocyanate may constitute one or preferably both of the reactive
groups. Alternatively, di-isocyanate could serve as the bonding agent and
as the coupling agent, and the spacer could be composed of a substance
with at least two groups which are capable of reacting, such as especially
amino and/or hydroxyl groups. The corresponding links are always stable.
To increase the occupation density it is moreover feasible that two or more
di-isocyanates, employed as bonding agents, are bonded with the coupling
agent through a substance containing at least three groups capable of
reaction especially amino- and/or hydroxyl groups.
In certain cases of application it is good policy to reduce the reactivity
of the isocyanate group of the vehicle substance using a deactivation
agent in order to arrive at a specific bonding effect. This is
particularly advantageous when proteins or enzymes in an aquaeous solution
are bonded with vehicle matter, because this reduces the rate of
hydrolysis of the isocyanate considerably.
The proposed method enables activated vehicle substances to be manufactured
in large quantities, using commercially produced cheap chemicals. In the
anhydrous state, the activated vehicle substances are stable, and they
bond covalently biologically active matter containing free -NH.sub.2 or
-OH-groups, in water or in organic solvents. The biological activity of
the substance is retained during the bonding process, the vehicle
substance causing steric interference of the biological activity is
effectively averted by the intermediate coupling of spacers. The covalent
linkage is chemically stable. The properties of the vehicle, especially
its porosity, flow rate, and melting temperature are not affected by the
method of activation. The method is particularly favourable where
immobilised enzymes are produced, and it yields agents which are
particularly valuable in flow reacters.
It is characteristic for vehicle substances for the immobilisation of
biologically active matter especially enzymes, that they have at least one
reactive group and that this reactive group is an isocyanate group.
Conditions are particularly favorable for the optimum efficiency of the
biologically active material which is later linked, when the reactive
isocyanate group is linked with the vehicle through a spacer, a coupling
agent being provided between the spacer and the vehicle. This coupling
agent may be a bonding agent with two reactive groups, whereby isocyanate
is preferentially employed as one, or both, of the reactive groups. When
isocyanates react with groups capable of reaction, one obtains exclusively
substituted urethane, or substituted urea.
The agent according to the invention containing biologically active matter
which is immobilised by means of a vehicle substance is distinguished by a
substituted urea resulting from the linkage of an amino group between the
biologically active matter. Alternatively a hydroxyl group of the
biologically active matter could be linked with the vehicle substance into
a substituted urethane to convert the isocyanate group. Also in this case,
the vehicle substance is distinguished by one or several of the
characteristics described above.
The examples for vehicle substances and agents which follow below
illustrate the importance of the invention:
(1a) Examples for the synthesis of vehicle substances capable of bonding
biologic matter, and using isocyanate groups:
(a) the isocyanate group provides the link with the vehicle: a vehicle, for
example cellulose, is activated with cyanogen bromide and converted with
the aid of phenylenediamine. The resulting compound reacts with phosgene,
forming isocyanate which in turn reacts with the biological matter. (See
FIG. 1)
(1b) The link between vehicle and biological material is provided by the
isocyanate group: Agarose, acting as a vehicle, is reacted with a bonding
agent which contains both a isocyanate group and an activated ester. The
isocyanate group combines with the vehicle, and the activated ester group
is able to condense with the biological substance, (See FIG. 2).
(1c) Both reactive groups of the bonding agent, are cyanate groups that is
to say the bonding agent is a di-isocyanate. (See FIG. 3).
(2a) Example for a vehicle substance of consisting of vehicle, coupling
agent, spacer, and bonding agent: All reactive groups in the chosen
example, whether in the bonding agent or in the coupling agent, are
isocyanate groups. A hydrophilic compound having an extended chain length,
acts as a spacer. Cellulose, constituting the vehicle, is converted in the
presence of the strong base sodium imidasolide, with toluene diisocyanate
and the resultant compound adds, as spacer, n-polyethyleneglycol. In a
further reaction with toluene di-isocyanate in the presence of the weak
base tri-ethylamine, the bonding agent reacts with the primary OH-group of
the spacer, while residual secondary hydroxyl groups of the vehicle no
longer react; thus the following substance evolves. (See FIG. 4).
(2b) Example for ramification caused by a polyamino compound: Reaction as
(2a), excepting the spacer which now is a polyethyleneimine. (FIG. 5).
(3a) Example for bondage of urea between a vehicle substance carrying
isocyanate groups, and a biological substance containing amino groups: A
vehicle substance, for example the agent resulting from the reaction
according to (2a), reacts in aqueous solution with lysin forming a
substituted urea (FIG. 6). Enzymes containing lysin react in the same
manner.
(3b) Examples for urethane bondage between a vehicle substance containing
free isocyanate groups and a biological substance containing free hydroxyl
groups: A substance as described in examples (2a) or 2b) is reacted with
chloramphenicol in diozane, whereby the following compound evolves (FIG.
7).
(4) Example for a deactivation:
A vehicle substance containing free isocyanate groups is reacted with
thiophenol in organic solutions (FIG. 8).
(5a) Vehicle Substance Prepared from Dextrane Gels.
5 g of dried Sephadex G 50 superfine (Pharmacia; microporous, cross-linked
dextrane gel) are suspended in 100 ml anhydrous tetrahydrofuran (THF).
While stirring, 1 ml of toluene di-isocyanate (commercial mixture of 2,4
and 2,6 isomers) and 0.5 ml of a catalyst in suspension are added,
followed by heating to the reflux temperature under rigorous stirring for
30 mins. The suspension is filtered off and scrubbed with anhydrous THF to
remove excess reagents.
The pearls are suspended in a solution of 2 g 2-Butyne-1,4-diol in dry THF,
and heated for 30 mins to reflux. The substance is extracted and carefully
scrubbed with anhydrous THF, after which the solvent is removed in the
vacuum, and the product is sealed in vials. When examined after storage
for several months, no decrease in the bonding capacity was detected.
The yellow pellets swell in water like the initial substance; similarly,
the appearance and flow resistance are the same.
Isocyanate concentration: 250 .mu.Moles/g
Lysine bonding power in aqueous solution: 4 .mu.Moles/g
Bondage of enzymes to the vehicle substance in accordance with 5a.
Alkaline Phosphatase from E. coli (Boehringer, 200 g) is dialysed against
0.2 M phosphate buffer (pH 8,0) and the volume of the enzyme solution is
increased to 400 .mu.l. The enzyme solution is mixed with 10 mg of the
vehicle substance according to 5a, and the solution is left standing for 2
hours under slight stirring, followed by shaking. 100 .mu.l of the
suspension are packed into a small column (diameter of the column 22 mm,
bed-volume of substance 10 .mu.l). This column is scrubbed with 0.05 H
tris-buffer (pH 7.5) followed by equilibration with substrate solution
(0.05 M glycine buffer, (pH 10.5), 5.5 mm p-nitrophenyle phosphate, 0.5 mM
MgCl.sub.2), The flow rate is adjusted at 4 secs. per column volume,
fractions being collected continuously. The reaction should adjust itself
to a steady state within a few seconds. The activity is calculated from
this level. 1 g of the agent has the activity of 1.3 mg free enzyme.
Secondary incubation of the collected fractions, maintained for several
hours, enables the contamination to be measured with detached enzyme. Such
contamination is immeasurably small.
After standing at room temperature for one week in a glycine buffer (pH
10.5), it was found that the activity of the agent had not diminshed.
Lysine Bondage Standard text: 1 mM lysine in 0.2 M of a buffer of potassium
phosphate, 50 mg vehicle substance per 1 ml solution. After reaction time
of 1 hour at 4.degree. C., the fixation of the radioactive lysine is
measured. Fixation of lysine when using other buffer conditions:
______________________________________
Standard condition 100%
0.02 M Phosphate pH 8.0 135%
0.02 M Phosphate pH 6.7 48%
0.05 MEPES pH 7.4 80%
0.05 Imidazol pH 8.0 90%
______________________________________
With neutral pH conditions, the resulting agent does not act as an
ion-exchanger. Measurements are taken of the amount of bound radioactive
ADP by the lysine substance. One g of the agent binds less than 1 p Mol
(10.sup.-12 Mol) ADP.
Using standard conditions, the following enzymes are bonded in their active
form with the vehicle substance:
Trypsin inhibitor from soya beans (Merck). The product has a reversible
bonding capacity of 3.1 mg Trypsin/g.
Lactate dehydrogenase from pigs muscles (Boehringer) The activity of the
product is too high for an accurate measurement of the surface density (50
E/g) to be carried out.
Apyrase from potatoes (Sigma)
Urease from Jack beans (Boehringer)
Diaphorase (Sigma)
The standard conditions are: 50 g of the vehicle substance are mixed with
the solution of 10 mg enzyme in 1 ml of an 0.2 M phosphate buffer (pH
8.0), and the suspension is incubated for 2 hours at 4.degree. C., under
occasional shaking. At least 2/3 of the excess enzyme activity are
recovered for use in subsequent bonding reactions.
(5b) Example for the use of polyethylene glycol as a spacer, the vehicle
being dextrane gels. The production of the vehicle substance from Sephadex
is analoguous to the example described above. However, the spacer solution
is replaced by a solution of 20 ml polyethylene glycol 200 in 200 ml THF.
The obtained product is very similar to the above; however, the bonding
capacity is usually below that of the previous example, the lysine bonding
capacity for example being 5.6 .mu.Mol/g, and the activity of the bonded
lactate dehydrogenase amounting to 16 units/g.
(6a) Vehicle Substance Made from Granular Cellulose.
(Cellulose for thin-layer chromatography (Merck) native, granulate).
2.5 g cellulose are vacuum-dried, and floated in a solution of 05 ml TDI
and 0.375 catalyst suspension in 150 ml THF, followed by heating the
reflux for 30 minutes under stirring. The product is added to a solution
of 2 g butine diol in 150 THF and heated to reflux for 30 mins. It is
again scrubbed and filtered, and the cake is floated in a solution of 2.5
TDI and 0.25 ml triethylamine in 150 ml THF. The substance is extracted,
thoroughly scrubbed in anhydrous THF and dried in the vacuum.
Characteristics: fine granulate, yellowish in colour.
Lysine bonding power in aqueous solution: 4.5 .mu.Mol/g.
Enzyme bondage with a vehicle substance according to 6a. Bondage takes
place under conditions described above.
Trypsin The activity of 1 g of the agent is equivalent to 1.8 mg enzyme.
Polynuleotide phosphorylase A partially enriched enzyme fraction of E.
coli. The activity of 1 g of the substance is equal to 0.8 mg of the
preparation. Measured was the formation of high polymer, poly-U from UDP.
In a continuous process, the half life amounts to approximately 14 days.
This means that after a reaction period of 14 days the throughput of a
flow reactor which is in continuous operation at room temperature, drops
to half the original value. The quantity dealt with during this period is
about 10,000 column volumes. Since no traces of enzymes were found in the
runs, the decrease is attributed to denaturation or desintegration of the
enzyme into its sub-units.
RNA Polymerase, a partially enriched preparation from E. coli. The
synthesis of poly-AU on a high-molecular matrix of poly dAT was measured.
The stability of the bonded enzyme amounted to a multiple of the stability
of free enzymes. The activity of 1 g of the agent is equivalent to 0.12 mg
of the free enzyme.
Guanylate Kinase, A partially enriched crude fraction from E. coli. The
column is suitable for enzymatic synthesis of GDP and GTP and it is
especially favourable for the synthesis of radioactively marked GDP, GTP,
IDP and ITP.
Nucleoside diphosphate Kinase, a partially enriched protein fraction from
E. coli. The agent is suitable for the synthesis of phosphate labelled
nucleoside- and desoxynucleoside triphosphates.
Diphospho-kinase and Polynucleotide Phophorylase. Partially enriched
protein fractions from E. coli are mixed followed by bonding. In the case
of nucleoside tri-phosphates and nucleoside di-phosphates the agent
exchanges specifically the .beta.-phosphate against added, radioactive
phospahte.
Prostatic, Acid Phosphatase, a partially enriched protein fraction from
human semen. The immobilised crude enzymes mentioned above are excellently
suited for the production of substrates for the synthesis of nucleic acid.
In free solution, these crude enzymes are unsuita | | |
