Note: Descriptions are shown in the official language in which they were submitted.
o
This invention relates to a method o~ and a device fox
determining the concentrat~on o~ low-molecular weight biological
substances by enzymatic react~on.
A chemical reaction catalyzed by specific enzymes is
often used for determining the concentration of low-molecular
weight biological substances. In this method, the test sub-
stance is mixed with suitable buffers and adjuvants, and the re-
action that makes the quantitative determination possible is
produced by the catalytic effect of the added enzyme. The de-
termination is usually done by measuring the optical absorption.
The enzymes necessary for this æe commercially available, but
are relatively expensive. In the known processes, the reaction
mixture is discarded after the measurement, so that the expen-
sive enzymes are lost.
The present invention in one aspect provides a method
of determining the conaentration of low-molecular weight biolo-
gical substances by enzymatic reaction, wherein the low-molecu-
lar weight reaction products obtained as a result of the enzy-
matic reaction, together with the low-molecular weight biologi-
cal substance are separated from the enzyme after the enzymatic
reaction by ultrafiltration, and wherein the enzyme used therein
which emerges as ~ concentrate from the ultrafilter separately
from the reaction mixture, is recycled and used again for enzym-
atic reaction with low-molecular weight biological substances,
the concentration of the biological substances being determined
by means of the measured concentration of the low-molecular
weight reaction products.
The method according to the invention makes full use of
the fact that all enzymes differ so greatly in molecular weight
from the adjuvants and test materials that they can be separated
from them by means of ultra-filtration. Thus the enzyme remains
~ - 2 -
o
ent~rely in the concentrate, wh~le the lo~molecular wei~ht com-
ponents in the filtrate and concentrate are present in equal
concentration. 5ince the amount of ~iltrate is significantly
greater than the amount of concentrate, the ratio of enzymes to
low-molecular weight substances in the concentrate is signifi-
cantly greater than in the reaction mixture. On the basis of
this fact, the re-use of the enzyme in the form of the concen-
trate is made possible.
In a preferred embodiment of the present invention
there is provided, in a method for determining the concentration
of low-molecular biological substances by reaction with an enzyme
including the steps of contacting a low-molecular biological
sample with an enzyme, thereby producing low-molecular reaction
products, quantitatively measuring the concentration ofthe low-
molecular reaction products and discarding the unreacted sample
and the low-molecular reaction products after measuring, the im-
provement of recovering a substantial portion of the enzyme com-
prising ~eparating the low-molecular reaction products and unre-
acted sample from the enzyme by a first ultra-filtration into a
~o filtrate containing substantially all of the low-molecular reac-
tion products and unreacted sample and a concentrate containing
the enzyme and a trace quantity of the low-molecular reaction
products and unreacted sample, diluting the concentrate and sub-
jecting the concentrate to at least one additional ultra-filtra-
tion, recovering the enzyme therefrom substantially devoid of
low-molecular reaction products and unreacted sample, and re-
using the thus separated enzyme in the continuous ~ncentration
determinations.
In a further embodiment of the present invention there
is provided an apparatus for quantitatively determining the con-
centration of low-molecular biological substan~es by reaction
~ - 2a -
4(3~t30
~ith an enz~me and product~on of lo~olecula~ reaction products
including:
a source of low-molecular biological sample;
a source of reactive enzyme;
pump means for delivering a predetermined quantity of
sample from the sample source to a reaction area;
pump means for delivering a predetermined quantity of
reactive enzyme from the enzyme source to a reac-
tion area;
means for mixing together the delivered smaple and the
delivered enzyme including therewith a reaction
area:
pipe means communicating the reaction area to a first
ultrafilter for separating a filtrate containing
substantially all of the low molecular reaction
products and unreacted sample from a concentrate
containing the enz~me and a trace quantity of the
same ahd low-molecular reaction products;
a second ultra-filter for further concentrating the
. enzyme and removing trace portions of sample and
low-molecular reaction products therefrom;
pipe means communicating the concentrate of the first
ultra-filter to a second ultra-filter;
a source of enzyme diluent intermediate the first and
second ultra-filters for delivery of enzyme diluent
to the concentrate passing from the first ultra-
filter to the second ultra-filter;
pipe means communicatecl from the outlet of the second
ultra-filter for delivering theseparated, diluted
enzy~me substantially devoid of sample to the
mixing means;
~ .
~ - 2b -
~ o~
pipe means ¢ommunicat~n~ the ~iltrate o~ the ~irst
ultra-filter to an outlet ~or discarding the
unreacted low~molecular sample and thelow~
molecular reaction products, and
means for determining the concentration of the low~
molecular reaction products ~or evaluation of:-
: the concentration o~ the low~molecular biological
sample.
- 2c -
g~4Q0~3~
After ultrafiltration, however, remains o~ the
low-molecular components of the reaction mixture can
still be found in the concentrate According to the
invention, the remains of dissolved low-molecular
components oi the reaction mi~ture left in the concentrate
after reaction and ultrafiltration can be determined
on the basis of the concentration measured after the
reaction~ and they san be considered in the calculation
of the next determination, using the enzyme le~t in
the conoentrate. The amounts of low-molecular substances
left in the concentrate can be determined from the
determination of the concentration of low-molecular
substances in a reaotion. This is done by seeing to it
that in the next reaction~ which is per~ormed with the
enzymcs left in the concentrate~ the amounts of low-mole-
oular substances left in the concentrate are considered
in the calculations, whereby the result of the determina-
tion ln the second reaction performed with the same
enzyme fraction can be corrected to such an extent, that
the measurement is exact again. In this way, the
determination of concentration can be performed with the
re-used enzyme in exactly the same manner as with a
fresh enzyme. The calculation can be performed simply
in an electronic computer~ in which the data concerning
the measurement performed previously with the same
enzyme fraction are used~ However~ the oalculation
of the fraction of low-molecular substances left in
the concentrate presupposes that a number of such
--3--
~.Q4~
determinations will be performed in direct sequenoe.
In accordance with a speoial mode of operation
of the invention, the proce~ can be perfor~ed in
such a way that the concentrate flowing off the
ooncentrate chamber of the ultrafilter can be-ultra-
filtered again after dilution, whereby this ~tep oan
be repeated ~everal times, and that the con¢entrate
from the last ultraflltration i9 again used for pro-
duoing low-molecular products by mean~ of enzyme reaction~
espeoially for enzymatic determination of low-moleoular
biologioal substanoes. In the ~irst ultra-filtration~
the amount of low-molecular substances left in the con-
oentrate is found from the ratio of concentrate to
filtrate. I~ for example~ the proportion of concentrate
to filtrate i9 1:10~ then only 1/10 of the low-molecular
substanoes oontained in the reaotion mixture i9 left in
the oonoentrate. If now the oonoentrate is diluted and
ultrariltered again~ then it is found that in the case
of the same proportion of oonoentrate to filtrate of 1:10
only 1/100 of the low-moleoular sub~tanoes contained in
the reaotion mixture are present in the concentrate.
In many oases~ such a fraction of low-molesular sub-
stances~ which affects the result~ oi the next determina-
tion~ using the same enzyme~ can be taken into account.
The fraotion of 1/100 low-moleoular substances is in
many cases already belo~ margin of error for the de-
termination~ 90 that the enzyme can be re-used regard-
less of the accompanying low-moleoular substances.
In any case, however~ this fraction is about equal again
in the second and each subsequent instance of use of the
enzyme~ so that consideration of an always constant
correction factor in the measurement i~ sufficient.
--4--
However, if the concentrate i9 ultrafiltered again
after dilution, then the smount of low-molecular
substances in the concentrate is only 1/1000 oi the
amount contained9 which i9 in nearly all cases lower
than the margin of error for the determination.
Since in the case of repeated ultrafiltration,
consideration of the fraction of low-molecular substan-
ces left in the ooncentrate is not necessary~ it is
also not neoessary in these ¢ases to take into account
a result o~ the previous determination~ and thus~ in
aocordance with the invention~ the concentrate of
the la~t ultrafiltration can be stored before re-use.
This is especially advantageous if a di~oontinuable pro-
oess i~ used.
Measurement of the reaction mixture can be performed
in the same way a~ was used in already known methode~
when the reaotion mi~ture was thrown away after measure-
ment. Aooording to the invention~ however~ the measure-
me~t i9 preferably taken on the filtrate flowing off
the flltrate ¢hamber of the fir~t ultrafilter. This
is easily pos~ible, beoause the low-moleoular substanoes
are present in the filtrate in the ~ame oonoentration
as in the reaotion mi~ture. This also of~ers the
advantage that the measurement is not fal~ified by
the high-moleoular substanoes. The measurement i8
usually done with a photometer~ whioh produoe~ a
heating of the material to be mea~ured. The faot
that measurement is performed on the filtrate ofiers
the advantage that the enzymes are not harmed by
the heat effeot~ whioh is of great signifioanoe~ as
these enzymes are to be re-used repeatedly.
--5--
~4~380
The method aooording to the invention is not
limited to a determination of low-molecular biologi¢al
~ubstances by means of enzyme reaotion but oan al30
be used to produce low-molecular products by an
enzyme reaction for other purposes.
In the device of the invention, for determining
the ooncentration of low-molecular biological sub~tances
by means of enzyme reaction, the enzyme i9 mi~ed with
the sample in the u~ual way at a mixing point~ whereby
the reaotion mi~ture is conveyed by means of pump-pressure
through a reaction ~tretch, through a pipe, into a measur-
ing point. In aocordance with the invention~ the
pipe conducting the reaation mixture is connected to the
entrance of the concentrate chamber of the ultraYilter~
whereby the exit of the ooncentrate ohamber is
oonneoted with the mixing point, and a disoharge pipe
i8 oonneoted with the filtrate ohamber. With suoh a
devioe~ it is possible to perform a great number of
oonoentration determinatlon~ in uninterrupted suooession~
whereby however~ i~ there is only one ultrafilter~ an
eleotronio oomputing and storing devioe is u~eful~ for
taking into aooount the fraotion of low-moleoular sub-
stanoes lert in the oonoentrate and thus in the re-u~ed
enzyme solution~ when viawing the previous determination
perfor~ed with this enzyme fraotion. Aooording to a
~peoial mode of operation of the invention, therefore~
the apparatus i9 arranged in suoh a way that the outlet
of the oonoentrate ohamber of the ultrafilter is connected
with the inlet of the oonoentrate ohamber of a seoond
ultrafilter, and a pipe for the solution agent i~
connected withtbe inlet of the conoentrate chamber of
the seaond ultrafilter, and that the outlet of the
--6--
~4(~ 0
oonoentrate chamber of this seoond ultraiilter is
oonne¢ted with the mixing point by means of a pipe~ ~nd
that a disoharge pipe is oonneoted to the filtrate cham-
ber of the seeond ultrafiltar. In thi~ case9 the fraction
of low-moleoular ~ubstances leit in the conoentrate can
generally be disregarded, 90 that an electronic oomputlng
and storing deYice i~ not necessary. ~en~ in aooordance
with the invention~ the pipe from the outlet Oir the oon-
oentrate chamber of the second ultraiilter leads to
the mixing point through the concentrate chambers oi
other ultrafilters9 to the inlets of which pipes ior
a solution agent are also connected~ then the aocuraoy
of the determination is even iurther in¢reased.
~ he mea~uring point can be plaoed in iront of
the inlet o~ the oonoentrate ohamber of the first ultra-
filter; however~ aooording to the ~nvention the
measuring point is plaoed within the disoharge pipe
leading out of the iiltrate ohamber of the iirst ultra-
~ilter~ whereby the advantages desoribed above are
aohie~ed~ in that the enzyme to be re-used i9 protected
against heating and thus grestly spared.
When several ultrafilters are used~ a storage
oontainer for the enzyme oan be in~erted into the pipe
leading from the concentrate ohamber of the last ultra-
iilter to the mixing point. In this tank~ the enzyme
or enzyme ~olution is stored before re-use.
In the drawing~ the invention is diagrammatioally
explained~ with reierenoe to examples.
Fig. 1 shows a devioe for determining the
oonoentration oi low-moleoular biologioal substanoe~ by
means of enzyme reaotion~ using an ultrafilter. Fig. 2
~hows this sort o~ devioa using several ultrafilter~.
--7--
~4(~U~O
In the oase of the dsvice represented in Fig. 1,
there are three peristaltic pumps 1~ 2 and 3. Peristal-
tic pump 1 draws in samples from the sample tank 4.
~he sample can be~ for example, a solution containing
glucose. Peristaltic pump 2 draws in an adjuvant for
the gluco~e to be determined~ e.g. a phosphate buffer
eontaining o-anisidi~ as a chromogen~ from the ad~u~ant
tank 5. Peristaltic pump draws in enzyme solution~
e.g. a solution o~ gluoose-oxydase and a peroxydase~
from the pipe 6~ which is connected with a ~torage tank 55
for fresh enzyme solution through a valve 53 and a pipe 54.
The solutions oonveyed by the peristaltio pumps~ operated~
for e~ample~ by synchronous motors~ are oonducted to a
~ixing point 8, and the mixture thus obtained is
oonveyed through a pipe 7~ in whioh the reaotion takes
plaoa. ~his pipe oan have~ for example~ an internal
diameter of about 1 mm and be a polyethylene tube about
15 m long. ~he pipe with the reaotion mixture 7 leads
into the inlet of the conoentrate chamber 21 of an
ultrafilter 10 with a membrane 9. To the outlet of
the oonoentrate ohamber 21 of the ultrafilter 10 there
is attaohed a pipe 6~ through whioh the enzyme-rioh
oonoentrate is oonduoted back to the mixing point 8
with the help of a peristaltio pump 3. At this time~
the valve 5~ is closed, whereafter all subsequent de-
terminations are performed on the concentrate of the
ultrafiltration, and taking into aocount the measure-
ments of previous determinations. The low-molecular
substanoes freed by the enzyme and those dye-containing
ones formed upon the enzymatic reaction~ flow through
a pipe 11 from the filtrate chamber 22 of the ultra-
filter 10 to a photometer 12 (lamp 12~, flow cell 12"
-8-
and photo resistanoe 12"~ he signal given of~ by
the photo resistor 12"t, whioh depend upon the ¢on-
oentration of dye in the solution flowing through the
flow oell 12"~ i9 registered in a logarithmic analog-
digital-transformer and tran~formed into a digital
~ignal that i9 registered in a slide register 14, in
the present oa~e a 16-stage slide register with 8 bits
per stage.
The following should be considered in the analysi~
of the measurements given by the logarithmio analog-
digital-transformer 13 and the measurement~ stored in
digital form in the slide register 14.
In the oase of the above-indicated diameter o~
ths pipe 7, whioh i9 essentially identioal to the pipe 11
the pipe system has a volume of about 15 to 20 ml between
the peristaltio pumps l, 2 and 3 on the one hand and the
photometer 12 on the other~ so that it is pos~ible that
a reaoted and there~ore oolored ~olution oould form
in the photometer ~rom the suotioned samplep still
during suotion of the substanoes ~rom the sample
oontainer 4. If we assume that in the ultrafilter 10
: the reaotion mixturep in a volume ratio of l:9p is
separated into an enzyme oonoentrate flowing away
through the pipe 6 and an ultrafiltrate flowing toward
the photometer and oontaining the oolored reaotion pro-
duot~ and that the volume of the pipe 6 is 90 great
that the enzyme solution used in the first test is re-
used for the fourth testp then care must be taken
that the value~ stored in the slide register 14 be
stepped up by a timing generator 15 90 ~astp that the
measurement entered into the slide regi3ter 14 for
the first te~t should appear at the e~it of thi~ ~lide
u~o
register 14 at the moment ~hen the measurement for
the fourth test i9 determinsd by the photometer 12.
Sinoe in the enzyme concentrate leaving the conoentrate
¢hamber 21 of the ultrafilter 10 and in the reacted
solution leaving the filtrate chamber 22 of the ultra-
filter 10 thro~gh the pipe 11 there i9 the same con-
centration o-f colored reaction product~ the measurement
appearing at the e~it of the slide register 14 for the
~irst te~t~ with a volume ratio of 1:9~ ~hould be
reduoed, and the differenoe between the measurement for
the fourth test and the reduced value ~or the first
test should be indicated. Toward thiY end~ the digital
signal that appears as an initial value in the logarith-
mio analog-digital-transformer 13 for the measurement
of the fourth test~ i8 transformed into an anulog signal
in a digital-analog-transformer 16~ and the digital signal
appeari~g simultaneously at the exit of the slide
register 1l~ ~or the measurement of the first test is
transformed into an analog signal in a digital-analog-
transformer 17. This last analog signal i~ reduced
in a reducer 18 acoording to the oited separation ratio
of 1s9~ whereupon the difierence i9 obtained ~rom the
analog signal giv.en by the digital-analog-transformer 16
and the signal given by the digital-analog-tran~former 17
and reduoed by the reducer 18. The differenoe i8
determined in a subtraotor 19. This difference is
indioated as an analog signal by means of a dial gauge 20,
but can also be transformed into a digital representation
by means of an analog-digital-transformer.
Sinoe as a rule the analog signals given off by
the photometer 12 for suooe~sive tests are of different
values~ there should be an appropriate look, so that
--10--
IV4(~ 0
a new output variable doss not appear in the output
of the logarithmio analog-digital-transformer 13 until
a constant absorbanoe value is indicated by the photo-
meter 12 for a long period o~ time.
Since the test fluid, the solution of ad~uvants
; and the enzyme solution are continuously being conveyed~
i$ is simple to set up the device represented diagramma-
tically in Fig. 1~ Only two periods need be measured
empirioally for this:
a) the period from drawing in of the sample to
appearance of the colored, reacted solution in the
photometer. This period remains the ~ame when advance-
ment of the material is ¢onstant~ and should be taken
into oonsideration in ooordinating the mea~urement~
with the individual tests.
b) the period from the appearanoe of the oolored,
reaoted solution to the reappearance of coloring of the
solution flowing through the photometer due to a re-
oiroulated enzyme solution, in the oase of oonstant
advanoement of the ad~uvant solution and oonstant ad-
vancement of water by means of the peristaltio pump 1.
The pulse frequenoy of the timing generator 15 should
be set up in suoh a way that a determined measurement
is advanced through all the levels of the slide
register ~ust a~ter termination of this period.
In Fig. 2~ another devi¢e for performing the pro-
oess o$ the invention i~ represented. A speoimen is
advanoed from a oontainer 23 by a peristaltio pump 24
an ad~uvant solution is advanoed from a oontainer 25
by a peristaltio pump 26~ and a first-filling enzyme
is advanoed ~rom a oontainer 27 by a peristaltic
pump 28 through pipes 29~30 and 31 to a mixing point 32.
--11--
~ o
The reaction mixture i~ then conduoted through a pipe 33
in which the reaction ta~es place~ to the inlet of the
con~ntrate chamber 349 of a ~irst ultrafilter 36.
Through a pipe 37~ whioh is oonneoted to the filtrate
chamber 35 of the ultrafilter 36~ the low-molecular
oomponents of the reaotion mi~ture emerge and are
¢onducted to the inlet of the flow oell 38 of a
speotrophotometer. Here, the light emitted by a lamp 39
up to a ¢ertain wave length is filtered~ and after
passing through the flow cell 38 comes into oontact
with a photo resistor 40~ whose resistance change is
indioated by a measuring apparatus 41 either in the
form of the light absorption or the extinction. The
solution that issues from the flow cell 38 can be thrown
away. A pipe 42 is oonnected with the outlet of the
oonoentrate chamber 34 of the ultrafilter 36 The puri-
fied enzyme is suotioned through this pipe by a pump 43
and at 44 it is mixed with a diluting agent pumped out
of a oontainer 45 by a pump 46. The enzyme solution~
whioh is ~Ow again diluted to about the original con-
oentration~ enters the conoentrate chamber 47 of
the ultrafilter 49 and is pumped from the outlet of this
ooncentrate ohamber 47 by a peristaltio pump 5~ through
a pipe 52 to the mixing point 32 and mixed with the
speoimen from the oontainer 23. The introduotion of
the en~yme from the container 27 through the pipe 31
can be stopped by shutting down the pump 28 as soon
as purified enzyme from the first test arrives at
the mixing point 32 through the pipe 52. The low-
molecular sub~tances that emerge from the filtratechamber 48 of the ~eoond ultrafilter 49 are conducted
through a pipe 51 out of the filtrate ohamber 48 and
-12-
oan be thrown awa~4~
In this mode of operation of the device Or the in-
vention~ purified enzyme is mixed with new te~t material
at the mixing point 32~ so that consideration oi the low-
molecular component~ leit in the oon¢entrate in oalcu-
lation~ ~rom pr3~ious determinations oan be omitted in
thi~ ea~e. Figo 2 shows this mode of operation only
diagra~matieally~ and it i9 equslly sel~-evident that
t~e test material ~rom the test container 23 need not
be the same in the case of several determination3 in
a series, snd that a great number of similar containers 23
can be ~illed with different test specimens~ the content~
of which are advanced ~orward in a oertain sequence by
the pump 24~ and the pipe 29 to the mixing point 32~
Moreover~ familiar devioes 08n be provided for separating
the individual test epecimen~ such as, for example~
air bubble ~eparator~. More concentrate chambers and~
if neoessary~ more places 44~ in which diluting agents
are mixed~ oan be added to the pipe 52, in addition
to the concentrate ohamber 47 of the second ultrafilter 4g.
Exampless
1.) Determination of pyruvate with repeated uee of lactate
dehydrogena~e
Pyruvate may be determined by its enzy~atic
reduoti~n to lactate, noting that an equivalent amount
; of NADH will be oxidized to NAD. ~he change oi the
concentration in ~ADH will be photometrically measured
at a wave len~th of 340 nm. The reaotion take~ place
aocording to the equation
pyruvate ~ NADH + H+ LD~ ~ lactate + NAD+
At a sufficient concentration of NAD~ (0~01 mMol)
the ohemical equilibrium i~ totally at the right hand
-13-
u~o
s~de o~ the e~uation.
The apparatus ~as assembled accord~n~ to Fi~ure 2.
As the pumps 24, 26 and 28, peristallic pumps o~ the type
"ISMATEC"*,mini were used toyether with a hose of a diameter of
0.8 mm and consisting o~ a plastics material "T~GON"*, said
pumps having a supply capacit~ of 0.75 ml per minute. The pump
46 used was equally a pujp of the type "ISMATEC"* mini being
equipped with a silicone hosè o~ a diameter of 1.5 mm and having
a supply capacity of 2.6 ml pex minute. The pumps 43 and 50
- 10 were of the type KLB 4912A having a supply capacity of 0.2 ml
per minute and being equipped with a silicone hose of a diameter
of 1 mm, In a tengential stream housing having a diameter of
90 mm, filters of the t~pe "AMICON PM"* 30 were used (ultrafil-
ters 36 and 49). The conduit 33 consisted of a polyethylene
hose having a diameter of 1 mm and a length of 30 m and having
a volume of 25 ml. All connecting conduits consisted of the
same hose material~ ,
The connecting components used were screwing fittings
consisting of "TEFLON"* and supplied by the firm Serva, Heidel-
berg. The conduit 33 was located within an ultrathermostat
according to Hoeppler and kept~ta temperature of 25C. The
total volume of the enzyme circuit was 37 ml including the fil-
trate portions. The photometer was of the type Zeiss PN2a with
through-flow cuvette 38.
Reagents:
By dissolving 46.6 g triethanolamine.HCl in 200 ml
water and adding 0.94 g complexon III and further adjusting
the pH to 7.6 by means of 2N-NaOH and adding water for a
total volume of 250 ml, a buffer solution having a pH of
7.6 was obtained. The solution of NADH
*,TRADEMARK
- 14
o
wa~ u~ed in a 0.33 millimolar oonoentration within
buffer solutionO The LDH was used in ~orm o~ a
suspension of oristals as can be obtained on the market.
At the beginning o~ the determinations, the
conduits 33~ 42 a~ 52 were ~illed with LD~ in bui~er
solution. For this purpose the cristal suspension
oontaining 300 pg LD~ uas diluted with 31 ml buifer
~olution and the solution obtained was sucked instead
of the ~ample. As ~oon as the enzyme circuit had besn
filled~ a ~olution of NADH and~ instead of the ~ample,
water wa~ supplied a~d the zero level was read from the
photometer. Subsequently~ the samples containing no
protein~ and maximally 0.1 yMol pyruvate per millilitre
were suoked. By separating air bubbles~ mixing of ad-
~acent samples, which were supplied in time intervals of
60 seoonds~ was avoided. The extinction wlll decrease
in proportion to the oontent in pyruvate aooording to
the equation
0,~14 . E340 = ~pyr(pMol/ml)
A oalibratlng ourve is not neoessary.
The enzyme solution remained fully active one
week also with permanent use of the solution within the
apparatus. With more extended worklng interval~ it is
to reoommend to discharge the enzyme solution by opening
a screwing connection and to store the ~olution at
a temperature of 4C.
2.) Determination o~ D-gluoose with repeated use o~
the gluoose-oxydase and the peroxydase.
Glucose will be o~idized to gluoonic acid and
hydrogen peroxide by GOD in the pre~ence of the oxygen
oi~ ambient air. The POD will transfer the oxYgen o~
the H202 ~nto a chromogene and the extinction of the
-15-
u~o
oolouring matter formed will be determined. The
react~ons take plaoe acoording to the equations:
~-D-gluco~e ~ ~2 ~ 2 - GOD ~ D-gluconic aoid ~ H202
~2 + D~2 ~ ~2 POD ~ 2 H20 ~ D
Both chemi¢al equilibrla are totally shifted to the
right hand side-of the above equations.
The apparatus used wa9 assembled in an analogous
ma~ner to the apparatus described in 1.). In partioular
the same pumps and the same oonnecting conduits were
used. However, the ultrafilters 36 and 49 used were
of the Amicon type PM 10. The ultrathermo~tat according
to Hoeppler was ad~usted to 35C in thi~ case.
Reagents:
A buffer solution having a p~ of 7 was prepared
by dig801ving 2.76 g Na2HP04.2~20 and 1.45 g Na~2P04.2~20
in iOO ml water. In this oase~ 100 mg ABTS (2~2~-azino-
di-(3-ethylbenzthiazolon)-6-sulfonate) was used as the
ohromogene in 100 ml buffer solution. ~he gluoose
o~ida~e as well as the pero~idase were used in solid
form (as oan be obtained on the m~rket)~ dissolved
in buf~er solutio~.
At the beginning of the determinations~ the
conduits 33, 42 and 52 ~ere filled with GOD and POD in
buffer solution. For this purpose 4 mg GOD and 1.5 mg
POD were dissolved in 31 ml buffer solution and this
solution was suoked instead of the sample~ thereby
sucking~ instead of a solution of ohromogene, pure
buffer ~olution. As soon as the enzyme circuit had
been filled~ the solution of the chromogene was
supplied and a calibrating ourve was established by
means of a plurality of standard solution~ of glucose
-16-
o
containing 1 to 20 pg glucose per milliliter. Sub-
sequently tha sampla~, whioh did not contain proteins
and oontained maximally 20 pg gluoose per milliliter~
were s~pplied. By separating aIr bubbles~ intermixing
of adjacent samples wa~ avoided, the samples being
~upplied with a time interval of 60 seoonds. The
extinotion is proportional to the content in gluoose.
Mea~urements were taken at a wave length of 420 nm and
tran~formed by means of the oalibrating ourve into the
~ought result~.
Thè enzyme solution remained fully active ona
week al~o with permanent use of the solution within the
apparatus. During working intervals it is to reoommend
to discharge the enzyme solution by opening a screwing
oonneotion and to store the solution at a temperature
of l~C
-17- 11.7.7~/li
