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US3977944A
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Enzyme-kinetic determination of the concentration of a substrate
Assignee: Eppendorf SE
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Filing: 1974-10-02
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Granted: 1976-08-31
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Expires: 1994-10-02
Abstract
Abstract Enzyme-kinetic determination of the concentration of an enzyme substrate which is acted on during an enzymatic reaction is carried out by adding at least one competitive inhibitor to an enzymatic reaction medium to bring the ratio of the substrate concentration C3 to the apparent Michaelis constant K'M to 0.2:1 or less, homogenizing the medium and determining the rate change of the enzymatic reaction at certain intervals of time.
Inventors
- 👤 Reinhard Muller-Matthesius
- 👤 Wolfgang Gruber
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 22:09
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/26; C12Q1/32; C12Q1/54; C12Q1/62
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
116 words
Method for the enzyme-kinetic determination of the concentration of a substrate wherein one or more specific enzymes are added and the rate of the enzymatic reaction is measured, the improvement which comprises adding at least one competitive inhibitor inhibiting the rate determining step of the enzymatic substrate reaction to bring the ratio of the substrate concentration c S to the apparent Michaelis constant K M ' of the inhibited enzymatic reaction to 0.2 : 1 or less, thereby establishing the conditions of a pseudo-first order reaction, homogenizing the mixture, determining the change in a reaction parameter specific for the reaction at certain intervals of time, and calculating the concentration of the substrate to be detected therefrom.
Claim 2
Dependent Claim
116 words
Method for the enzyme-kinetic determination of the concentration of a substrate wherein one or more specific enzymes are added and the rate of the enzymatic reaction is measured, the improvement which comprises adding at least one competitive inhibitor inhibiting the rate determining step of the enzymatic substrate reaction to bring the ratio of the substrate concentration c S to the apparent Michaelis constant K M ' of the inhibited enzymatic reaction to 0.2 : 1 or less, thereby establishing the conditions of a pseudo-first order reaction, homogenizing the mixture, determining the change in a reaction parameter specific for the reaction at certain intervals of time, and calculating the concentration of the substrate to be detected therefrom.
Claim 3
Dependent Claim
32 words
Method of claim 1 wherein the ratio of the substrate concentration c S to the apparent Michaelis constant K M ' is brought to a value of 0.05 : 1 or less.
Claim 4
Dependent Claim
19 words
Method of claim 1 wherein a competitive inhibitor is used which reversibly binds the enzyme without being chemically reacted.
Claim 5
Dependent Claim
24 words
Method of claim 3 wherein a competitive inhibitor is used which is chemically similar to one of the starting products of the measuring reaction.
Claim 6
Dependent Claim
29 words
Method of claim 1 wherein a competitive inhibitor is chosen to result in a ratio of the concentration of substrate to enzyme permitting a sensitivity suitable for analytical measurement.
Claim 7
Dependent Claim
17 words
Method of claim 1 wherein the sensitivity of the measurement is increased by increasing the enzyme concentration.
Claim 8
Dependent Claim
23 words
Method of claim 1 wherein the determination is performed immediately after homogenizing the mixture, waiting for the end of any abrupt temperature rise.
Claim 9
Dependent Claim
18 words
Method of claim 1 wherein any extended creep that may occur is corrected by recording a blank value.
Claim 10
Dependent Claim
34 words
Method of claim 1 wherein a buffer adapted to the measuring mixture is used, the pH value of said buffer coinciding substantially with the pH-rate-maximum of the total reaction rate of the measuring mixture.
Claim 11
Dependent Claim
45 words
Method of claim 1 wherein within the range of the pseudo-first order reaction, in all measurements in a series of analyses, the measurement is performed in an equal period of time after a certain chosen time after the start of the reaction ("time fixed" technique).
Claim 12
Dependent Claim
55 words
Method of claim 1 wherein within one series of analyses, one performs, after the certain chosen time, an averaging of the measures values determined in the measuring period, or a kinetic two-point measurement at the beginning and end of the time period, or a determination of the momentary rate at a set point in time.
Claim 13
Dependent Claim
13 words
Method of claim 1 wherein the initial rate is determined ("initial rate" technique).
Claim 14
Dependent Claim
36 words
Method of claim 1 wherein the competitive inhibitor or inhibitors and the enzyme are dissolved in a buffer adapted to the measuring mixture, this solution is added to the serum, and thereafter a co-substrate is added.
Claim 15
Dependent Claim
22 words
Method of claim 1 wherein a solution of the substrate to be determined is used, which already displays a competitive inhibiting effect.
Claim 16
Dependent Claim
23 words
Method of claim 14 wherein the competitive inhibitor is added in an amount which produces a 60 to 95% inhibition of the enzyme.
Claim 17
Dependent Claim
17 words
Method of claim 14 wherein a substrate solution is used which inhibits the enzyme up to 50%.
Claim 18
Dependent Claim
16 words
Method of claim 1 wherein said substrate is dissolved or suspended in blood serum or plasma.
Claim 19
Dependent Claim
32 words
Method of claim 1 wherein the ratio of the substrate concentration c S to the apparent Michaelis constant K M ' is brought to a value of 0.01 : 1 or less.
Claim 20
Dependent Claim
19 words
Method of claim 1 wherein a competitive inhibitor is used which reversibly binds the substrate without being chemically reacted.
Claim 21
Dependent Claim
23 words
Method of claim 3 wherein a competitive inhibitor is used which is chemically similar to one of the end products of the reaction.
Claim 22
Dependent Claim
17 words
Method of claim 1 wherein the sensitivity of the measurement is increased by increasing the reaction temperature.
Claim 23
Dependent Claim
25 words
Method of claim 1 wherein the determination is performed immediately after homogenizing the mixture and waiting for the cessation of any creep that may occur.
Claim 24
Dependent Claim
22 words
Reagent for an enzyme-kinetic determination of the concentration of an enzyme substrate consisting essentially of uricase, buffer, potassium thiocyanate and lithium carbonate.
Claim 25
Dependent Claim
22 words
Reagent for an enzyme-kinetic determination of the concentration of an enzyme substrate consisting essentially of uricase, buffer, potassium thiocyanate and lithium carbonate.
Claim 26
Dependent Claim
27 words
Reagent for an enzyme-kinetic determination of the concentration of an enzyme substrate consisting essentially of phosphate buffer; sulfosalicyclic acid; Edta; nad; glucose; glucose-dehydrogenase; potassium thiocyanate; and phloretin.
Claim 27
Dependent Claim
27 words
Reagent for an enzyme-kinetic determination of the concentration of an enzyme substrate consisting essentially of phosphate buffer; sulfosalicyclic acid; Edta; nad; glucose; glucose-dehydrogenase; potassium thiocyanate; and phloretin.
Claim 28
Dependent Claim
24 words
Reagent for an enzyme-kinetic determination of the concentration of an enzyme substrate consisting essentially of borate buffer; uric acid; uricase; potassium thiocyanate; and xanthine.
Claim 29
Dependent Claim
24 words
Reagent for an enzyme-kinetic determination of the concentration of an enzyme substrate consisting essentially of borate buffer; uric acid; uricase; potassium thiocyanate; and xanthine.
References Cited (Backward Citations)
US3627645A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1979-08-31 | 1980-03-01 | 1980-08-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1983-08-31 | 1984-03-01 | 1984-08-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1987-08-31 | 1988-03-01 | 1988-08-31 | $7700.00 | Window Closed / Lapsed |