Method for simultaneous determination of enzymatic activities of enzymes

• US 3,925,162 A
• Filed: 08/06/1973
• Issued: 12/09/1975
• Est. Priority Date: 08/17/1972
• Status: Expired due to Term

• Current Assignee: Takashi Kanno
• Original Assignee: Takashi Kanno
• Inventors: Kanno, Takashi
• Primary Examiner(s): Monacell, A. Louis
• Assistant Examiner(s): Fan, C. A.
• Application Number:
  US05/385,815
• Time in Patent Office: 855 Days
• Field of Search: 195/13.5R
• US Class Current: 435/16
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/46 involving cholinesterase
• CPC Class Codes: C12Q 1/52 involving transaminase
• CPC Class Codes: C12Q 2337/30 Naphthyl amides, e.g. beta-NA, 2-NA, 4-methoxy-beta-naphthylamine, i.e. 4MNA

Abstract

A method for the simultaneous determination of enzymatic activities of a plurality of enzymes in a single reaction medium which comprises adding a substrate of each of the enzymes to be determined and optionally reagents required for the measurement of the enzymatic activity to an aqueous solution containing a plurality of enzymes to be determined to allow the enzymatic reactions to proceed simultaneously under the same conditions, measuring simultaneously changes in the absorbance or fluorescence of the resulting reaction system with the lapse of time at a plurality of optional wavelengths which are different from each other and the number of which is equal to the number of the enzymes to be determined, formulating simultaneous equations of the first degree by taking advantage of the proportional relationship, which is independent with respect to each of the enzymes, between the measurement values and the enzymatic activities and determining the enzymatic activity of each of the enzymes by the thus formulated equations is disclosed.

Claims

1. A METHOD FOR THE SIMULTANEOUS DETERMINATION OF ENZYMATIC ACTIVITIES OF A PLURALITY OF ENZYMES INA SINGLE REACTION MEDIUM, COMPRISING:

ADDING A SUBSTRATE FOR EACH OF THE ENZYMES TO BE DETERMINED, EACH SUBSTRATE BEING SELECTED SUCH THAT IT IS CAPABLE OF GIVING AN ENZYMATIC REACTION WITH ITS RESPECTIVE ENZYME, IT DOES NOT INTERACT WITH THE OTHER SUBSTRATES REACTION PRODUCTS OR REAGENTS USED AND IT DOES NOT ADVERSELY AFFECT THE MEASUREMENT OF THE ENZYMATIC ACTIVITY, TO AN AQUEOUS SOLUTION CONTAINING A PLURAITY OF ENZYMES TO BE DETERMINED, THE ENZYMES BEING SELECTED SUCH THAT THE ENYZMATIC CONDITIONS OF EACH ARE SIMILAR TO EACH OTHER AND THEY CAN BE REACTED WITH THEIR RESPECTIVE SUBSTRATES UNDER THE SAME REACTION CONDITIONS;

SIMULTANEOUSLY REACTING EACH OF THE ENZYMES WITH THEIR RESPECTIVE SUBSTRATES UNDER THE OPTIMUM REACTION CONDITIONS WITH RESPECT TO EACH OF THE ENZYMES UNDER WHICH THE ENZYMATIC REACTIONS OF THE PLURALITY OF ENZYMES TO BE DETERMINED TAKE PLACE SIMULTANEOUSLY;

MEASURING SIMULTANEOUS CHANGES IN THE ABSORBANCE OR FLUORESCENCE OF THE RESULTING REACTION SYSTEM WITH THE LAPSE OF TIME AT A PLUTALITY OF OPTIONAL WAVELENGTHS WHICH ARE DIFFERENT FROM EACH OTHER AND THE NUMBER OF WHICH IS EQUAL TO THE NUMBER OF ENZYMES TO BE DETERMINED;

FORMULATING SIMULTANEOUS EQUATIONS OF THE FIRST DEGREE BY TAKING ADVANCING OF THE PROPOTIONAL RELATIONSHIP, WHICH IS INDEPENDENT WITH RESPECT TO EACH OT THE ENZYMES, BETWEEN THE MEASUREMENT VALUES AND THE ENZYMATIC ACTIVITIES;

AND DETERMINING THE ENZYMATIC ACTIVITY OF EACH OF THE ENZYMES BY THE THUS FORMULATED EQUATIONS.

2. A method according to claim 1 wherein the plurality of enzymes is a combination of leucine aminopeptidase or cholinesterase and a dehydrogenase or a transaminase, the enzymatic activity of which can be determined in coupling with a dehydrogenase.

3. A method according to claim 2 wherein said dehydrogenase is selected from the group consisting of lactate dehydrogenase and 2-oxobutyrate dehydrogenase.

4. A method according to claim 2 wherein said transaminase is selected from the group consisting of glutamate oxalacetate transaminase and glutamate pyruvate transaminase.

5. A method for the simultaneous determination of enzymatic activities in a single reaction medium, comprises:

adding a. L-leucyl- Beta -naphthylamide as a substrate for the measurement of the enzymatic activity of leucine aminopeptidase, b. acetylthiocholine as a substrate and 4,4'"'"''"'"' dithiopyridine or dithionitrobenzoic acid as an aid for the measurement of the enzymatic activity of cholinesterase, c. a substrate for the measurement of the enzymatic activity of a dehydrogenase, and d. a substrate of a transaminase, and a dehydrogenase which is capable of being coupled with the transaminase plus NADH as aids for measurement, for the measurement of the enzymatic activity of the transaminase, to an aqueous solution containing leucine aminopeptidase or cholinesterase and a dehydrogenase or a transaminase, the enzymatic activity of which can be determined in coupling with said hydrogenase;

allOwing the two enzymatic reactions to proceed in a single reaction medium simultaneously under the optimum reaction conditions with respect to each of the enzymes under which the enzymatic reactions of the plurality of enzymes to be determined take place simultaneously;

measuring changes in the absorbance or fluorescence at optional different two wavelengths for a period of predetermined time with the lapse of time;

formulating simultaneous equations of the first degree by taking advantage of the proportional relationship between the absorbance at each of the wavelengths or changes in the fluorescence with the lapse of time and the each of the enzymatic activities; and

determing the enzymatic activity of each of the enzymes by the thus formulated equations.

6. A method according to claim 5 wherein said dehydrogenase is selected from the group consisting of lactic dehydrogenase and 2-hydroxybutyrate dehydrogenase.

7. A method according to claim 5 wherein said transaminase is selected from the group consisting of glutamic oxalacetic transaminase and glutamate pyruvate transaminase.

8. A method according to claim 5 wherein the enzymatic activity of each of the enzymes is calculated by the following equations from the measurement values of changes in the absorbance at optional different wavelengths or of changes in the fluorescence with the lapse of time:

9. A method for the simultaneous determination of enzymatic activities of the enzymes LAP and lactate dehydrogenase in a single reaction medium, comprising:

adding a. L-leucyl- Beta -naphthylamide as a substrate for detecting LAP and b. a pyruvate and a reduced type of niacine adenine dinucleotide (NADH) as substrates for detecting dehydrogenase to a sample of an aqueous enzyme solution containing LAP and lactate dehydrogenase;

allowing the two types of enzymatic reaction to proceed simultaneously in a single reaction medium under the optimum reaction conditions with respect to each of the enzymes under which the enzymatic reactions of the plurality of enzymes to be determined take place simultaneously;

measuring the changes in absorbance or fluorescence of the reaction system with the lapse of time at wavelengths 335 and 355 m Mu ;

formulating simultaneous equations of the first degree by taking advantage of the proportional relationship, which is independent with respect to each of the enzymes, between the measurement values and enzymatic activities of the enzymes present in said enzyme solution; and

determining the enzymatic activity of each of said enzymes by the thus formulated equations.

10. A method according to claim 9 wherein said enZymatic reaction is conducted at a pH value of 7.2 at a temperature of 37*C.

11. A method for the simultaneous determination of enzymatic activities of the enzymes LAP and 2-hydroxybutyrate dehydrogenase in a single reaction medium, comprising:

adding a. L-leucyl- Beta -naphthylamide as a substrate for detecting LAP and b. 2-oxobutyrate and a reduced type of niacine adenine dinucleotide (NADH) as substrates for detecting 2-hydroxybutyrate dehydrogenase to a sample of an aqueous enzyme solution containing LAP and 2-hydroxybutyrate dehydrogenase;

allowing the two types of enzymatic reaction to proceed simultaneously in a single reaction medium under the optimum reaction conditions with respect to each of the enzymes under which the enzymatic reactions of the plurality of enzymes to be determined take place simultaneously;

measuring the changes in absorbance or fluorescence of the reaction system with the lapse of time at wavelengths 335 and 355 m Mu ;

formulating simultaneous equations of the first degree by taking advantage of the proportional relationship, which is independent with respect to each of the enzymes, between the measurement values and enzymatic activities of the enzymes present in said enzyme solution; and

determining the enzymatic activity of each of said enzymes by the thus formulated equations.

12. A method according to claim 11 wherein said enzymatic reaction is conducted at a pH value of 7.2 at a temperature of 37*C.

13. A method for the simultaneous determination of enzymatic activities of the enzymes LAP and glutamate oxalacetate transaminase in a single reaction medium, comprising:

adding a. L-leucyl- Beta -naphthylamide as a substrate for detecting LAP and b. 2-oxoglutarate and L-asparate as substrates for detecting oxalacetate transaminase as well as malate dehydrogenase and a reduced type of niacine adenine dinucleotide (NADH) as aids for detection of the enzyme to a sample of an aqueous enzyme solution containing LAP and glutamate oxalacetate transaminase;

allowing the two types of enzymatic reaction to proceed simultaneously in a single reaction medium under the optimum reaction conditions with respect to each of the enzymes under which the enzymatic reactions of the plurality of enzymes to be determined take place simultaneously;

measuring the changes in absorbance or fluorescence of the reaction system with the lapse of time at wavelengths 335 and 355 m Mu ;

formulating simultaneous equations of the first degree by taking advantage of the proportional relationship, which is independent with respect to each of the enzymes, between the measurement values and enzymatic activities of the enzymes present in said enzyme solution; and

determining the enzymatic activity of each of said enzymes by the thus formulated equations.

14. A method according to claim 13 wherein said enzymatic reaction is conducted at a pH value of 7.2 at a temperature of 37*C.

15. A method for the simultaneous determination of enzymatic activities of the enzymes LAP and glutamate pyruvate transaminase in a single reaction medium, comprising:

adding a. L-leucyl- Beta -naphthylamide as a substrate for detecting LAP and b. 2-oxoglutarate and L-alanine as substrates for detecting glutamate pyruvate transaminase as well as lactate dehydrogenase and a reduced type of niacine adenine dinucleotide (NADH) as aids for detection of the enzyme to a sample of an aqueous enzyme solution containing LAP and glutamate pyruvate transaminase;

allowing the two types of enzymatic reaction to proceed simultaneously in a single reaction medium under the optimum reaction conditions with respect to each of the enzymes under which the enzymatic reactions of the plurality of enzymes to be determined take place simultaneously;

measuring the changes iN absorbance or fluorescence of the reaction system with the lapse of time at wavelengths 335 and 355 m Mu ;

formulating simultaneous equations of the first degree by taking advantage of the proportional relationship, which is independent with respect to each of the enzymes, between the measurement values and enzymatic activities of the enzymes present in said enzyme solution; and

determining the enzymatic activity of each of said enzymes by the thus formulated equations.

16. A method according to claim 15 wherein said enzymatic reaction is conducted at a pH value of 7.2 at a temperature of 37*C.

17. A method for the simultaneous determination of enzymatic activities of the enzymes choline-esterase and lactate dehydrogenase in a single reaction medium, comprising:

adding a. acetylthiocholine as a substrate for detecting choline-esterase and 4,4'"'"''"'"'-dithiopyridine or dithionitrobenzoic acid as an aid for measurement of choline-esterase activity, and b. pyruvate and a reduced type of niacine adenine dinucleotide (NADH) as substrates for detecting lactate dehydrogenase to a sample of an aqueous enzyme solution containing cholineesterase and lactate dehydrogenase;

allowing the two types of enzymatic reaction to proceed simultaneously in a single reaction medium under the optimum reaction conditions with respect to each of the enzymes under which the enzymatic reactions of the plurality of enzymes to be determined take place simultaneously. measuring the changes in absorbance or fluorescence of the reaction system with the lapse of time at wavelengths 325 and 340 m Mu ;

formulating simultaneous equations of the first degree by taking advantage of the proportional relationship, which is independent with respect to each of the enzymes, between the measurement values and enzymatic activities of the enzymes present in said enzyme solution; and

determining the enzymatic activity of each of said enzymes by the thus formulated equations.

18. A method according to claim 17 wherein said enzymatic reaction is conducted at a pH value of 7.2 at a temperature of 37*C.

19. A method for the simultaneous determination of enzymatic activities of the enzyes choline-esterase and 2-hydroxybutyrate hydrogenase in a single reaction medium, comprising:

adding a. acetylthiocholine as a substrate for detecting choline-esterase and 4,4'"'"''"'"'-dithiopyridine or dithionitrobenzoic acid as an aid for measurement of choline-esterase activity, and b. 2-oxobutyrate and a reduced type of niacine adenine dinucleotide (NADH) as substrates for detecting 2-hydroxybutyrate dehydrogenase to a sample of an aqueous enzyme solution containing cholineesterase and 2-hydroxybutyrate dehydrogenase;

allowing the two types of enzymatic reaction to proceed simultaneously in a single reaction medium under the optimum reaction conditions with respect to each of the enzymes under which the enzymatic reactions of the plurality of enzymes to be determined take place simultaneously;

measuring the changes in absorbance or fluorescence of the reaction system with the lapse of time at wavelengths 325 and 340 m Mu ;

formulating simultaneous equations of the first degree by taking advantage of the proportional relationships, which is independent with respect to each of the enzymes, between the measurement values and enzymatic activities of the enzymes present in said enzyme solution; and

determining the enzymatic activity of each of said enzymes by the thus formulated equations.

20. A method according to claim 19 wherein said enzymatic reaction is conducted at a pH value of 7.2 at a temperature of 37*C.

21. A method in accordance with claim 1 wherein said reactions conditions are under a pH of approximately 7.0 at a temperature of from 25* to 40* C.

22. A method in acCordance with claim 5 where said reactions conditions are under a pH of approximately 7.0 at a temperature of from 25* to 40* C.

23. A method in accordance with claim 1 wherein said adding step further includes the addition of other reagents useful as aids for the measurement of the enzymatic activity, which reagents are selected such that they do not interact with the substrates, reaction products or other reagents used and they do not adversely affect the measurement of the enzymatic activity.