Stopping agents for enzyme reactions of dehydrogenase systems

Page 1 — bibliographic record

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for the former enzyme or al-ketoglutaric acid and L-ala

STOPPING AGENTS FOR ENZYME REACTIONS nine for the latter, and a reduced form of NAD (herein OF DEHYDROGENASE SYSTEMS after referred to as NADH) in either case. Oxaloacetic acid derived from aspartic acid with GOT (in case of

This invention relates to a reaction stopping agent S GPT, pyruvic acid derived from alanine, the GPT case utilizable for the measurement of activity of serum being hereinafter referred to in the parenthesis) is con enzymes which are thought of markers for diagnosis of verted to malic acid (lacetic acid) with malic dehydro liver, heart and muscle diseases in human beings. genase (lactic dehydrogenase). and NADH concur More specifically, it is concerned with a new applica rently with oxidation of NADH to NAD, which results tion or use of an aqueous alkali solution of cyclohex O in decrease of absorbance at 340nm arising from ylaminopropanesulfonic acid having specified ranges of NADH. An amount of the oxaloacetic acid (pyruvic the concentration and pH value thereof as a stopping acid) produced by GOT (GPT) is stoichiometrically agent for stopping an enzyme reaction of a dehydroge equal to that of the NADH to be oxidized and a GOT nase or a dehydrogenase system in a spectrophotomet (GPT) activity can be, therefore, determined from an ric assay method of an activity of serum enzyme in the 15 oxidized amount of NADH which is obtained from an above enzyme or enzyme system. absorbance decrease at 340nm. Still more particularly, it is concerned with a method ALD activity in serum is raised in case of liver dis for stopping an enzyme reaction of a dehydrogenase or eases, progressive muscular dystrophy or coronary a dehydrogenase system in a spectrophotometric assay heart infarction. .

method of an activity of serum enzyme in said enzyme In measuring ALD activity, one can carry out by or enzyme system which comprises applying as a stop adding to a reaction medium fructose-1,6-diphosphate ping agent for said enzyme reaction an aqueous alkali which is a substrate, togethe with triose phosphate solution of cyclohexylaminopropanesulfonic acid hav isomerase (hereinafter referred to as TMI) and glyce ing a concentration of 0.1 - 2M and a pH value of 10.5 25 rol-1-phosphate dehydrogenase (hereinafter referred - 12.5. to as GDH). In this system, the products by ALD are In the spectrophotometric assay method of an activ converted to dioxyacetone phosphoric acid, which is ity of serum enzyme in a dehydrogenase or a dehydro then converted to glycerol-1-phosphate by the action genase system which may be used in this invention, of GDH and NADH, NADH is simultaneously oxidized there may be included various assay methods com 30 to NAD. Hydrolysis of 1 mole of fructose-1,6-diphos monly employed in the art for such purposes, for in phate with ALD results in oxidation of 2 moles of stance, an ultraviolet absorbance assay method (here NADH.

inafter referred to as “ultraviolet assay method”) CPK in serum is increased along with injury of skele wherein an activity of glutamic - oxaloacetic transam tal muscle or heart muscle. For determination of CPK inase (hereinafter referred to as GOT), glutamic - py activity, creatine phosphoric acid and adenosine-5'- ruvic transaminase (hereinafter referred to as GPT), 35 diphosphate (hereinafter referred to as ADP) are em aldolase (hereinafter referred to ALD), creatinephos ployed as substrates and further hexokinase (hereinaf phokinase (hereinafter referred to as CPK), lactic de ter referred to as HK), glucose, glucose-6-phosphate hydrogenase (hereinafter referred to as LDH), alcohol dehydrogenase (hereinafter referred to as G6PDH) dehydrogenase or sorbitol dehydrogenase in human and NADP are added to the reaction medium. By the serum is determined by measuring an absorbance 40 action of CPK, ADP is converted to adenosine-5'-tri change at 340nm of nicotinamide-adenine dinucleotide phosphate (hereinafter referred to as ATP), while glu (hereinafter referred to as NAD) or nicotinamide ade cose in the reaction medium is converted to glucose-6- nine dinucleotide phosphate (hereinafter referred to as phosphate by the action of the ATP and HK. Thereaf NADP) added in the reaction medium either alone or ter, the glucose-6-phosphate is converted to 6-phos in combination with an appropriate dehydrogenase 45 phogluconic acid by the action of G6PDH and NADP (system); a fluorometric assay method (hereinafter and NADP is converted to a reduced form of NADP referred to as "fluorometric assay method') wherein a (hereinafter referred to as NADPH), along with the serum enzyme activity of the above-mentioned en conversion, which results in increased absorbance at zymes in human serum is determined by measuring a 340mm. The ATP amount produced by CPK is stoichio change in fluoroescence intensity of NAD or NADP 50 metrically equal to that of NADP to be reduced. . excited with 340nm beam, said NAD or NADP being In case of other serum enzymes than those as named added to a reaction medium either alone or in combi above, similar reaction system is employable. In case of a serum enzyme itself being a dehydrogenase as LDH, nation with a dehydrogenase; and the like. .

This invention will be more fully disclosed with par an enzyme activity is determined by direct dehydroge ticular reference to the ultraviolet assay method for 55 nation of NADH or NADPH with a substrate so as to convenience' sake, but it should be noted that the stop change absorption intensity at 340mm. ping agent of this invention is satisfactorily applicable As stated above, an ultraviolet assay method has a to other assay methods such as the fluorometric assay characteristic in that measurements can be effected by method and so on. the use of a dehydrogenase reaction to couple the reac Increase in the GOT or GPT activity in serum occurs 60 tion of aforementioned serum enzyme with oxidation along with some liver diseases or coronary heart infrac reaction of NADH (or NADPH) or reduction reaction tion, then measurement of the activity is considered of NAD (or NADP) in every case.

useful for diagnosis of these diseases. . . . . An ultraviolet assay method does not require color For measuring serum GOT or GPT activity, the reac ing procedures and can be effected by extremely simple tion can be carried out through addition of malic dehy 65 determination procedures, differing from other assay drogenase to the reaction medium in case of GOT and methods wherein colorimetric determination should be LDH in case of GPT, together with the enzyme sub made after coloration of the reaction product or resid strates such as a-ketoglutaric acid and L-aspartic acid ual substrate.

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An absorption intensity of NADH or NADPH at kits. Then, the prior solution seems to be of no practi 340nm is also completely proportionable to a concen cal use.

tration thereof and a molar extinction coefficient is As a result of our earnest studies to supply deficien sufficiently high. Remarkably high precision, accuracy cies of such stopping agents, it has been found that a and reproducibility is obtainable by the ultraviolet wide variety of dehydrogenases can be completely in assay method, as compared with other known methods. hibited by the use of an appropriate amount of an aque Indication of an activity value (unit), which has been ous alkali solution having a specific concentration of recently recommended by The International Union of cyclohexylaminopropanesulfonic acid (hereinafter re Biochemistry, is easily available by multiplying change ferred to as CAPS) and a specific pH value to show a in absorbance at 340nm by a coefficient. In view of the 10 strong buffer action within an alkaline pH range which additional advantage as mentioned just above, an ultra does not cause changes in absorption of NAD or NADP violet assay method previously applied in a basic bio around 340nm and also that the present stopping agent chemical research has been ultimately expected to has ideal characteristics as a stopping agent, without become practicable in routine assay of serum enzymes 15 any influence on turbidity of serum of normal healthy in a clinical diagnostic laboratory. human beings as well as of lipemic serum. This inven However, the prior ultraviolet assay methods which tion has been completed upon the above findings. are manually worked have not yet been widespread, It is, accordingly, a primary object of this invention to since it is difficult to assay a large number of serum provide a new method for stopping an enzyme reaction samples owing to impracticability of a stopping agent 20 of a dehydrogenase or a dehydrogenase system in a useful for the abovementioned enzyme reaction and, in spectrophotometric assay method by the use of an case where many serum samples are to be daily deter aqueous alkali solution of CAPS.

mined as done in a clinical diagnostic laboratory, a Other objects and advantages of this invention will specific photometer developed particularly for an ul become apparent from the following descriptions. traviolet assay method should be employed, for exam By the use of the present stopping agent, it becomes ple, such apparatus as LKB-8600 type Reaction Rate 25 feasible to conduct simultaneous reactions of many Analyzer (LKB Co., Sweden), Rotochem II Fast Analy serum samples, stop the rection by addition of the stop zer (Aminco Co., U.S.A.) and so on. More specifically, ping agent after a given period of time and then deter the ultraviolet assay method in manual procedures mine an enzyme activity by measuring absorption at employing a conventional photometer is conducted by 340nm. According to such procedures, it is feasible to placing a reaction mixture and a serum sample into a 30 determine the respective activities of 100 - 200 serum cell and measuring change with time of absorbance at samples per day by one operator. In case where an 340nm over 10 - 30 minutes, while according to this activity is to be determined with lapse of time in a cell, time consuming method the number of serum samples it is impossible to reduce a volume of the reaction to be tested per day is limited to at most 10-20 and an mixture to a given one due to necessity for a cell capac operator is very tired with all day working for measure 35 ity, whereas it is possible, according to the procedures ment. Moreover, the above-mentioned specific pho which use the present stopping agent, that an amount tometer is a highly expensive apparatus with a cost of of the reaction mixture to be reacted is reduced by adjusting an amount of the stopping agent to be added, ten million - thirty million yen and, therefore, it is not as prevalent to middle and small clinical diagnostic labo 40 the it is sufficient that the final volume after addition of ratories. stopping agent is beyond the necessary capacity for In the art, there have been proposed a considerable measuring. In general, it is easy to reduce the volume of number of stopping agents for ordinary enzyme reac a reaction mixture so small as 1/3 - 1/6 of the conven tion, but they are not utilizable for the ultraviolet assay tional manual method. A blank test for serum can be method from the following reasons. Deproteinizing performed without addition of the reaction mixture and agents such as trichloroacetic acid, perchloric acid, 45 a reagent blank is determined separately. A blank value methaphosphoric acid, sulfuric acid - tungstenic acid for the assay is estimated as a sum of the above both and the like make disappeared absorption of NADH blank values. Thus, the reagent is not wasted for such (or NADPH) at 340nm and other acids, when incorpo blank correction, which meets requirements from clini rated in an amount to stop the reaction, make disap 50 cal laboratory to save an amount of the reagent. peared absorption at 340nm. On the other hand, when invention The findings which are concerned with the present an aqueous solution of alkali such as Sodium hydroxide tively and the present method will be more illustra is added in an amount to stop the reaction, absorption explained hereinbelow. of NAD (or NADH) at 340nm is apt to change irregu 1. INHIBITORY ACTION OF AN ALKALINE larly. If the enzyme reaction is stopped by heat treat 55 BUFFER SOLUTION ON DEHYDROGENASE ment, the reaction mixture becomes turbid owing to ACTIVITY AND INFLUENCE ON ABSORPTION serum protein and ordinary centrifugation of 3000 INTENSITY OF COENZYME AT 340NM. r.p.m. or lower can only remove turbidity partially and not provide a clear solution for measurement. As a result of our studies on phosphates buffer solu It is also reported in the art that an aqueous alkali 60 tion, glycine buffer solution, arginine buffer solution, solution of p-chloromercurybenzoate is applicable as a guanidine buffer solution, and CAPS buffer solution, it stopping agent for dehydrogenase reaction (E. Raabo, has been found that other buffer solutions than CAPS buffer solution have some drawbacks and are unable to

Scandinavian Journal of Clinical & Laboratory Investi gation, 17, (1965), 265). However, this solution has be utilized as a stopping agent.

been confirmed to give inhibition of dehydrogenase 65 Phosphate buffer solution has an action to promote reaction only in case of restricted buffer concentration autoxidation of NADH remarkably. This action may be prevented by the addition of an appropriate amount of of a reaction mixture and further to give only partial inhibition of dehydrogenase reaction in case of using a ethylenediaminetetraacetic acid (EDTA), while ad reaction mixture of commercially available diagnostic sorption at 340nm of NAD is highly increased by the

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addition of a phosphate buffer solution containing of coenzyme at 340nm to some extent, though more or EDTA to an enzyme reaction mixture containing an less differences may be observed upon the concentra amino acid, e.g., glycine, thereby causing non tion and pH of the buffer solution. However, increases enzymatic change in NAD. Similar change to the above in absorbance of oxidized form and reduced form of may be caused when a glycine buffer solution or an 5 coenzyme are approximately equal per unit u mole arginine buffer solution is added to an enzyme reaction and, accordingly, when working curves are made up by mixture containing EDTA, which leads to increase in measuring absorbance at 340nm at varied ratios of the absorption of NAD at 340nm. A guanidine buffer solu oxidized form to the reduced form where a total tion does not show such action, but the solution itself amount of coenzyme is kept constant, there is obtained may increase absorption of NAD at the concentration 10 a working curve, in case of the addition of CAPS buffer and pH value to inhibit an action of an enzyme. Such solution, which has a slope equal to that obtained with increasing action on absorption of NAD may continu out any addition of the buffer and a shape transferred ously vary with the lapse of time and it is, therefore, in parallel with upwards by increased absorption inten difficult to correct influence caused therefrom. sity.

2. INHIBITORY ACTION OF CAPS BUFFER In ultraviolet assay method, an enzyme activity is to SOLUTION ON DEHYDROGENASE REACTION be determined from differences in absorbance at 340nm before and after reaction proceeding and,

Four enzymes which are widely employed for exami therefore, an absorption increasing effect by CAPS nation in clinical diagnosis among various serum en zymes, were tested on the inhibitory actions by CAPS 20 bufferii.

solution is eliminated.

Influence upon stability of absorption intensity buffer solution. Some of our experiments are summa rized in the Table 1 wherein changes in absorbance at As stated above, we have found that absorption of 340nm during the whole process of the reaction are coenzyme at 340nm is increased to some extent upon automatically recorded and expressed in terms of inhi dissolution by the addition of CAPS buffer solution bition degree by adding serum to a reaction mixture for 25 according to this invention. Some of our experiments proceeding the reaction and then adding thereto CAPS about stability of absorption after dissolution are sum buffer solution with varied concentrations, pH values marized in Table 2 which shows changes in absorption and amounts added. intensity at 340nm over 3 hours after dissolution of

; : CAPs buffer soln. Serum enzyme 1) * Amount Concentration added 2) (M) 3) PH 3) GOT GPT ALD CPK 0.5 2.0 C 4) C C C

% Vol. 11.0 80 C C C

2. 10.5 65 C C C ..

0.5 1.0 85 C C C

Vol. . l 10.5 70 C C C

2 10.5 C C C C

2 Vol. 0.125 11.5 80 C C C. 0.5 10.5 85. C C C

0. 1.5 C C. : C C 4 Vol.

0.25 110 C C C C

Complete inhibition concentration with aqueous

NaOH soln. (Final concentra tion) 1) Diagnostic kits available from Calbiochcm U.S.A., used for measuring GOT, GPT and CPK and diagnostic kits available from Boeringer-Manheim, West Germany, used for mcasuring ALD, standard human scrum (Caltrol-Abnormal) available from Calbiochem used for scrum.

2) volumcratio of thc added buffer solution to thc enzymc reaction mixture. For instance, a volumc is meant to show that % volumc is added per volume of thc reaction mixture.

3) Concentration and pH of the addcd buffer solution.

4) crepresents 100% complete inhibition and figure (%) represents partial inhibition.

3. INFLUENCE OF ADDITION OF CAPS BUFFER

SOLUTION UPON ABSORPTION INTENSITY AND

ITS STABILITY OF COENZYMEAT 340mm i. Influence upon absorption intensity

By the addition of CAPs buffer solution to a coen zyme solution is usually increased absorption intensity coenzyme in CAPS buffer solution.

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Table 2

CAPS )

buffer solution NAD NADH NADP NADPH

0.5M.pH1 1.5 g . O.D. change not not not not more than changed changed changed

0.33M,pH 11.5 O.D. change not FF more than 0.25M, pH 1 1.5 not changed 3) FF

(containing not changed 3) g

0.005A/hricm F. ff

not more than

0.25M,pH 12.5 O.D. change not f O.D. change PF

1) Cocnzymc dissolved in the indicated buffer solution to 1.1 x 10 M.

2) Changc in absorbancc pcr hour, given whcin mcasurcd by the usc of a cell with optical path lcngth

3) Changcs in absorbance of t0.002A or lcss over 3 hours immediately aftcr dissolution arc expresscd

As seen from Table 2, all coenzymes are stable in hydroxides, e.g., potassium hydroxide, sodium hydrox buffer solutions having a concentration of 0.25M or ide, lithium hydroxide; ammonium hydroxide and the less and a pH value of 115 or less and also stable when 2. like.it is to be noted that other alkaline substances than EDTA contained at 12.5 mM or less. On the other the above may be utilized, provided that they do not hand, when the concentration is 0.33M of more or pH adversely affect the assay conditions, the assay results is 12 or higher, stability of NAD is somewhat reduced and the like.

and, when pH is 12.5 or higher, stability of NADP is It is desirable that a water-miscible aliphatic alcohol also reduced, whereas stability of a reduced form of is further added to an aqueous alkali solution of CAPS. coenzyme is not influenced. As the alcohol which may be employed in this inven If NAD or NADP is dissolved in an aqueous solution tion, there may be mentioned, for example, methanol, of a strong alkali, e.g., sodium hydroxide or potassium ethanol, n-propanol, isopropanol and the like. An hydroxide, absorption intensity at 340nm is increased amount of the alcohol to be added is not essential fea with the lapse of time to a peak after 10 - 20 minutes 35 ture, but it is usual and preferable to use the alcohol in and then gradually decreased. Such changes continu: an amount of 10 - 30% (v/v). Generally, methanol or ously occur over several hours and the higher an alkali ethanol may be practically used in an amount of 30%, concentration is, the greater change is. It is, therefore, 10% for n-propanol and 20% for isopropanol being extremely difficult to find out such a condition that an 40 used respectively.

enzyme reaction is stopped without any influence upon This invention will be concretely illustrated by the absorption of coenzyme, when an aqueous solution strong alkali having no buffer action is employed.

of a following thereof.

examples, but they are not limiting the scope 4. INFLUENCE UPON SERUM TURBIDITY EXAMPLE 1.

In determining an enzyme activity, absorbance be- 45 Measurement of GOT activity in serum fore reaction is ordinarily measured by adding serum to a. Two test tubes A and B were used for one serum reaction mixture and immediately adding CAPS buffer sample. A reaction mixture (pH 7.4) containing the solution for reaction stopping. However, the following following convenient technique may be utilized by simplifying poured intoingredient at the indicated amount was each test tube in 1.0 ml. portion.

the above-mentioned procedures, since the addition of 50

CAPS buffer solution does not affect turbidity of the In units ofand preparation the values as shown below, other ingredients an enzymein are indicated

Sc. o Absorbance of the reaction medium having CAPS terms of international unit and u mole, respectively. buffer solution incorporated therein is determined beis 55 forehand and then absorbance of serum at 340nm 1S Tris(hydroxymethyl)aminomethane 100 added thereto, thereby absorbance before reaction -Ketoglutaric acid 7.5 being defined. According to this technique, saving of CEid : reagents is feasible, since reagents are not wasted for Sodium hydrogencarbonate 0.8 everyti serum in order to determine absorbance before 60 Reduced form of nicotinamide

reaction. Malic acid dehydrogenase 0.33 unit In practising the present invention, the concentration Lactic acid dehydrogenase 0.33 unit of CAPS in its aqueous alkali solution should be within a range of 0.1 - 2M, preferably 0.1 - 0.5M and most preferably 0.5M. The pH value of the CAPS aqueous The test tubes were incubated in a water incubator at alkali solution should also be within a range of 10.5 - 65 30° C. and, after 5 minutes, 25 ful. of serum was added 12.5, preferably 11.5 - 12.5 and most preferably 11.5. into the one test tube A and well mixed. After subse As the alkali which may be employed in this invention, quent incubation for 60 minutes, each 1.0 ml. of 0.5M there may be mentioned, for example, alkali metal - CAPS buffer solution (pH 1 1.5) was added into the

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both test tubes A and B and then 25 pl. of serum was . (pH 11.5) was added into the test tubes A and B and added into the test tube B. Two test tubes were tho then 30 ul. of serum was added into the test tube. B. rougly shaken. Within 3 hours after addition of the Both test tubes were thoroughly shaken and CAPS buffer solution, absorbance of the solution in the absorbance was measured at 340mm. . . . . respective tubes A and B was measured at 340nm by. The respective absorbances are defined as A and B means of a cell with an optical path length of 1 cm. and activity is calculated according to the following GOT activity was calculated according to the follow equation. . ... . . . . ing equation.

The absorbance of the solution in the test tube A is (B-A) x 90.6 = milliunit/ml. of serum defined as A and that in the test tube B as B. O

(B-A) X 217 = milliunit (international unit)/ml. of b. Following the same procedures as in the above (a) Su except that a 0.2M - CAPS - 10%(v/v) n-propanol b. Following the same procedures as in the above (a) buffer solution (pH 11.5) was employed instead of the

0.5M CAPS

except that a 0.2M - CAPS - 30% (v/v) methanol 15 similar buffer solution, there can be . obtained

buffer solution (pH 11.5) was employed instead of the 0.5M CAPS buffer solution, there can be obtained EXAMPLE 4 similar results. Measurement of CPK activity in serum

Two test tubes were used for one serum sample and

Measurement of GPT activity in serum. 1 ml. of a reaction solution (pH 74) of the following. a. Two test tubes A and B were used for one serum formulation was poured into the respective tubes. Units sample. A reaction solution (pH 7.4) having the follow for the values are as defined in Example 1 (a). ing formulation was poured into each test tube in 1.0 ml. portion. Units for the values are as defined in Ex 25 N,N'-Bis(2-ethanesulfonic acid)piperazine, ample 1 (a). W sodium salt 50 *-

Disodium creatiniephosphate 20

Trilithium adenosine-5'-diphosphate S

Tris(hydroxymethyl)aminomethane OO Glucose 5. on-Ketoglutaric acid 8.17 Magnesium aspartate O L-Alanine 6.5 30 Adenosine-5'-phosphoric acid O Succinic acid 35 Glutathione 8.7 Sodium hydrogencarbonate 0.8 Tetrasodium ethylenediaminetetraacetate 1.66 Reduced form of nicotinamide Oxidized form of nicotineamide-adenine. adenine.dinucleotide O. dinucleotide phosphate 0.6 Lactic acid dehydrogenase 0.33 unit Hexokinase 1 unit Glucose-6-phosphoric acid dehydrogenase 0.33 unit

The test tubes were incubated in a water incubator at 30°C. and the subsequent procedures were carried out C.Testfor tubes were incubated in a water incubator at 30 5 minutes and then each 50 ul. of serum was in the same manner as in Example 1 (a). GPT activity added into the test tubes A and B and mixed. Incuba was calculated by using the same equation as in Exam 40 tion was continued and, after 15 minutes and 60 min- . ple 1 (a). . . . . . . . . . utes, each 2 mi. of 0.1M - CAPS - 5 mM EDTA b. Following the same procedures as in the above (a) except that a 0.2M - CAPS - 20%(v/v) isopropanol buffer solution (pH 11.5) was added to the test tubes A and B, respectively, to cease the reaction proceedings.

buffer solution (pH 11.5) was employed instead of the The period of time when absorbance at 340nm is to be 0.5M CAPS buffer solution, there can be obtained 45 measured after addition of the CAPS buffer solution, is similar results.

arranged to be substantially the same with regard to

EXAMPLE 3. both test tubes A and B.

Measurement of ALD activity in serum Absorbances of the liquids in test tubes A and B are defined as A and B, respectively, and an activity is a. Two test tubes A and B were used for one serum 50 calculated according to the following formula. sample. A reaction solution (pH 7.4) containing the following formulation was poured into each test tube in (A-B) x 218 = milliunitml. of serum What is claimed is:

1 ml. portion. Units of the values are as defined in

Example 1 (a). 1. A method for stopping an enzyme reaction of a 55 dehydrogenase or a dehydrogenase system in a spectro photometric assay method of an activity of serum en

Collidine buffer 56 zyme in said enzyme or enzyme system which com Monoiodoacetic acid

Fructose-1,6-diphosphoric acid

3 prises applying as a stopping agent for said enzyme

Reduced form of nicotineamide reaction an aqueous alkali solution of cyclohex 60 ylaminopropanesulfonic acid having a concentration of

Glycerol-1-phosphoric acid dehydrogenase 0.35 unit 0.1 - 2M and a pH value of 10.5 - 12.5. Triose phosphate isomerase 2 units 2. A method according to claim 1 wherein said assay method is effected by measuring a change in absor bance at 340nm of nicotinamide adenine dinucleotide

Test tubes were incubated in an incubator at 30°C. 65 or nicotinamide adenine dinucleotide phosphate. and, after 5 minutes, 30 pil. of serum was added into the 3. A method according to claim 2 wherein said aque test tube A and well mixed. After incubation for 60 ous alkali solution further contains a water-miscible minutes, each 1 ml. of 0.5M - CAPS buffer solution aliphatic alcohol.

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4. A method according to claim 3 wherein said alco 14. A method according to claim 13 wherein said hol is methanol, ethanol, n-propanol or isopropanol. alcohol is methanol, ethanol, n-propanol or isopropa 5. A method according to claim 3 wherein said alco no.

hol is contained in an amount of 10 to 30%. 15. A method according to claim 13 wherein said 6. A method according to claim 3 wherein said alco alcohol is contained in an amount of 10 to 30%. hol is isopropanol and said isopropanol is contained in alcohol ismethod 16. A according to claim 13 wherein said isopropanol and said isopropanol is con an amount of 20%. tained in an amount of 20%. 7. A method according to claim 3 wherein said con 17. A method according to claim 13 wherein said centration is 0.2M, said pH value is 11.5 and said alco 10 concentration is 0.2M, said pH value is 11.5 and said hol is isopropanol in an amount of 20%. alcohol is isopropanol in an amount of 20%. 8. A method according to claim 3 wherein said alco 18. A method according to claim 13 wherein said holis methanol or ethanol in an amount of 30%. alcohol is methanol or ethanol in an amount of 30%. 9. A method according to claim 3 wherein said alco 19. A method according to claim 13 wherein said hol is n-propanol in an amount of 10%. 15 alcohol is n-propanol in an amount of 10%. 10. A method according to claim 2 wherein said 20. A method according to claim 12 wherein said concentration is 0.1 - 0.5M and said pH value is 11.5- concentration

21. A method according to claim 12 wherein said 11. A method according to claim 2 wherein said 20 concentration concentration is 0.5M and said pH value is 11.5. is 0.5M and said pH value is 11.5. 12. A method according to claim 1 wherein said aqueous 22. A method according to claim 1 wherein said assay method is effected by measuring a change in ble aliphatic alkali solution further contains a water-misci alcohol.

fluorescence intensity with exciting light of 340nm of 23. A method according to claim 1 wherein said nicotinamide adenine dinucleotide or nicotinamide 25 concentration is 0.1 - 0.5M and said pH value is 11.5- adenine dinucletide phosphate. 12.5.

13. A method according to claim 12 wherein said 24. A method according to claim 1 wherein said aqueous alkali solution further contains a water-misci concentration is 0.5M k and

saidk pHk value is 11.5.

ble aliphatic alcohol.