JPS5982398A - Method for stabilizing coenzyme - Google Patents

Abstract

PURPOSE:To stabilize a coenzyme, e.g. nicotinamide adenine dinucleotide, etc. conjugative with a dehydrogenase, and useful for clinical diagnosis, etc., by adding a chelating agent and azide compound to a buffer solution within a specific pH range containing the above-mentioned coenzyme in the oxidized or reduced form. CONSTITUTION:A chelating agent, e.g. trisodium ethylenediaminetetraacetate, and an azide compound, e.g. sodium azide, are added to a buffer solution within 6.5-9.0pH range containing one or more coenzymes, e.g. nicotinamide adenine dinucleotide and nitotinamide adenine dinucleotide phosphate, in the oxidized or reduced form to stabilize th coenzymes. Preferably, the amount of the chelating agent to be used is 0.01-10W/V%, and the amount of the azide to be used is 0.001-1W/V%.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing a substantially neutral bath solution containing NAD and NADP.

NAD (cotinamide adenine dinucleotide) & NAJ) (P) nicotinamide adenine dinucleotide phosphate) are coenzymes that conjugate with various dehydrogenases, and are important compounds in the field of clinical chemistry. .

As a dehydrogenase conjugated with NAD'& NADP,
Lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glycerol dehydrogenase, formaldehyde dehydrogenase,
glutamate dehydrogenase, cholesterol dehydrogenase,
Examples include malic acid dehydrogenase, hydroquinosteroid dehydrogenase, leucine dehydrogenase, and diaphorase.

Therefore, by combining these dehydrogenases with the above-mentioned coenzymes, or by combining them with other appropriate enzymes depending on the abundance, for example, glucose, glycerin, neutral fat, urine h1, creatinine, creatine, Ammonia, urea electrons, cholesterol, bile acids, steroids,
Measuring the content of lactic acid, pyruvic acid, etc., or measuring twance aminase activity or aminopeptidase activity
I can be determined.

From the opposite point of view, it is also possible to measure dehydrogenase activity by combining the coenzyme with an appropriate group 'R'.

These measurements are performed in front of a water bath, and the pH is adjusted to near neutrality, but generally the pH is adjusted to around neutrality.

NAD and oxidized NADP& are stable under acidic solutions, while the original form NAI)H and the reduced form NADPH
is stable in the alkaline region F, and it is thought that the V constant value in the vicinity of neutrality is at most tX- for one day. The above-mentioned coenzyme group, which is in a solid state, is
It is known that the coenzyme exhibits good storage properties as long as water, oxidizing agents, reducing agents, etc. are excluded, and the coenzyme is provided in a solid form. Therefore, when using it, set it as the water-soluble nf, adjust it to the optimum p II suitable for the measurement system, adjust W and '4, and use it as a standard. Either discard it or change the oxidized type to acidic, and the reduced type to alkaline to increase storage stability, and temporarily return to the optimal PHK near neutrality. Ta.

However, such a method is foolish because the /1β◇ffl operation for measurement becomes ρJ y++; more than necessary, and if the storage conditions are incorrectly set, the stability of the coenzyme may decrease.
This has the disadvantage that the 111 accuracy itself is reduced.

The present invention solves this situation with D! The purpose of this study was to improve the stability of the coenzymes in a solution near neutrality. However, Akira Kokuma's drawing ['17 element'] game determination method, which is suitable for this purpose, is
pH6 containing D or NADP in oxidized or reduced form
, chelating agent and azide c in a slow solution of 5 to 9.0
The gist of this is to add a ``mu'' stabilizer (hereinafter sometimes referred to collectively as a ``mu''stabilizer'').

Application of the present invention As is clear from the details in Section iiJ, the coenzymes that are the subject of application of the present invention are NAD of the oxidized type and the J/I type, as well as the coenzymes of the NAD The pH of the enzyme-containing aqueous surface was set at 6.5 to 9.0 because the optimum pH of the dehydrogenase falls within this range, and the M'S pH
This is because within this range, the stability of the coenzyme is low, and the stability-improving effect of the chelating agent and azide becomes significant.

In order to maintain the p II of the water bath solution within the range of 11. It may have any composition as long as it can be used by anyone. Examples of particularly suitable materials include, for example, phosphoric acid tα, halogen acid, and tris chloride B (U〆f If the liquid is thin or inorganic, the liquid may be
For 1 m degree of use of e II m, lO ~ 500 t
Approximately nM is appropriate.

The chelating agent used in the present invention may be of any type, as long as it is a general metal chelating agent for A11 and can capture and inactivate gold-14 ions. Typical examples include ethylenediaminetetraacetic acid and its fruit. Next, regarding azides, there are those that have the function of preventing il (alteration and deterioration of enzymes) caused by mixed microorganisms, and have a dramatic +S'p bactericidal effect on microorganisms, or those that inhibit the activity of microorganisms. I believe that you can use any kind of material as long as it is activating.
It was specially selected from among those that had been tested with azides, antibiotics, sulfa drugs, and other anti-inflammatory agents. That is, among the compounds vA group, antibiotics, sulfur
It was found that V-heavy effect was hardly obtained with V-1 and other commonly used home remedies, and only azide was effective; examples of such azide include sodium azide and potassium azide. be done.

However, the effects of chelating agents and azides were insufficient even when used alone, and results were obtained only when used in combination. In such a case,
The strength of the V-electrifying effect or the chemical stability of the female-determining agent itself can be determined by taking into consideration the combination of the above-mentioned ``determining agents'', pH, etc., but the general As a guideline, chelating agent is 0.01-10W/V'J, azide is 0.00
1 to IW/V%, and if it is less than the above range, the hair fixing effect will be insufficient, and if it is in excess, it will only be uneconomical for the nails and is not desirable.

In NIM preparation of the stabilizer-containing trap INN cumulative surface as described above, the stabilizer is dissolved in a buffer solution with a pH of 6.5 to 9.0, and then the coenzyme is added in the form of a coenzyme or Either by pre-mixing the coenzyme and the stabilizer in solid or solution form, or by pre-mixing the coenzyme and the stabilizer in solid or solution form.
Next, it is advisable to add a soothing solution with a p value of 116.5 to 9.0 to moisten it. The liquid thus prepared may be stored as it is in the hoof shape, but if necessary, it can also be converted into a solid form by freeze drying or blow drying and then stored.

Since the present invention is made of the above-mentioned cedar, the pH is 6.5~
The stability of the coenzyme in a buffer solution of 9.0% is excellent, making it easy to measure each component in body fluids using hydrogenated enzymes. As a result, he has been able to make significant contributions to the field of clinical diagnosis.

The present invention will be explained below with reference to Examples.

Example 1 0,1 M phosphoric acid 11 tur(i (p II 7.5
) NA to 100me) (oxidized type) 50 mf
Add and dissolve the coenzyme solution, add the additives listed in Table 1, adjust the pH to 7.5 for 4 times if necessary, store the coenzyme solution at room temperature, and adjust the residual activity of each to 11]. Established. The results are shown in Table 1, and the storage stability was significantly improved when a chelating agent and azide were used together, whereas the storage stability was significantly improved when each was used alone or in combination with other general-purpose preservatives. No improvement in storage stability was observed for
.

The measurement of NADi in this example was determined by applying alcohol dehydrogenase to ethanol as a substrate and determining the change in absorbance JJt (840 nm) when the remaining NAD was converted to NAD)I.

Example 2 0.05M) Squirrel buffer i&(p 118.0) 10
Add 15fng of NADPH (reduced form) to 0me and 1f
l If L, the M5 additive shown in Table 2 was added thereto, and the coenzyme solution was stored at room temperature after adjusting to +1 II 8.0 if necessary.

The residual rate of each NADP II was determined by directly measuring 10 absorption traces of 840 songs. The results are shown in Table 2, and only the combination of a chelating agent and azide was able to suppress the headache.

Day Example 3 An enzyme preparation having the following composition was prepared. .

Creatine amidinohydrolase 20 +1
11th place (from Pseudomonas 1) Sarcosine oxidase 80-Tli
(from Corynebacterium) Formaldehyde dehydrogenase 8 units (from Pseudomonas) N A, D (j'Wi type)
7.2*+y Nonionic surfactant
I Q mq Sodium azide 1
0 mqEDTA-8Na 1
0mg of this was dissolved in 0.05M phosphoric acid solution (PH7,5) to make the total amount 10mg. The nonionic surfactant in the above composition was blended with the aim of stabilizing creatinamidinohydrolase, but a surfactant with one extra amount of sodium azide and E J> T A -8Na was prepared in the same manner from the above composition. , 0 to 05 M phosphoric acid to make 10 mg of whole violet. Each solution was incubated at 25°C for 6
The one stored for 1m per day and the one made by 11j, 1
50ml of a sample containing creatine was added to
The reaction was carried out at 87°C for 10 minutes. After the reaction, the NADft, which changed in proportion to the amount of creatine in the sample, was
The amount of creatine in the sample was determined by determining ff1J as the absorption of H at N (840 nm) i.

-Prepare sample φ5 (for sample blank) obtained by removing creatinamidinohydrolase from Manjo if enzyme agent, and
The sample blank value was determined by processing j7 in the same manner as ν, and the amount of creatine in the page was determined from the difference.

The results are as shown in Table 8. 1. If the present invention is satisfied, the results will not stop even after 16 days. Il! Those who have given a fixed result but do not satisfy the conditions of tree 1 to light, 6 days later fIjl
1゜958- where J constant was not 1-1f function

Claims (1)

[Claims] A chelating agent is added to a laxative solution having a pH of 6.5 to 9.0 containing one or more trapping molecules selected from nicotinamide adenine dinucleotide 1 and nicotinamide adenine dinucleotide phosphoric acid in oxidized or reduced form. and a method for V-polymerization of coenzymes, characterized by containing an azide.