JPS6057838B2 - L-lysine quantitative reagent - Google Patents

Description

[Detailed description of the invention] The present invention relates to a reagent for quantifying L-lysine.

Specifically, the present invention provides reagents for quantitative analysis of L-lysine contained in foods, natural products, clinical test specimens, culture fluids, and the like. A feature of the present invention is that it utilizes L-lysine-α-ketoglutarate-E-transaminase (hereinafter referred to as enzyme I) and L-glutamate dehydrogenase (hereinafter referred to as enzyme Ⅰ), which are highly specific for L-lysine. Therefore, even in the presence of other amino acids, L-lysine alone can be easily extracted.
The purpose of this method is to perform colorimetric determination in a short period of time and with great accuracy.

Conventional methods for quantifying L-lysine include microbioassay method, chromatography/ninhydrin method, and pressure detection method using L-lysine decarboxylase.
They have the disadvantage of reacting with both D- and L-lysine,
It has the disadvantage that the operation is very complicated. The present inventors have conducted various studies on analytical reagents for use in colorimetric assays that do not have the above-mentioned drawbacks, can quantify only L-lysine, and are easy to operate. The present invention was completed by discovering that L-lysine can be quantified with high accuracy by using the reagent of the present invention containing the following.

Specifically, the present invention involves subjecting an analyte containing L-lysine to a transaminase action with Enzyme I and a sufficient amount of α-ketoglutaric acid in the presence of the coenzyme pyridoxal-5'-phosphate. α-aminoadipic acid δ-semi, aldehyde and L-glutamic acid are produced. The generated L-glutamic acid is produced by enzyme ■ and coenzyme (nicotinamide adenine dinucleotide (hereinafter referred to as NAD) or NAD7 osfade (hereinafter referred to as NADP)).
The dehydrogenation reaction in the presence of .alpha.-ketoglutaric acid, NADH2 or NADPH, and NH3 are produced.

generated NADH.

or NADPH. is an electron transfer intermediate, such as phenazine methosulfate (Phena2ir) emeth
sulfate) (hereinafter referred to as PMS) to generate reduced PMS.

The generated reduced PMS is tetrazolium (Tetra
2olium) salt to form bluish-purple to reddish-purple formazan (Form2an), which is quantified colorimetrically.

That is, in the presence of a sufficient amount of α-ketoglutaric acid,
Since an equimolar amount of L-glutamic acid is produced from L-lysine, and an equimolar amount of formazan is also produced, the amount of L-lysine in the analyte can be measured by measuring the absorbance of formazan.

This can be explained using a chemical formula as follows. The formazan produced here has a reddish-purple to bluish-purple color that is easy to identify, and since it is colorimetrically quantified, highly accurate analytical values can be obtained.

This is a unique effect of the present invention not found in other analytical methods.

In other words, in the conventional colorimetric method that uses yellow color development,
In the case of analysis of biological samples, etc., the blind value becomes high due to the bilirupin pigment in blood of similar color, which leads to a decrease in analysis accuracy. However, the present invention solves this problem. We succeeded in completely eliminating the defects.

As the tetrazolium salt used in the present invention, INT)
There are MΠ and NTB, and their chemical formulas, other names, and measurement wavelengths are summarized in Table 1.

In addition to the above-mentioned INT)MΠ and NTB, examples of tetrazolium salts that can be used in the present invention include neo-tetrazolium chloride (NeO-TetrazOliumchlOr
ide), m-NitroBlueTetrazolium chloride (m-NitroBlueTetrazOliumc)
hlOride), Tetra Nitro Blue Tetrazolium Chloride (TetraNitrOBBlueT)
etrazOliumchlOride), 2, 3, 5
-triphenyltetrazolium chloride (2,3,
51TriphenylTetrazOliumchl
Oride). The electron transfer intermediate used in the present invention is phenazine methosulfate (Phe
nazinemethOsulfate) or MeldOlaBlue can be effectively used. Enzyme 1 used in the present invention is Achromobacter liquidum (AhrOmOba) which has enzyme 1-producing ability.
cterIquidum) IFO3O8tun Flavobacterium fuscum (FlavObacterium)
fuscurll) IFOl2997, Flavobacterium flavescens (FlavObacteriumf
lavescens) is cultured in liquid, and Enzyme 1 is collected from the cells.

The form of use thereof may be a purified crystallized standard, or a partially purified enzyme may be used for any purpose. Enzyme 1 is EC class 2,6,1,36.

The enzyme ① used in the present invention is of microbial origin, N, AD
EC class (EC class) is used as a coenzyme.
s) 1, 4, 1, 2, also of microbial origin, N
Among those that use ADP as a coenzyme, E. Sea class (EC
class) 1, 4, 1, 4.

Furthermore, there are EC classes 1, 4, 1, and 3, which are derived from animals and use NAD as a coenzyme, and are commercially available as reagents.

All three types can be effectively used in the present invention.

The reaction temperature when using the reagent of the present invention is enzyme 1 and
The optimum temperature is preferably 30 to 40°C, especially 3rC.

The reaction time is preferably 6 times the required reaction time for enzymes 1 and 2, and the pH is 7.
．． 5~&5, especially PH8.2 is desirable. The quantifiable range of L-lysine hydrochloride using the reagent of the present invention is 0.05 to 1.0 μMole in the case of a standard reaction solution composition,
In this range, a linear relationship is established between the L-lysine content and the absorbance. In the case of the standard reaction solution composition used in the present invention, it is necessary to add a sufficient amount of α-ketoglutaric acid, and the quantitative range of L-lysine hydrochloride is 0.05 to
In the case of 1.0 μMole, α-ketoglutaric acid is 2
It is desirable to add 0 μM Moles. Enzyme 1 requires pyridoxal-5'' monophosphate as a coenzyme, so the reaction solution composition contains (the required amount of pyridoxal-5'')
``Add monophosphoric acid.

Further, necessary amounts of NAD or NADP and PMS are added to the reaction liquid composition in order to conjugate the coloring reagent tetrazolium salt. The pH of the reaction solution is maintained at 8.2 with a 0.2M potassium phosphate buffer. After the required reaction time has elapsed,
To stop the reaction, add 2N HCl in the required amount. As a result of the reaction, the coloring substance formazan is produced in proportion to the amount of L-lysine originally present in the analyte.
The maximum absorption value of formazan is determined depending on the type of tetrazolium salt used at that time.

By measuring the absorption value of this formazan at that wavelength using a spectrophotometer, the content of L-lysine in the analyte can be quantified.

Next, the operation of a colorimetric reaction using the analytical reagent of the present invention will be explained in more detail with reference to Examples.

Example 1 The composition of the enzyme reaction solution was adjusted as shown in Table 2 below, and INT was used as the coloring reagent tetrazolium salt.

Claims (1)

[Claims] 1 L-lysine-α- in the presence of α-ketoglutaric acid
L-, which is characterized by containing ketoglutarate-ε-transaminase, L-glutamate dehydrogenase, supplementary oxygen, an electron transfer intermediate, and a tetrazolium salt.
Reagent for quantifying lysine.