JPS608800B2 - How to measure peroxidase activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel and highly sensitive method for measuring peroxidase activity using crab photoanalysis.
Various peroxidases derived from plant and animal tissues are already known as enzymes that catalyze the oxidation reaction of
Lactoberoxidase (Lacto) contained in milk
Cytochrome C peroxidase (Cytochrome C peroxidase contained in yeast)
oxidase), peroxidase produced by enterococci, and weld peroxidase (Verdoperxjdase) contained in leukocytes and eosinophilic cells of the bone marrow.

The peroxidases used in the present invention include these various types of peroxidases, but plant-derived peroxidases are particularly important. Plant peroxidase is widely distributed in various plant kingdoms, such as the roots of horseradish, radish, turnips, leaves of fava beans and spinach, and the stems of wheat. Furthermore, regardless of the type of plant that is the source of plant peroxidase, the prosthetic group heme is all protoheme, and based on the electrostatic state of the enzyme protein part, it can be acidic, neutral, or basic. The properties of peroxidases, such as being broadly classified into enzymes, are extremely common, and in particular, peroxidase derived from horseradish (hereinafter abbreviated as HRP) is usually considered to be the most representative.

Plant peroxidase For example, HRP has been extracted, separated and purified since the earliest times, and has been used in biochemical analysis methods for the direct quantification of H202 and substrates used, as well as &02 from measurement targets such as sugars, amino acids, organic bases, and cholesterol. It is widely used in the field of clinical chemistry to aim at determining the characteristics of a measurement target by combining the oxidase reaction produced and the quantification using peroxidase.

Therefore, by being able to measure peroxidase activity with high sensitivity, we can not only detect peroxidase itself with high sensitivity, but also use the measurement principle to determine the correlation between peroxidase itself and its inhibitory power depending on the substrate and necessity. It has become possible to measure the amounts of various enzyme inhibitors and the like, and also to measure ultra-trace amounts of the above-mentioned target analytes based on trace amounts measured on a day-202 basis, which is of great significance in terms of clinical diagnosis. On the other hand, enzyme-linked immunoassay and enzyme-linked immunoassay for detecting antigens using immunohistochemical techniques, which have been rapidly developed in recent years as a method for measuring ultra-trace amounts of in-vivo components, are used to detect antigens and antibodies. There are many cases in which peroxidase such as HRP is utilized as a labeling enzyme. In the former enzyme immunoassay method, as is clear from the measurement system, if the activity of the labeled enzyme can be measured with high sensitivity, it is possible to measure the amount of the labeled enzyme, and as a result, the amount of the labeled enzyme can be measured. Ultratrace quantification of antigen and antibody is achieved. This logic also applies to enzyme antibody methods that use plant peroxidase such as HRP as the labeling enzyme, and is useful when tracking the localization of surface antigens and internal antigens in microscopic tissues in more detail in smaller samples. is also helpful. Establishing a highly sensitive measuring method for peroxidase activity will not only facilitate the precise measurement of the amount of peroxidase enzyme, but also facilitate the measurement of minute amounts of oxidizable substrates or enzyme inhibitors. As described above, a wide range of applications is possible in the field of biochemical or enzyme immunochemical measurement of biologically detected components, the field of immunohistochemistry, etc. The conventional method for measuring the activity of various peroxidases including HRP is the visible absorption method, but a common characteristic of the visible absorption method is that its measurement sensitivity is generally quite low. was there.

On the other hand, Guilbout et al. previously reported a new crab optical analysis method using homovalinic acid as a substrate [Guilbout et al.
,et,al:Anal,Chem,39(No2)2
71 (1967), ibid, 40 (Nol) 190~
196 (19 porridge), ibid, 40 (No8) 1256
~1263 (1968)] This crab photoanalysis method involves converting homovanillic acid, a stable non-carifluorescent substrate, into a highly photochromic substance by the coexistence of 202 in a neutral to weakly alkaline solution. Peroxidase activity is determined by measuring the intensity of light, and although the measurement sensitivity is generally higher than when using the conventional visible absorption method, it is still insufficient in many cases where peroxidase is used. It was there.

Under these circumstances, the inventor of the present invention aimed to improve the shortcomings of the above-mentioned known methods.As a result of intensive research, the inventors of the present invention have discovered that a halophotogenic substance is produced by the action of peroxidase on a non-caerilescent substrate in the coexistence of hydrogen peroxide. In a method for measuring peroxidase activity using crab photoanalysis, whose measurement principle is to determine peroxidase activity based on the halo intensity shown by For example, compared with the conventional method, for example, the most representative method in this field, the method of Gilbout et al., by performing a reaction in which homovanillic acid is used as a substrate and peroxidase is activated in the presence of hydrogen peroxide to produce a haloscopic substance. The inventors have now discovered that peroxidase activity can be measured with extremely high sensitivity, accuracy, and reproducibility of results, leading to the completion of the present invention. That is, the enzyme reaction in the present invention is performed under acidic conditions.

In this case, the pH should be 7 or less, but in order to obtain the highest sensitivity, it is preferable to select a specific pH range under acidic conditions. pH 3.5 to 5.0 is adopted depending on the type. The protein used in the present invention does not have peroxidase activity,
Any protein may be used as long as it does not have an inhibitory effect on peroxidase, such as serum proteins derived from humans and animals (alpmin and various globulins), animal proteins (casein, gelatin, egg albumin, etc.), and protein materials. The protein is selected from among enzymes (egg white lysozyme, aldolase, chymotrybusinogen A, etc.), other plant proteins, proteins produced by microorganisms, etc., but bovine serum albumin (abbreviated as plaque A) is usually used. Often.

Similarly, the peptide may be any peptide as long as it does not have peroxidase activity and has an inhibitory effect on the expression of peroxidase activity, and may be either chain or cyclic peptides. . In practice, a partially hydrolyzed protein, a chemically synthesized protein, or a peptide antibiotic as a peptide material is used. The amount of these proteins and peptides used can be extremely small, and although the amount used varies slightly depending on the type of protein or peptide used, the time of addition, etc., there are usually several amounts in the test tube for measurement. It is sufficient that the amount is present in an amount of 1.5 g or more, and no particular disadvantage is observed even if an extremely large excess amount is used. The proteins and peptides used here may be used alone or in combination of two or more. There is also no particular restriction on the timing of making these proteins and peptides present; for example, they may be added just before the enzymatic reaction is completed, or they may be added in advance at the beginning of storage of the peroxidase solution or enzyme preparation and kept at a constant level. After storage for a period of time, the solutions and preparations may be used as they are in carrying out the present invention.The details of the effects of adding proteins and peptides, which form the gist of the present invention, will be shown in Examples below. Experimental example 1: Relationship between enzyme action PH and effectiveness of plaque A addition 0.1M - citrate - Na 407 buffer (pH 4.0) 0.08M - Na ratio P04 - NaoH buffer (pH 7.0) 0.1M - Tris(hydroxymethyl)aminomethane one day CI buffer (
pH 8.50) 500 yen HRP (Type V1)
, RZ: Approx. 2.75, Activity: Approx. 26 Misaki MPMO Hoko 11i
n Units / 9 ri SigmaChemi
CalCo (purchased from the US) 1 unit/private liquid 10〃
In the evening, add 20 liters of water solution or pure water with an AI of 2.5,
0.25% homovanillic acid aqueous solution 50 overnight, 0.05%
37. The reaction mixture containing 50 m2 of 202 liquid per day. 1. After incubating for 30 minutes while slowly incubating the monkey with o.
After adding 50 r of 25% KCN solution to stop the reaction and adding 0.1N NaOH2.5', the crab light intensity was measured using a Hitachi spectrophotometer model 204 ( The results shown in Table 1 were obtained using an excitation wavelength of 32° and an excitation wavelength of 405°.

Table 1 Experimental example 2: Effect of addition of various proteins 0.1M-citric acid-Na2B407 buffer (PIH.
0) or 0.09M-NaQP04-NaOH buffer (pH 4.5) 500 μM, HRPI shown in Experimental Example 1
．． 1921 Nitsutonokite [liquid 10 butsuyu, protein solution 12.
The same operation as in Experimental Example 1 was performed using 20 pieces of rice on the 5th evening, and the results shown in Table 2 were obtained.

Table 2 ☆1: Purchased from Pharma Pharmaceutical (USA) ☆2: Purchased from Boehringer Mannheim (West Germany) Experimental example 3: Addition amount of protein 0.1M citric acid Na2B
Using 407 buffer solution (pH 4.0) with 500 ml of buffer solution, add 20 ml of a solution containing bovine serum albumin or milk casein in the amount listed in the table below, and then perform the same operation as in Experimental Example 1 to obtain the solution shown in the table below. An HRP activity value of 3 was obtained.

Table 3 Experimental example 4: Effect of addition of Satoshi A during storage of HRP solution
ymeGrade l-C, RZ: 3.4, activity: 36
Fertilizer mpmo saddle mnUni$/female) 1×10-1 to 1×
10-3 units/solution and BSA were added to this HRP solution at a final concentration of 11.2-12.4 and both were heated at 4℃.
After leaving the samples in a refrigerator for 6 hours, the HRP activity of the 10 samples was measured according to Experimental Example 1, and the results shown in Table 4 were obtained.

However, when measuring HRP activity, correction was made by adding so that the final concentration in the reaction mixture was 250 peaks/test tube for each group. Table 4 Experimental example 5: Relationship between incubation time and effect of adding plaque A using a pH 4.0 buffer solution and 250 mg of BSA/
The activity was measured over time in the same manner as in Experimental Example 1 with and without addition of the test tube, and the values shown in Table 5 below were obtained.

Table 5 As is clear from the results in Tables 1 to 5, the effect of adding protein during peroxidase activity measurement is remarkable when the enzyme action is carried out at an acidic pH; As long as it has a buffering capacity that can maintain pH, the buffering capacity will be exhibited essentially regardless of the type of acids and alkalis used to prepare the buffer.

On the other hand, under neutral or alkaline conditions based on the report by Gilbout et al. mentioned above, not only was there no significant addition effect, but the addition of the protein actually acted as an inhibitor.
The proteins or peptides used here include serum proteins such as BSA, animal-derived proteins, enzymes as protein materials, proteins with biological activity such as peptide antibiotics, and proteins with special activities. The source of the protein, its molecular weight, chemical structure, lot difference, and preparation method for protein preparations do not matter. Furthermore, it is also possible to use a suitable combination of two or more types of proteins. However, the effects observed with proteins cannot be expected from individual amino acids or mixtures of amino acids constituting these various proteins and peptides, and even inorganic ammonium salts. Furthermore, since pigment proteins containing metal atoms in their molecules, such as ferritin, myoglobin, and cytochrome C, generally act as inhibitors, it is desirable to reduce their use. The optimal amount of these proteins to be added varies slightly depending on the type of protein or peptide used, but usually 1 to 6 g or more in each test tube is sufficient, but even if extremely excessive amounts are used, There is no particular inconvenience.

Next, regarding the timing of addition of these proteins, as mentioned above, it is customary to add them when preparing the reaction mixture for measuring peroxidase activity, but there are also cases where they are added when storing the peroxidase solution or sample. . Particularly in peroxidase solutions and specimens to be stored, the amount of enzyme is more dilute, and the more dilute the enzyme is, the more advantageous it is to pre-add it during storage. In any case, it is sufficient that proteins and peptides are present at the time of measuring peroxidase activity, and as a result, a two-pronged effect of increasing peroxidase activity and suppressing deactivation can be expected. The phenomenon of increasing the sensitivity of peroxidase activity detection by adding protein or peptide 1, which is a constituent factor of the present invention, is due to the RZ value reflecting the degree of purification of the enzyme, prosthetic group heme protein, etc. In addition to various peroxidases and their respective isozymes, as well as native peroxidase itself, artificially chemically modified ones,
Furthermore, the same effect is observed with peroxidase labeled with various antigens and antibodies, and with immobilized peroxidase, as well as with various substrates such as p-oxyaromatic carboxylic acids such as homovanillic acid, tyramine, and tyrosine. . In practice, if this method is combined with other measures for measurement operations, such as using a microcell instead of the single cell that is normally used when measuring the intensity of light, even smaller amounts of samples can be measured. Therefore, the earlier application [Tokubuta 52-16]
By implementing the present invention in addition to [6518], a special high-performance, high-sensitivity crab light analyzer is no longer necessary, and an ordinary crab light analyzer installed in ordinary clinical laboratories etc. can be used. You can easily measure peroxidase activity, as well as various biological signature components, etc., with easier operation and in a shorter time.
The clinical significance of the present invention is extremely great as it allows for better measurement with better sensitivity and reproducibility.

Next, the present invention will be explained in more detail with reference to Examples.

However, the measurement examples shown below are merely examples of implementing the present invention, and do not limit the scope of application of the present invention as described above. Example 1: Measurement of HRP amount 0.1M-citric acid-NaB07 buffer (pH 4.0)
500 mU, HRP (same as used in Experimental Example 1)
1 x 10-2 to 1 x 10-3 units/10 tablets of desk fluid,
BSA (Fraction V, Annour Pharma
Purchased from the US to ceutje-aICo) 12.5 claws 9
A reaction mixture containing an aqueous solution or pure water 20 ㎃, the same substrate solution as in Experimental Example 1, and a 202 ml solution was incubated for 55 minutes according to Experimental Example 1, and further a predetermined amount of KCN and NaOH solutions were added. After adding , the luminous intensity was measured using a 1.0-meter cell and the values shown in Table 6 below were obtained.

In other words, even under enzyme action pH 4.0, the HRP measurement limit in the protein-free plot was 5 x 10-5 units, whereas in the protein-added plot it was 1 x 10-5 units (38.5 pg in weight). It was possible to measure up to a very high level of HRP (equivalent to 20%). This value corresponded to an increase in sensitivity several times compared to the additive-free plot, and about 100-odd times as much as the previously described method of Gilbert et al. Table 6 Example 2: Determination of day 202 0.1M-citric acid-Na2B407 buffer (pH 4.
0) 500 liters of HRP (RZ: 3.4 standard in Experimental Example 4) 1 x 10-1 units/10 liters of mother A aqueous solution or pure water shown in Example 1 20 So, 0.
50 ml of 25% homovanillic acid solution and the following amount of H20
The same operation as in Experimental Example 1 was performed on an enzyme reaction mixture consisting of 50 test solutions containing 2, and the values shown in Table 7 below were obtained.

At this time, contain 0.225.01g/test tube and contain BSA.
The activity of each other group was shown as a relative value, with the crab luminosity when no additives were added as 100. That is, by carrying out the present invention, the detection sensitivity of HRP is generally 40 to 60% higher.It is also possible to easily and reproducibly measure a uniform amount of 10-8 g, compared to known measurement methods. Compared to both, it showed extremely superior measurement sensitivity and measurement accuracy.

Claims (1)

[Claims] 1. A method for measuring peroxidase activity by fluorescence analysis, which comprises carrying out an enzymatic reaction under acidic conditions and in the presence of a protein or peptide that does not have peroxidase activity.