JPWO2011058958A1 - Composition for stabilizing hemoprotein and method for stabilizing hemoprotein using the same - Google Patents

Abstract

SUMMARY A novel method for stabilizing a hemoprotein and a composition therefor have been disclosed in which the effect of stabilizing a hemoprotein in a specimen is superior to known methods. A composition to be added to a sample that may contain heme protein to stabilize the heme protein contained in the sample has a dissociation constant (Kd1) of less than 10-9 M as an affinity for haptoglobin or heme protein. A derivative thereof and a low binding substance having a dissociation constant (Kd1) of 10-9 M or more as an affinity for heme protein, and the low binding substance is converted into a dissociation constant ratio in a molar concentration of the haptoglobin or a derivative thereof. It is contained between the molar concentration multiplied by 1/100 of (Kd2 / Kd1) and the molar concentration multiplied by 100 of the dissociation constant ratio (Kd2 / Kd1).

Description

  The present invention relates to a composition for stabilizing a heme protein contained in a sample such as stool and a method for stabilizing a heme protein using the same.

  In the fecal occult blood test, the subject himself or herself often dissolves the stool in a predetermined suspension and leaves it for several days. At this time, it may be exposed to high temperatures, and there is an urgent need to completely prevent denaturation / degradation of fecal hemoproteins at high temperatures.

In order to prevent degeneration / degradation of heme protein, a method of adding defatted albumin or a modified product thereof to a sample containing heme protein (Patent Document 1), a method of adding haptoglobin (Patent Document 2), other than human There is a method of adding the animal serum (Patent Document 3).
In addition, in order to prevent denaturation / degradation of heme protein, a method of adding non-human animal hemoglobin (Patent Document 4), a method of adding iron protoporphyrin, hematin, heme, and hemin (Patent Document 5), transferrin Various additives for stabilizing heme proteins have been invented, such as a method of adding ferritin (Patent Document 6) and a method of adding ferrocyan compound (Patent Document 7). However, the stabilization effect of heme protein in the specimen by a known stabilization method is not always satisfactory.

JP 2003-194825 A Japanese Patent Laid-Open No. 10-132824 JP-A-4-145366 JP-A-2-296149 Japanese Patent Laid-Open No. 5-281227 JP-A-8-262020 JP 11-166932 A U.S. Pat. No. 2,765,299 Japanese Patent No. 2683944

J. Am. Chem. Soc, 68, 459-475, 1946

  An object of the present invention is to provide a novel method for stabilizing a heme protein and a composition therefor, which are superior to known methods in stabilizing the heme protein in a specimen.

  As a result of diligent research, the inventors of the present application have (1) binding to haptoglobin and (2) heme protein, but the binding property is lower than that of haptoglobin and has an affinity for heme protein expressed by a dissociation constant. The present inventors have found that heme protein in a sample can be highly stabilized by adding a composition containing a substance having a specific range in a specific concentration ratio to the sample, thereby completing the present invention.

That is, the present invention is a composition for adding to a specimen having a possibility of containing a heme protein to stabilize the heme protein contained in the specimen, and a dissociation constant (as an affinity for haptoglobin or heme protein). and its derivatives Kd1) is less than 10 ^-9 M, dissociation constant as an affinity for the hemoprotein (Kd1) is contained low binding substance which is 10 ^-9 M or more, the low binding substance, said haptoglobin or Heme protein containing between the molar concentration obtained by multiplying the molar concentration of the derivative by 1/100 of the dissociation constant ratio (Kd2 / Kd1) to the molar concentration by multiplying by 100 of the dissociation constant ratio (Kd2 / Kd1) A stabilizing composition is provided. The present invention also provides a method for stabilizing the heme protein contained in the specimen, which comprises adding the composition of the present invention to a specimen having a possibility of containing heme protein.

  According to the present invention, a method for stabilizing a heme protein and a composition therefor are provided, which are more effective in stabilizing a heme protein in a specimen than known methods. By applying the stabilization method of the present invention, the heme protein in a specimen such as stool, which can be left at room temperature or a temperature higher than room temperature, is highly stabilized, particularly in occult blood tests and the like. More accurate test results can be obtained.

  As described above, the composition of the present invention contains haptoglobin or a derivative that satisfies specific properties (described later) as the first essential component. Haptoglobin is a protein having a function of preventing free hemoglobin from being lost in urine by binding to free hemoglobin in blood, and its amino acid sequence is also known. The amino acid sequence of human haptoglobin is described in, for example, GenBank Accession No. AAA88080. This amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing.

In the present invention, it is also possible to use a haptoglobin derivative having a dissociation constant (Kd1) of less than 10 ⁻⁹ M as an affinity for heme protein instead of or together with haptoglobin. The dissociation constant for heme protein (hereinafter simply referred to as “dissociation constant”) can be measured, for example, by a known method described in Non-Patent Document Iwanami Biology Dictionary 4th Edition (Iwanami Shoten). Specifically, it can be calculated from numerical values obtained by a dialysis equilibrium method, an ELISA equilibrium method, or a protein-protein interaction measurement method. Generally, the dissociation constant of human haptoglobin for human heme protein is 10 ⁻¹⁰ M.

As derivatives of haptoglobin,
(1) a protein having a sequence identity of 90% or more, preferably 95% or more, more preferably 99% or more, with the amino acid sequence of human haptoglobin shown in SEQ ID NO: 1;
(2) A protein having an amino acid sequence in which one to several amino acids are substituted or deleted, or one to several amino acids are inserted or added in the amino acid sequence of human haptoglobin shown in SEQ ID NO: 1 ,
(3) a protein comprising the protein of (1) or (2) as a partial region,
(4) The protein of the above (1) to (3) subjected to commonly used protein modification methods such as binding a polyethylene glycol chain to increase stability,
And the dissociation constant is less than 10 ⁻⁹ M, preferably 10 ⁻¹⁰ M or less.

  Regarding the derivative of (1) above, “sequence identity” means that both amino acid sequences are aligned so that the amino acid residues of the two amino acid sequences to be compared match as much as possible. What is divided by the number of amino acid residues is expressed as a percentage. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such alignment of sequences can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the homology (%) is the total number of amino acid residues in the longer sequence, and the number of amino acid residues matched. Is calculated by dividing. The 20 amino acids constituting the natural protein are neutral amino acids having low side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), and neutral amino acids having hydrophilic side chains (Asn , Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, Trp) It is known that protein properties often do not change if substitution is made between these groups. Therefore, when substituting amino acid residues in haptoglobin, substitution between these groups yields a high-affinity derivative for heme protein that satisfies the above-mentioned requirements for the dissociation constant for heme protein. Is likely to be.

  The concentration of haptoglobin or a derivative thereof (the total thereof when two or more are included) in the composition is preferably 0.01 to 0.2 μg / mL, more preferably 0.01 to 0.1 μg / mL. When the concentration of haptoglobin or a derivative thereof is within this range, the effect of stabilizing the hemoprotein in the specimen is particularly high, which is preferable.

The composition of the present invention comprises, as a second essential component, a low-binding substance having a binding property to heme protein but having a binding property lower than that of haptoglobin and a dissociation constant of 10 ⁻⁹ M or more. Including. Dissociation constant for the low binding agent, 10 ^-9 M or more, preferably, 10 ^-9 M~10 ^-5 M.

  Preferable examples of such a low binding substance include at least one substance selected from the group consisting of albumin, hemopexin, α-hemoglobin stabilizing protein (AHSP), and derivatives thereof. All of these proteins are well known, and their amino acid sequences are also well known. For example, the amino acid sequence of bovine serum albumin is described in GenBank Accession No. CAA76847 and the like, the amino acid sequence of human hemopexin is described in GenBank Accession No. AAA58678, and the amino acid sequence of human AHSP is GenBank Accession No. It is described in NP_057717 etc. Since various commercial products are available for these proteins, commercial products may be used. However, if the dissociation constant is in the above range, other heme protein-binding proteins can be used.

These protein derivatives can also be used as low-binding substances in the composition of the present invention as long as the dissociation constant is 10 ⁻⁹ M or more. Here, examples of the “derivative” include those similar to (1) to (4) described above for haptoglobin (however, the reference is a known amino acid sequence of each protein).

  As the low-binding substance, albumin or a derivative thereof is preferable, and among these, mammalian (except human) serum albumin such as bovine serum albumin, which is commercially available at a relatively low price, is preferable. As methods for preparing serum albumin, a heat shock method (for example, Patent Document 8) and a corn fraction V method (for example, Non-Patent Document 1) are known. In the heat shock method, a fatty acid such as caprylic acid is added at a final concentration of 0.0075 to 0.02 M, and then heated at 65 to 71 ° C. for 20 minutes or more to precipitate other proteins including antibodies and collect only serum albumin. . On the other hand, the corn fraction V method is a method in which serum albumin is extracted with ethanol without heating. In the present invention, when serum albumin is used, it is preferable from the viewpoint of the effect of stabilizing the heme protein that serum albumin prepared by the corn fraction V method, which does not receive a thermal history and has no fatty acid binding, is used. . Since serum albumin prepared by the corn fraction V method is also commercially available, a commercially available product can be preferably used.

  The composition of the present invention has a low binding substance at a molar concentration of haptoglobin or a derivative thereof (the total concentration when two or more thereof are included, the same shall apply hereinafter) of 1 dissociation constant ratio (Kd2 / Kd1) Contained between the molar concentration multiplied by / 100 and the molar concentration multiplied by 100 of the dissociation constant ratio (Kd2 / Kd1) (hereinafter, the concentration of the low binding substance is dissociated to the molar concentration of haptoglobin or its derivative) The value multiplied by the constant ratio is sometimes referred to as “dissociation constant ratio product ratio” for convenience). Here, kd1 is the dissociation constant of haptoglobin or a derivative thereof contained in the composition (when two or more of these are included, the average dissociation constants measured by the method described in the following examples) ), Kd2 is the dissociation constant of the low-binding substance (when two or more of these are included, the average dissociation constant measured by the method described in the examples below). The concentration of the low binding substance is determined by multiplying the molar concentration of haptoglobin or its derivative by 1/100 of the dissociation constant ratio (Kd2 / Kd1), and multiplying by 100 of the dissociation constant ratio (Kd2 / Kd1). Between concentrations, preferably between a molar concentration multiplied by 1/10 of the dissociation constant ratio (Kd2 / Kd1) to a molar concentration multiplied by 10 of the dissociation constant ratio (Kd2 / Kd1), more preferably Is between the molar concentration multiplied by 1/2 of the dissociation constant ratio (Kd2 / Kd1) to the molar concentration multiplied by 2 of the dissociation constant ratio (Kd2 / Kd1). When the concentration of the low binding substance is within this range, a high stabilization effect of the hemoprotein can be obtained.

  As described above, serum albumin can be preferably used as the low-binding substance, but the composition of the present invention preferably does not contain serum. Since serum contains various substances in addition to serum albumin, there is a possibility that the stabilizing effect of heme protein may be reduced by those substances, and since serum has quality variations among lots, This is not preferable because the reproducibility of the stabilizing effect of the heme protein is lowered.

  The composition of the present invention preferably further contains sodium azide and / or a protease inhibitor as an optional component in addition to the above-described essential components. Sodium azide is a compound commonly used in biochemical reagent compositions as a bactericidal agent. The concentration of sodium azide in the composition is usually about 0.01% (w / v) to 0.5% (w / v), preferably about 0.05% (w / v) to 1% (w / v). .

  Protease inhibitors are used to prevent degradation of haptoglobin, albumin, and the like, which are essential components of the composition of the present invention, by various proteases that may be contained in a specimen. Protease inhibitors include aprotinin, urinastatin, hirudin, soybean protease inhibitor, lima bean protease inhibitor, corn protease inhibitor, gabexate mesylate, camostat mesylate, nafamostat mesylate, 4- (2-aminoethyl) benzenesulfonyl hydrochloride Salt (AEBSP), E-64, leupeptin hemisulfate-hydrate, disodium ethylenediaminetetraacetate (EDTA), bestatin, pepstatin A, benzamidine, phenylmethylsulfonyl fluoride, antipine, chymostatin, phosphoramidon, etc. It can be illustrated. Protease inhibitor cocktails in which a plurality of protease inhibitors are mixed are sold by Nacalai Tesque, Cosmo Bio, Roche, etc., and are similarly used. The concentration of the protease inhibitor in the composition is usually about 0.001 μg / mL to 5 mg / mL, preferably about 0.002 μg / mL to 1 mg / mL.

  The composition of the present invention includes an aqueous medium as a medium for dissolving the various components described above, and the aqueous medium preferably includes various buffer solutions containing physiological concentrations of sodium chloride, such as phosphate buffered saline. Can be mentioned. Examples of the buffer solution include phosphate buffer solution, acetate buffer solution, citrate buffer solution, borate buffer solution, tartaric acid buffer solution, Tris buffer solution and the like. The pH of the buffer is preferably neutral (about 6.5 to 7.5).

  The composition of the present invention is added to a specimen having the possibility of containing a heme protein and used to stabilize the hemoprotein contained in the specimen. Examples of such specimens include, but are not limited to, stool, urine, tears, saliva, nasal discharge, semen, and the like. Among these specimens, the composition of the present invention has a high possibility of degeneration of heme protein. The specimen is collected in a specimen such as stool, urine, saliva, nasal discharge, semen, etc. It is particularly effective for stabilizing heme proteins. Examples of hemoproteins include hemoglobin, myoglobin, cytochrome, catalase, and the like. Usually, many tests require stabilization of hemoglobin.

  The amount of the composition of the present invention to be added to the specimen is appropriately selected according to the nature of the specimen, etc., but is usually about 0.001 to 0.1, preferably about 0.005 to 0.02, with respect to the specimen 1 in weight ratio. . The addition may be simply by adding the liquid composition of the present invention to the specimen.

  As specifically described in the following examples, by adding the composition of the present invention to a specimen, hemoproteins such as hemoglobin contained in the specimen are stabilized, and a more accurate test than in the past is performed. Is possible.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 and Comparative Example 1
(1) Preparation of fecal extraction solution
0.15M phosphate buffered saline (pH 7.2), 0.1% bovine serum albumin (Calbiochem, Corn Filaction V), 0.1μg / mL haptoglobin (from human, Sigma) at a predetermined concentration, 121 ° C Dissolve gelatin heated to 60 minutes for 0.2% (w / v), sodium azide 0.09% (w / v), and polyethylene glycol 6000 0.0025% (w / v), and extract this by stool extraction A solution was obtained.

(2) Preparation of test solution In the fecal extraction solution prepared in (1) above, human hemoglobin (manufactured by Sigma) is dissolved (or suspended) so that the stool of healthy persons is 0.3 mg / mL. The solution was stored on the day (day 0) at 30 ° C. and 35 ° C. for 3 days or 7 days.

(3) Measurement method Human hemoglobin in the test solution was measured by an aggregation method using commercially available anti-human hemoglobin antibody-sensitized magnetic particles. This method is a known method described in Patent Document 9, for example, and can be performed using a commercially available kit. After antigen-antibody reaction of human hemoglobin and anti-human hemoglobin antibody-sensitized magnetic particles in the test solution, the particles are collected magnetically at the bottom of the well of the microplate, and the state of the particle mass when the plate is tilted This is a method for determining a change. When human hemoglobin is contained in the test solution, particle aggregation occurs due to the antigen-antibody reaction, and when the plate is tilted, the state of the particle mass remains almost unchanged, but the test solution remains When human hemoglobin is not contained therein, particle aggregation does not occur due to antigen-antibody reaction, so that a particle mass flows out. Since the flow-out state of the particle mass correlates with the amount of human hemoglobin contained in the test solution, human hemoglobin in the test solution is quantified using this as an index. The smaller the amount of human hemoglobin in the test solution, the greater the assay count.

25 μL of the test solution prepared in (2) above and 25 μL of 0.2% anti-human hemoglobin antibody-sensitized magnetic particles (manufactured by Fujirebio) were added to a predetermined container, and stirred for 5 minutes with a plate mixer. Next, the container containing the test solution and the particle suspension was moved onto the magnetic table and attracted with a magnet for 1 minute. Immediately, the microplate was tilted at 60 degrees for 2 minutes, and the flow length of the particles was measured. The hemoglobin concentration in the specimen was calculated from a calibration curve created from the measured particle length of the flow of each standard hemoglobin solution. In Example 1, the dissociation constant ratio (Kd2 / Kd1) was 10 ⁻⁴ .

(4) Results The results are shown in Table 1 below (the cut-off value was 208). In the case of the test solution containing neither bovine serum albumin nor haptoglobin, the count increased at 3 ° C. or more at both 30 ° C. and 35 ° C., and was judged negative. In the case of a test solution containing bovine serum albumin and haptoglobin, the counts did not increase even at 7 ° C. for 7 days and remained positive. Addition of haptoglobin to bovine serum albumin improved the stability of fecal hemoglobin, and an effect that could not be achieved by adding bovine serum albumin alone was observed.

Examples 2-6, Comparative Examples 2-6
(1) Preparation of test solution To the stool extraction solution of Example 1, human hemoglobin (manufactured by Sigma) was added to 20 ng / mL, and haptoglobin (human-derived, Sigma) was added to 0 to A test solution added with 2.5 μg / mL was prepared.

(2) Measuring method It carried out similarly to Example 1.

Results The results are shown in Table 2 below. In this hemoglobin measurement, when the haptoglobin concentration was 0 to 0.16 μg / mL, the assay count did not increase and remained positive, but when the haptoglobin concentration was 0.31 μg / mL or more, the assay count increased and was judged negative. It was thought that this measurement system itself was suppressed by the addition of excess haptoglobin.

(1) Preparation of extraction solution
0.15M phosphate buffered saline (pH 7.2), 0.1% bovine serum albumin (manufactured by Calbiochem, Corn Filaction V), haptoglobin (human, Sigma) concentration up to 0.2μg / mL Dissolve gelatin to 0.2% (w / v), sodium azide 0.09% (w / v), and polyethylene glycol 6000 0.0025% (w / v) at different concentrations and heat-treated at 121 ° C for 60 minutes. This was used as a stool extraction solution.

Examples 7-12, Comparative Examples 7 and 8
(2) Preparation of test solution In the fecal extraction solution prepared in (1) above, human hemoglobin (manufactured by Sigma) is dissolved (or suspended) so that the stool of healthy persons is 0.3 mg / mL. The stool extraction solution prepared in the above (1) or the stool-extracted solution containing only 40 ng / mL of human hemoglobin (Sigma), the solution without stool on the day (0 day), 3 days or 7 days at 35 ° C What was preserve | saved for the day was made into the test liquid.

(3) Measuring method It carried out similarly to Example 1.

Results The results are shown in Table 3 below. By adding haptoglobin, the count did not increase even at 35 ° C. for 7 days regardless of the presence or absence of stool at any haptoglobin concentration (0.05 μg / mL to 0.2 μg / mL). Not only the stability of hemoglobin in feces but also the stability of fecal extract itself could be improved at least up to 35 ° C. for 7 days. The effect of adding haptoglobin was observed.

Examples 13 and 14, Comparative Examples 9 and 10
(1) Preparation of extraction solution
0.15M phosphate buffered saline (pH 7.2), 0.2% (w / v) gelatin, heat-treated gelatin at a haptoglobin (human origin, Sigma) concentration of 0.1 μg / mL at 121 ° C for 60 minutes Prepare a solution without adding 0.1% bovine serum albumin (Calbiochem, Corn Filaction V) or bovine serum albumin to 0.09% (w / v) sodium and 0.0025% (w / v) polyethylene glycol 6000. Was used as a stool extraction solution.

(2) Preparation of test solution In the fecal extraction solution prepared in (1) above, human hemoglobin (manufactured by Sigma) is dissolved (or suspended) so that the stool of healthy persons is 0.3 mg / mL. The stool extraction solution prepared in the above (1), or human hemoglobin (manufactured by Sigma) with only 40 ng / mL, a solution without stool, on the day (0 day) at 35 ° C for 3 days or The test liquid was stored for 7 days.

(3) Measuring method It carried out similarly to Example 1.

Results The results are shown in Table 4 below. When the stool extract was added only to haptoglobin, the assay count increased at 35 ° C. for 7 days without stool and a negative determination was made, and deterioration of the stool extract itself was observed with haptoglobin alone. When not only haptoglobin but also bovine serum albumin is added, the count does not increase even at 35 ° C for 7 days regardless of the presence or absence of stool, it remains positive, and not only the stability of stool hemoglobin but also the stool extract itself Stability could also be improved. The addition of both haptoglobin and fraction V bovine serum albumin was necessary for the stability of fecal hemoglobin and the stability of the fecal extract itself.

Example 15
It has been shown that a stabilizer for hemoglobin is effective when a mixture of haptoglobin having a high binding constant (1.00E + 10 L / mol) and serum albumin having a low binding constant (1.00E + 6 L / mol) is used. The complex formation rate was calculated from the initial hemoglobin concentration, the initial haptoglobin concentration or the initial serum albumin concentration and the binding constant. In addition, the molecular weight of hemoglobin is 64,500, and the initial hemoglobin concentration is shown in Table 6-1. The molecular weight of haptoglobin was 430,000, and the initial haptoglobin concentrations were 0.1 μg / ml and 0.05 μg / ml. The binding constant k of haptoglobin is 1E + 10 (1 / M).

Results When the haptoglobin concentration that contributes very strongly to the stabilization of hemoglobin in feces was 0.1 μg / mL, the complex binding rate moved from 30 to 64% according to the fecal hemoglobin concentration. In addition, even when 50% of haptoglobin was reduced in activity or bound to another substance, the complex binding rate moved from 16 to 45%. In addition, the molecular weight of hemoglobin is 64,500, and the initial hemoglobin concentration is shown in Table 6-1. The molecular weight of serum albumin was 68,000, and the initial haptoglobin concentration was 1 mg / ml. The binding constant k of serum albumin is 1E + 6 (1 / M).

Results When the serum albumin concentration contributing to the stabilization of hemoglobin in stool was 0.1% (1 mg / mL), the complex binding rate was almost unchanged at 94% regardless of the stool hemoglobin concentration.

  When haptoglobin and serum albumin contributing to the stabilization of hemoglobin in feces are mixed and used, it is considered that a complex of hemoglobin and haptoglobin or a complex of hemoglobin and serum albumin is formed. When only one of the complexes is extremely increased, it is assumed that it becomes an effect of a single stabilizer. Therefore, it is necessary to consider both the coupling constants and the concentration used.

Reference example 1
(1) Preparation of elution solution
0.25% (w / v) gelatin, 0.15M phosphate buffered saline (pH 7.2) heat-treated at 121 ° C for 60 minutes, 0.09% (w / v) sodium azide, 0.0025% polyethylene glycol 6000 (W / v) with 0.1% bovine serum albumin (Calbiochem, Corn Filaction V) or 0.1% bovine serum albumin (Sigma, heat shock) added or without bovine serum albumin added A solution was prepared and used as a stool extraction solution.

(2) Preparation of test solution In the fecal extraction solution prepared in (1) above, human hemoglobin (manufactured by Sigma) is dissolved (or suspended) so that the stool of healthy persons is 0.3 mg / mL. The stool extraction solution prepared in the above (1) or the stool-extracted solution containing only 40 ng / mL of human hemoglobin (Sigma), the solution without stool on the day (0 day), 3 days or 7 days at 35 ° C What was preserve | saved for the day was made into the test liquid.

(3) Measuring method It carried out similarly to Example 1.

Results The results are shown in Table 6 below. Compared to the stool extract without serum albumin, the increase in assay count of feces (-) at 35 ° C for 3 days was suppressed by the addition of heat shock bovine serum albumin. Furthermore, by using fraction V bovine serum albumin, the increase in assay count at 35 ° C. for 3 days was suppressed, and a positive determination was made. Fraction V bovine serum albumin was more effective than heat shock bovine serum albumin. Not only the stability of hemoglobin in feces but also the stability of fecal extract itself could be improved. However, the assay count increased at 35 ° C. for 7 days under any condition, and both were negative, and not only the fraction V serum albumin but also the stability of fecal extract itself as well as the stability of hemoglobin in feces It was insufficient.

Claims (9)

A composition for stabilizing a heme protein contained in a sample having the possibility of containing heme protein and having a dissociation constant (Kd1) of 10 ⁻⁹ as an affinity for haptoglobin or heme protein A derivative thereof having a dissociation constant (Kd1) of 10 ⁻⁹ M or more as an affinity for heme protein, and the low-binding substance in a molar concentration of the haptoglobin or a derivative thereof. A composition for stabilizing a heme protein comprising a molar concentration multiplied by 1/100 of the dissociation constant ratio (Kd2 / Kd1) to a molar concentration multiplied by 100 of the dissociation constant ratio (Kd2 / Kd1) .   The composition according to claim 1, comprising the haptoglobin or a derivative thereof in a concentration range of 0.01 to 0.2 µg / mL.   The composition according to claim 1 or 2, comprising haptoglobin.   The low-binding substance is at least one substance selected from the group consisting of albumin, hemopexin, α-hemoglobin stabilizing protein (AHSP), and derivatives thereof. Composition.   The composition according to claim 4, wherein the low binding substance is albumin.   The composition according to claim 5, wherein the albumin is bovine serum albumin prepared by the corn fraction V method.   The composition according to any one of claims 1 to 6, which does not contain serum.   The composition according to any one of claims 1 to 7, further comprising sodium azide and / or a protease inhibitor.   A method for stabilizing the heme protein contained in the specimen, comprising adding the composition according to any one of claims 1 to 8 to a specimen having a possibility of containing heme protein.