JPH06116293A - Protein, antibody and hair modifier - Google Patents

Abstract

PURPOSE:To obtain an antibody useful for diagnosing damaged hair and to provide a hair modifier effective for protecting or improving hair injury, improving combability, softness, touch, etc. CONSTITUTION:This protein is an antigen of an antibody exhibiting immunological activity against a matrix protein existing in human hair and has the following physical properties and chemical constitution. The antibody is a protein induced by this protein as its antigen and the hair modifier contains the antibody. The antigen: 1. Molecular weight: 14,000-16,000. 2. Amino acid composition: Cys 4.5-14.1, Asx 1.5-5.5, Thr 5.8-16.2, Ser 12.1-16.1, Glx 9.2-13.0, Pro 8.5-16.5. Gly 6.8-15.2, Ala 2.2-4.0, Val 14.9-9.7, Met 0.0-1.5, Ile 2.3-3.1, Leu 1.6-5.6, Tyr 0.9-2.8, Phe 0.0-2.0, Lys 0-2.5, His 0-1.1 and Arg 6.1-12.0.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel protein, antibody,
And more particularly to a hair modifier, which more specifically relates to a hair modifier having the properties of protecting or improving damage to hair and improving combability, suppleness, texture and the like.

[0002]

2. Description of the Related Art Hair is damaged by physical and chemical causes such as shampoo, brushing, hair dryer, perm, and hair color, and the surface structure of hair is destroyed. By this destruction, split ends and split hair are produced, and the protein, which is a constituent component of hair, is eluted from the destroyed portion and gradually disappears from the hair. Thus, when the protein is eluted, the hair becomes thin and weak and becomes fragile. However, once the hair is damaged, it cannot return to its original state by its own power, and if left unattended, the hair damage progresses and split ends tend to become split ends.

Therefore, in order to keep beautiful and healthy hair, it is necessary to prevent damage and to repair the hair if damaged.

For this purpose, various protein hydrolysates derived from natural products and their derivatives have hitherto been used as hair protection components in hair cosmetics such as rinses and treatments. However, although these substances are highly safe, their adsorptivity to hair is extremely small, and since they are water-soluble, they are washed out and a sufficient effect cannot be obtained.

On the other hand, US 3987161 (1976)
Discloses a method of using an antiserum obtained by using hair particles themselves as an antigen to give a body sensation and improve the set holding power. However, even this method cannot repair damaged hair. In addition, there is a problem that the comb passage is deteriorated.

By the way, the hair is composed of three types of cells: a cuticle that wraps and protects the hair fiber from the outside, a cortex that is the hair fiber, and a central medula (hair medulla). The main constituents of cuticle and cortex include fibrous “keratin type intermediate filament constituent protein (or α-keratin fiber protein)”, non-fibrous “matrix protein”, and “cell membrane peripheral protein”. There are three.

There are three types of matrix proteins: an ultra-high sulfur-containing protein having an extremely high amino acid composition of cystine of 30% or more, a high sulfur-containing protein of about 20%, and a high tyrosine protein having a relatively high glycine and tyrosine content. It is known that most of them have a molecular weight of several thousand to 30,000.

This matrix protein, which is usually insoluble in water, is a protein that is solubilized by a reducing agent, a denaturing agent such as urea or SDS, or a chemical modification, and is also known to be present in nails, feathers and the like. Has been.

It has also been pointed out that this matrix protein is eluted from the inside of the hair when the hair is damaged by a perm or the like, as a result.

As a method for preparing the matrix protein, the disulfide bond of the matrix protein in the raw material is cleaved by reduction or oxidation to solubilize it, and then iodoacetic acid is added to the sulfhydryl group to make it water-soluble. A method of removing α-keratin by treatment such as spot precipitation is known.

Said et al. Separated these solubilized matrix proteins by C4 reverse phase chromatography [J. Chromatogr. , 324 (198
5), p. 65].

In addition, Katuumi et al. [Arch. De
rmatol. Res. , 281 (1989), 49
5 page], hair proteins were solubilized after S-carbamoylmethylation, and matrix proteins were separated by two-dimensional electrophoresis.

However, each of the separated proteins is a modified protein and is not a natural protein itself. Therefore, most antibodies that use these proteins as antigens recognize the modified portion of the protein, and have the drawback of low binding to hair.

A report on an antibody obtained using a matrix protein as an antigen has been published by Frater, “Aust. J. Biol. Sci., Vol. 29 (197).
6), p. 453], but this report discusses the reactivity of the antibody at the hair follicle and the root of the keratinization incompletely, and it is considered that the antibody does not bind to the completely keratinized portion. ing.

Lynch et al. Prepared monoclonal antibodies against matrix proteins, but recognized a wide range of proteins with a molecular weight of 10,000 to 25,000 and had low specificity.

Hair modifying agents using antibodies include those for hair particles [USP 3987161] and those for keratin [JP-A-4-29912, JP-A-4-414].
13], but since it is not a matrix antibody, it has weak specificity for matrix proteins.

At present, there is no index indicating the degree of hair damage. However, it can be used as an index of the degree of damage by using an antibody against each matrix protein that is eluted at an early stage of damage.

[0018]

Therefore, an object of the present invention is to provide a matrix protein purified without solubilization by modification of a protein such as carboxymethylation, an antibody having the protein as an antigen, and the antibody. It is to provide a hair modifier containing the same.

[0019]

[Means for Solving the Problems] The above-described object is to provide a protein which is an antigen of an antibody having an immunological activity against a matrix protein present in human hair, and which has the following physical characteristics, and the protein: It is achieved by an antibody as an antigen and a hair modifier containing the antibody.

Note 1. Molecular weight: 14,000-16,000 (14-16K) 2. Amino acid composition; Cys 4.5 to 14.1, Asx 1.5 to 5.5,
Thr 5.8 to 16.2, Ser 12.1 to 16.1, Glx 9.2 to 13.0, Pr
o 8.5 ~ 16.5, Gly 6.8 ~ 15.2, Ala 2.2 ~ 4.0, Val
4.9 to 9.7, Met 0.0 to 1.5, Ile 2.3 to 3.1, Leu 1.
6 to 5.6, Tyr 0.9 to 2.8, Phe 0.0 to 2.0, Lys 0.0
~ 2.5, His 0 ~ 1.1, Arg 6.2 ~ 12.0

In the present invention, examples of the raw material of the matrix protein include human body hair and hair, wool, feathers, nails and the like, but human body hair and hair are preferable in view of species differences, and hair is particularly preferable.

In order to purify the matrix protein from these raw materials, a known method may be used, and a method in which the disulfide bond of the matrix protein in the raw material is cleaved by reduction or oxidation treatment, solubilized and extracted is generally used. is there. In the case of the reduction treatment, various denaturing agents such as urea and guanidine hydrochloride are added in advance as solubilizing aids under alkaline conditions for solubilization. An insoluble substance is removed from these by filtration or centrifugation, and the matrix protein fraction can be obtained by collecting the fractions from which α-keratin has been removed from the resulting supernatant by gel filtration or ultrafiltration. .

The obtained fraction can be further separated by using an ion exchange resin or the like according to the difference in isoelectric point to purify the target protein. More preferably, it can be purified and recovered by a method such as eluting the target protein from the gel using the SDS-polyacrylamide precise molecular weight fractionation method.

The molecular weight of the protein of the present invention can be determined by, for example, SDS.
-Polyacrylamide gel electrophoresis (SDS-PAG
The value measured by E) is 14000-16000.

The amino acid composition of the protein of the present invention can be analyzed, for example, after acid hydrolysis by the post-column method using o-phthalaldehyde as a fluorescent chromophore.

The purified protein can be used as an antigen. At that time, it is preferable to appropriately reduce or remove low-molecular impurities such as denaturing agents by dialysis or the like. Further, an antigen may be obtained by insolubilizing this protein into an aggregate.

Further, the above protein may be fragmented with a proteolytic enzyme such as trypsin to be used as an antigen. As a result, when the antigen is reduced in molecular weight and the antigenicity is lowered, the antigen is used as a hapten by a known method, for example, a method of binding to bovine serum albumin or the like via a cross-linking agent such as carbodiimide. And it is sufficient. Also in this case, it is preferable to remove low-molecular impurities to obtain an antigen by dialysis or the like.

Next, the obtained antigen is used to immunize mammals, birds, etc. by a known method to produce an antibody.

Animals used for the production of antibodies include
Appropriate livestock such as cows, horses, sheep, goats, rabbits, mice and chickens can be selected.

The immunization can be performed by a conventional method such as subcutaneous injection, intraperitoneal injection, intramuscular injection, intravenous injection or the like, or a method such as nasal drop, eye drop or oral administration. The dose and period of the protein as an antigen may be appropriately selected so that the desired titer is obtained and the animal is not adversely affected.

Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) as required
Such an adjuvant may be used in combination with the antigen.

The antibody can be obtained from normal milk or colostrum of these animals, serum or egg yolk by a known method.

The antibody of the present invention may be a monoclonal antibody produced by a hybridoma obtained by a cell fusion technique. Further, it may be a product of a microorganism or a cultured cell into which an immunoglobulin gene cloned from the hybridoma is introduced.

The antibody obtained as described above may be an antibody fragment obtained by treating with an enzyme such as papain or pepsin to remove the Fc portion of immunoglobulin. Further, it may be a product of a microorganism or a cultured cell into which a fragment of immunoglobulin gene cloned from spleen cells or lymphocytes of an immunized animal has been introduced.

The antibody of the present invention is preferably a purified antibody, but may be an unpurified product containing other components (eg, serum, colostrum, etc.).

The antibody or fragment thereof obtained as described above is provided as an aqueous solution or a dried product obtained by operations such as freeze-drying and spray-drying.

The hair modifier comprising such an antibody can be prepared into various types of hair compositions depending on the form of application to the hair. That is, components that are usually used can be added within a range that does not impair the effects of the hair modifier of the present invention, and the form of the composition for hair includes shampoo, conditioner, styling foam, hair conditioner, and hair treatment. , Hair packs, hair liquids, hair tonics, permanent wave agents and the like.

The amount of the protein of the present invention or the antibody prepared from the protein in the composition for hair may be appropriately selected according to the administration form, and for example, 100 or more antibodies per 0.1% by weight concentration. What has a valency is 10
The value of 0 can be about 0.00001 to 10% by weight.

[0039]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but various analysis methods used prior to this will be described.

(SDS polyacrylamide gel electrophoresis) SDS-PAGE is carried out by a method such as Laemmli. That is, a crude purified sample or a purified sample (0.2 to 35 μg) described below was individually added to 2% SDS,
Treatment is carried out in 62.5 mM Tris-HCl buffer (pH 6.8) containing 5% 2 mercaptoethanol and 20% glycerol at 100 ° C. for 3 minutes.

Electrophoresis contains 0.1% SDS 7.
10% at room temperature in 5% separating gel and 4% concentrated gel
A 2 hours. In addition, as a molecular weight marker, phosphorylase (phosphorylas)
e, molecular weight 97400; 97K), bovine serum albumin (bovine serum albumin, molecular weight 66000; 66K), ovalbumin (ovalbu)
min, molecular weight 45,000; 45K), carbonic anhydrase
e, molecular weight 29000; 29K), soybean trypsin inhibitor (soy bean trypsin i)
nhibitor, molecular weight 20100; 20K), α-
Lactalbumin (α-lactalbumin, molecular weight 14200; 14K) (all manufactured by Pharmacia)
The band can be detected by staining with Coomassie Brilliant Blue (CBB).

(Reverse Phase Chromatography) Reverse phase chromatography is performed by a method such as Said. That is, proteins are carboxymethylated with iodoacetic acid, and Hi-
C4 high-speed reverse phase chromatography is performed using a Pore RP-304 column (manufactured by Bio-Rad). The eluent used for chromatography is 0.1% trifluoroacetic acid containing 0.1% trifluoroacetic acid and 90% acetonitrile, and the protein is eluted by a linear gradient.

(Western blotting) Western blotting is performed by the method of Towbin et al.
That is, the protein was developed by 18% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (manufactured by Atto). The antiserum used as the primary antibody was diluted 10 6 times. Blotting detection kit (Amersham, RPN23 streptavidin.
Rabbit IgG detection kit using biotinylated alkaline phosphatase complex) was used according to the protocol specified in the kit.

Example 1 (1) Protein Purification 400 mg of normal human hair was washed with a 2% aqueous solution of sodium polyoxyethylene lauryl sulfate (3 EO), and then 8
M urea, 0.2 M Tris-HCl buffer (pH 9.5) 50
The mixture was added to ml and stirred at 50 ° C. for 1 hour while passing nitrogen gas. This was ground using a Teflon homogenizer, and the extraction operation was further carried out for 1 hour. 20,000
After centrifugation at X g for 30 minutes, the supernatant was collected and passed through a 5 μm filter to obtain an extract containing matrix protein.

This was concentrated with an ultrafiltration membrane, and 2 m of Sephacryl S-200 equilibrated with a 50 mM Tris-hydrochloric acid buffer solution (pH 9.5) containing 8 M urea and 0.2 M-2 mercaptoethanol that had been passed through nitrogen gas. It was applied to the column. All the low-molecular-weight fractions (molecular weight of several thousand to 40,000) eluting after the α-keratin fraction of the main peak were collected. By repeating this operation, α-keratin is completely removed,
A matrix protein fraction was obtained.

The obtained matrix protein fraction was dialyzed against a 20 mM sodium acetate buffer (pH 4.3) containing 8M urea and 0.2M2 mercaptoethanol, and 20M acetic acid containing 8M urea and 0.2M2 mercaptoethanol containing nitrogen gas. Equilibrated with sodium buffer (pH 4.3). It was applied to a 10 cm CM Sepharose fast flow column. After applying to the column and washing with the same equilibration buffer,
Fractions eluting with equilibration buffer containing 1M NaCl were collected. Crude protein was obtained by this operation.

The crude protein was concentrated with an ultrafiltration membrane having a molecular weight of 3,000 and separated by SDS polyacrylamide gel electrophoresis. The separated gel was immersed in an aqueous solution of 0.25 M potassium chloride, and a band of 14 to 16 K (molecular weight 14000 to 16000) clouded by precipitation of SDS was cut out and ground using a glass homogenizer, and 8 M urea passed through nitrogen gas, 50 mM Tris-HCl buffer containing 0.2 M2 mercaptoethanol (pH 9.
Extracted all day and night in 5). By this operation, the protein of the present invention was obtained.

This was again concentrated with an ultrafiltration membrane and dialyzed against a physiological phosphate buffer solution (PBS) containing 7.4M urea and 0.1M2 mercaptoethanol to obtain a protein antigen.

(2) Amino acid analysis The results of amino acid analysis of the protein of the present invention are shown in Table 1.
The protein of the present invention includes Cys, Thr, Ser, Glx,
The protein was rich in Pro, Gly, and Arg.

[0050]

[Table 1]

(3) Reversed Phase Chromatography The results of subjecting the protein of the present invention to reverse phase chromatography (C ₄ column) are shown in FIG. The acetonitrile concentration was increased from 24% to 57% over 16 minutes and kept for 5 minutes. The protein of the present invention was recognized as a single peak at an elution time of 12 to 16 minutes.

Example 2 (1) Preparation of antiserum Using the protein of the present invention purified in Example 1 as an antigen, it was adjusted to 2 mg / ml in PBS containing 7.4M urea and 0.1M2 mercaptoethanol. And was mixed with FCA at a ratio of 1: 1 to obtain a water-in-oil type (hereinafter abbreviated as W / O type) emulsion. This was subcutaneously administered to the backs of two rabbits in an amount of 1 ml per bird. Two weeks later, the adjuvant was changed to FIA and the same administration was performed. E increase in antibody titer
After confirmation by LISA, 2 weeks after the final administration (8th week), blood was collected from the heart and centrifuged to collect serum (hereinafter referred to as anti-hair matrix protein antiserum).
Serum was also collected from non-immunized rabbits and used as a control antiserum. An antibody fraction was obtained from serum by the ammonium sulfate fractionation method according to a conventional method.

(2) Western blotting The results of western blotting with antiserum are shown in FIG. From this, it was observed that the antiserum prepared from the protein of the present invention specifically reacts with a protein of 14 to 16K (molecular weight 14,000 to 16000).

Comparative Example 1 (1) Preparation of Antigen Normal hair of a 28-year-old man was washed with a 2% sodium polyoxyethylene lauryl sulfate (3EO) aqueous solution and then air-dried. This was shredded with an electric razor, and fine hair powder was obtained through a 150 μm mesh.

50 mg / m of the above hair powder in physiological saline
l / suspended at a concentration of 1: 1 and mixed with FCA at a ratio of 1: 1 W /
It was an O-type emulsion. This was administered to 2 rabbits at a dose of 4 ml per animal intramuscularly in the buttocks, and after 1 week and 2
The same administration was carried out after a week. Two weeks after the final administration (4th week), blood was collected from the heart and centrifuged to collect serum.
Thus, an antiserum against hair powder like USP 3987161 (hereinafter abbreviated as anti-hair particle antiserum) was prepared. By this method, an antiserum having a desired antibody titer against the protein of the present invention could not be obtained.

Test Example 1 (Hair-binding property of antibody) It was proved by the indirect fluorescent antibody method that the antibody of the present invention binds to completely keratinized hair (hair shaft).

Female normal hair was cut to an appropriate length and frozen. A 6 μm-thick section was cut out from the frozen block with a cryostat and fixed on a non-fluorescent slide glass.

To this, an antiserum of the protein of the invention obtained in Example 2 or a control antiserum obtained from a rabbit that was not immunized was diluted 40-fold with PBS containing 10% goat serum and reacted. After that, it was washed with PBS three times for 10 minutes.

A rhodamine-labeled goat anti-rabbit IgG antibody was reacted as a secondary antibody and examined under a microscope.

Fluorescence was observed throughout the hair only in the specimen reacted with the protein antiserum of the invention (FIG. 3). This indicates that the antibody binds to keratinized hair.

Test Example 2 (Hair Breaking Strength Enhancement Effect) Whether or not the antibody prepared from the protein of the present invention enhances hair breaking strength was tested according to the following method.

Hair that has been permeated twice according to the usual method is treated with PBS or 0.5% antiserum solution for 3 times.
Immerse at 0 ℃ for 1 hour, wash with water for 10 minutes, then at 45 ℃, 1
It was dried for 2 hours. Hair tester (manufactured by LB Chemical Company) under the environment of 25 ° C. and humidity of 65%
Was used to measure the breaking strength. The results are shown in Fig. 4.
The antiserum prepared from the protein of the present invention clearly enhanced the breaking strength of hair significantly as compared with the control group (PBS, non-immune serum).

Test Example 3 (Diagnosis of Damaged Hair) The hair of a woman was bundled (18c
m, 5g), perm treatment is repeated 3 times according to the usual method,
Permed hair was created.

Next, 0.5 g was cut from the hair bundle,
Put in 10 ml of 07M phosphate buffer (pH 9.0),
The protein in hair was eluted by leaving it at 5 ° C. for 1, 2, 5, 24 hours. For comparison, normal hair was used and the other treatments were performed in the same manner as the above method.

The protein eluted from each hair over time was
Using the antibody of Example 2, it was quantified by the above-mentioned ELISA method. The results are shown in Fig. 5. It was found that the protein of the invention elutes more in perm hair than in normal hair, which indicates that the damaged hair diagnostic method using this antibody is highly sensitive and effective.

Test Example 4 (Effect of improving sensory aspect of hair) Bunch of female hair (18
cm, 5 g) and the perm treatment was repeated 3 times according to a conventional method to prepare perm-treated hair. Next, the perm-treated hair was immersed in 50 ml of a 0.3% PBS solution of the antiserum of Example 2 for 1 hour, washed with running water for 10 minutes, and air dried for 24 hours.

The obtained antiserum-treated hair was subjected to a sensory evaluation on combability, suppleness, and texture, which was better than the hair before treatment. As a result, the antiserum prepared from the protein of the present invention was found to be combed on , Suppleness, having the effect of improving the texture, anti-hair particle antibody, control antibody,
It had excellent properties not found in collagen hydrolyzate and PBS solution.

Application Example 1 (Shampoo) A shampoo was prepared using the antibody fraction obtained in Example 2. The composition is shown in Table 2. The values in the table indicate% by weight.

[0069]

[Table 2]

Compared to the shampoo having the same composition as in Table 2 except that the antibody of Example 2 was not added, this shampoo did not have dryness, could be easily combed, and had a good finish in smoothness and touch. .

Application Example 2 (Hair Treatment) A hair treatment was prepared using the antibody of Example 2. The composition is shown in Table 3. The values in the table indicate% by weight.

[0072]

[Table 3]

After being washed with a commercially available shampoo, when treated with the above hair treatment, compared to when treated with a hair treatment having the same composition as in Table 3 except that the antibody fraction was not blended, the hair was combed well. It had a good and moist feeling, and was particularly effective in repairing and improving damaged hair.

[0074]

EFFECTS OF THE INVENTION According to the present invention, it is possible to bind to completely keratinized hair with a strong affinity and specifically, and as a result, to increase the breaking strength of hair, and to make it useful for combing, suppleness, and texture. There is provided a hair modifier that can be modified to protect or improve hair damage. The antibody of the present invention is also useful for diagnosing damaged hair.

[Brief description of drawings]

FIG. 1 is a diagram of reverse phase chromatography of the protein of Example 1.

FIG. 2 is a diagram showing Western blotting with the antiserum of Example 2. In addition, (a) shows the results of dye staining, (b) shows the results of antibody staining, lane 1 shows 15K protein, and lane 2 shows hair keratin.

FIG. 3 is a diagram showing the results of a hair binding test of an antiserum (a) and a control serum (b) of Example 2.

FIG. 4 is a view showing the hair breaking strength of the antiserum of Example 2 (1; PBS, 2; non-immune serum, 3; antiserum of the present invention).

FIG. 5 is a graph showing a protein elution amount of healthy hair and permed hair, which was examined using the antibody of Example 2.

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[Procedure amendment]

[Submission date] August 23, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 3

[Correction method] Change

[Correction content]

FIG. 3 is a fluorescence photograph (drawing substitute photograph) showing the results of the hair binding test of the antiserum (a) and the control serum (b) of Example 2.
Is.

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 3

[Correction method] Change

[Correction content]

[Figure 3]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display area C07K 15/14 8517-4H

Claims (3)

[Claims] 1. A protein which serves as an antigen of an antibody having an immunological activity against a matrix protein present in human hair and which has the following physical properties. Note 1. Molecular weight: 14,000 to 16,000 2. Amino acid composition; Cys 4.5 to 14.1, Asx 1.5 to 5.5,
Thr 5.8 to 16.2, Ser 12.1 to 16.1, Glx 9.2 to 13.0, Pr
o 8.5 ~ 16.5, Gly 6.8 ~ 15.2, Ala 2.2 ~ 4.0, Val
4.9 to 9.7, Met 0.0 to 1.5, Ile 2.3 to 3.1, Leu 1.
6 to 5.6, Tyr 0.9 to 2.8, Phe 0.0 to 2.0, Lys 0.0
~ 2.5, His 0 ~ 1.1, Arg 6.2 ~ 12.0 2. An antibody having an immunological activity against a matrix protein present in human hair, which antigen is a protein having the following physical properties. Note 1. Molecular weight: 14,000 to 16,000 2. Amino acid composition; Cys 4.5 to 14.1, Asx 1.5 to 5.5,
Thr 5.8 to 16.2, Ser 12.1 to 16.1, Glx 9.2 to 13.0, Pr
o 8.5 ~ 16.5, Gly 6.8 ~ 15.2, Ala 2.2 ~ 4.0, Val
4.9 to 9.7, Met 0.0 to 1.5, Ile 2.3 to 3.1, Leu 1.
6 to 5.6, Tyr 0.9 to 2.8, Phe 0.0 to 2.0, Lys 0.0
~ 2.5, His 0 ~ 1.1, Arg 6.2 ~ 12.0 3. A hair modifying agent comprising an antibody having an immunological activity against a matrix protein present in human hair, which antigen is a protein having the following physical properties. Note 1. Molecular weight: 14,000 to 16,000 2. Amino acid composition; Cys 4.5 to 14.1, Asx 1.5 to 5.5,
Thr 5.8 to 16.2, Ser 12.1 to 16.1, Glx 9.2 to 13.0, Pr
o 8.5 ~ 16.5, Gly 6.8 ~ 15.2, Ala 2.2 ~ 4.0, Val
4.9 to 9.7, Met 0.0 to 1.5, Ile 2.3 to 3.1, Leu 1.
6 to 5.6, Tyr 0.9 to 2.8, Phe 0.0 to 2.0, Lys 0.0
~ 2.5, His 0 ~ 1.1, Arg 6.2 ~ 12.0