JP2828426B2 - Isoelectric focusing marker for fluorescence detection - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a marker for fluorescence detection isoelectric focusing used in a fluorescence detection isoelectric focusing method.

[0002]

2. Description of the Related Art When a particle having a charge on the surface of a molecule is suspended in an electrolyte solution and an electric current is passed, the particle moves in a direction opposite to the respective charges. And Linder, S .; E. FIG. The electrophoresis phenomenon has been used as a means of separating and analyzing substances since it was observed by Tis in 1937.
Since the completion of the device by elius, its application has rapidly expanded to this day.

[0003] In contrast to general electrophoresis, which utilizes the difference in charge state at a specific pH, in 1966, Vesterberg, O. et al. And Svensson, H .;
Since the development of ampholine, which is a synthetic ampholyte, the isoelectric focusing method using the phenomenon that the effective charge of each ampholyte becomes zero at a certain pH value and does not migrate was established. .

Here, the pH value at which the effective charge becomes zero is called the isoelectric point of the substance. In isoelectric focusing,
The sample is concentrated at a position equal to its isoelectric point in the pH gradient formed on the carrier for electrophoresis and stands still.

As described above, since a sample is concentrated and separated by focusing, it is used for separation and analysis of substances as an electrophoresis method having extremely high resolution.

In recent years, electrophoresis has been performed with an inner diameter of 5 to 100 μm.
Capillary electrophoresis, which is performed in a capillary made of fused silica with a length of 30 to 100 cm, has been devised. Since it has a high resolution for a very small amount of analytical sample, it can be used not only for proteins but also for inorganic ions. And the separation and analysis of low-molecular-weight compounds and nucleic acids.

Attempts have also been made to quantify the concentration by attaching a detector to one end of the capillary and detecting the separated sample components.

[0008]

Conventionally, as a detection method, a method of irradiating mainly with ultraviolet light or visible light and detecting from a change in the amount of light due to absorption of the light by a sample component (ultraviolet light) Visible detection method) is common.

However, this method is difficult to use in the above-described capillary electrophoresis method because the inner diameter of the capillary corresponds to the optical path length.

[0010] As a more sensitive detection method, a detection method in which an analysis sample is labeled in advance with a fluorescent substance, the separated sample components are irradiated with light, and the emitted fluorescence is detected to quantify the concentration (fluorescence) Detection method).

To label an analysis sample with a fluorescent substance for this purpose, when the analysis sample is a protein, a method of chemically bonding an amino group of the protein to a fluorescent substance is generally used. As a fluorescent substance to be bound, fluorescein isothiocyanate (fluo)
rescein isothiocyanate, FI
TC) or tetramethylrhodamine isothiocyanate (tetramethylrhodamine isothiocyanate)
thiocyanate, TRITC) and the like are known (Genori Ono, Yuichi Kanaoka, Fumio Sakiyama, Hiroshi Maeda, Chemical Modification of Proteins (below), Gakkai Shuppan Center, 1981).

On the other hand, even in the above-mentioned capillary isoelectric focusing method, the sample components separated by this fluorescence detection method can be quantified sufficiently, and their high resolution and detection sensitivity make it an ultra-sensitive protein analysis technique. Likely to be.

However, in isoelectric focusing, since the isoelectric point (pI) is estimated from the rest position of the separated sample in the carrier for electrophoresis, the pH formed on the carrier for electrophoresis is determined. It is necessary to define the gradient, for which a standard marker is required.

For this purpose, for example, proteins whose pI is known from the past (for example, trypsinogen (pI = 9.30), lentilectin (pI = 7.8)
0, 8.00, 8.20), human hemoglobin C (p
I = 7.50), human hemoglobin A (pI = 7.1)
0), horse myoglobin (pI = 7.00), human
Carbonic anhydratase (pI = 6.50), bovine carbonic anhydratase (pI = 6.0)
0), β-lactoglobulin B (pI = 5.10), phycocyanin (pI = 4.65), amyloglucosidase (pI = 3.50), etc. as such as the above markers can be generated from these proteins themselves. It is extremely difficult because the fluorescence obtained is very weak.

Therefore, it is considered necessary to provide a method of labeling the above-mentioned protein having a known pI with an appropriate fluorescent substance.

However, since the pI of a protein depends on the type of amino acids constituting the protein and the N-terminal amino group (Seismological Chemistry Laboratory Lecture 2, Protein Chemistry (above), The Biochemical Society of Japan, 1987) ), By chemically modifying the N-terminal amino group of a commonly used protein with a fluorescent substance, the isoelectric point of the protein itself is greatly changed.

Further, since many amino acids capable of reacting with the fluorescent labeling substance are present in the protein, the number and position of the fluorescent labeling substance to be bound are unspecified. As a result, although one protein has a plurality of pIs, (Flatmark, T., Veste)
rberg, O.M. , Acta Chem. Scan
d. , 20, 1497-1503, 1966),
There is a problem that it is difficult to accurately determine the pH region formed in the carrier for electrophoresis.

Further, the tertiary structure of the protein is changed by the fluorescent substance labeling (Williamson, AR,
Kreth, H .; W. , Ann. N. Y. Acad. S
ci. , 209, 211-224, 1973), and the chemical stability of the protein itself deteriorates.

This poses a problem in terms of storage stability as a marker.

Therefore, in fluorescence detection isoelectric focusing, it is not possible to use, as a marker, a conventional protein having a known pI and labeled with a fluorescent substance.

The present inventors have conducted intensive studies in order to solve the above problems, and found that an oligopeptide having a fluorophore dye is
It shows a unique pI, covers a wide range of pI, and has excellent storage stability.
The present invention has been completed.

[0022]

SUMMARY OF THE INVENTION The present invention provides a method for isoelectric focusing by fluorescence detection, in which an appropriate oligopeptide is selected in order to accurately identify the pH region formed in a carrier for electrophoresis. The present invention relates to a fluorescent detection isoelectric focusing marker characterized by exhibiting a uniform pI by labeling with an appropriate fluorescent substance and having high storage stability.

That is, the present invention relates to a marker for fluorescence detection isoelectric focusing used in a fluorescence detection isoelectric focusing method, wherein the marker is an oligopeptide containing a fluorophore dye. It relates to a marker for electrofocusing.

The present invention also relates to a marker for fluorescence detection isoelectric focusing, characterized in that it is an oligopeptide having a fluorophore dye bonded to the N-terminal amino group via a bonding group. is there.

Further, the present invention relates to a marker for electrofocusing such as a fluorescence detection capillary, wherein the binding group is based on amide, thioamide, sulfonamide, urea, thiourea, urethane bond or the like. is there.

Further, in the present invention, the oligopeptide is at least an amino acid having a group having a negative charge by releasing a proton, and the acid dissociation constant of the group having a negative charge by releasing a proton is represented by K _j . n _j number of amino acids or an amino acid having a group with a positive charge by receiving a proton, and a n _i number of amino acids to the acid dissociation constant of the group with a positive charge by receiving the proton and K _i, Z = Σ
_i (n _i / (1 + K _i / [H ⁺ ])) − Σ _j (n _j /
(1+ [H ⁺ ] / K _j )), | Z | <0.0
The present invention relates to a fluorescent detection isoelectric focusing marker characterized by satisfying 3 <pI <11, where -log ([H ⁺ ]) when satisfying 1 is pI.

The present invention also relates to a marker for fluorescence detection isoelectric focusing, wherein the oligopeptide is angiotensin or a derivative thereof.

Further, the present invention provides an oligopeptide wherein Arg-
Arg-Val-Tyr-Ile-His-Lys, Arg-Arg-Lys-His-Tyr, Arg-
Arg-His-Tyr, Arg-Lys-His-Tyr, Arg-Arg-Val-Tyr-Ile-
Tyr, Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, Asn-Arg
-Val-Tyr-Val-His-Pro-Phe, Asp-Arg-Val-Tyr-Ile-His-
Pro-Phe-His-Leu, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
And Gly-Arg-Gly-Asp, which are at least one oligopeptide selected from the group consisting of Gly-Arg-Gly-Asp.

Further, the present invention provides a fluorophore dye selected from the group consisting of rhodamine, fluorescein, cyanine, indocyanine, indocarbocyanine, pyronin, lucifer yellow, quinacrine, squaric acid, coumarin, fluoroanthenylmaleimide and anthracene. The present invention relates to a marker for fluorescence detection isoelectric focusing, which is characterized in that

Further, the present invention relates to a marker for fluorescence detection capillary isoelectric focusing, which is a fluorescence detection capillary isoelectric focusing method as a fluorescence detection isoelectric focusing method.

Further, the present invention relates to the present invention, wherein the oligopeptide comprises at least Tyr-His-Lys-Arg-Arg, Tyr-His-Lys-Arg,
Asp-His-His-Arg, Asp-His-Arg, Arg-Arg-Val-Tyr-Il
e-His-Lys, Arg-Arg-Lys-His-Tyr, Arg-Arg-His-Tyr, A
rg-Lys-His-Tyr, Arg-Arg-Val-Tyr-Ile-Tyr, Arg-Val-
Tyr-Ile-His-Pro-Phe-His-Leu, Asn-Arg-Val-Tyr-Val-
His-Pro-Phe, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-
Leu, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Gly-Arg-Gl
y-Asp, Tyr-His-Arg-Arg, Tyr-Tyr-His-Arg-Arg, Ty
r-Tyr-His-Lys-Arg, Glu-His-Lys-Arg, Tyr-Tyr-His-
His, Tyr-His-His, Asp-Glu-Tyr-His-Lys-Arg, Tyr-
His, Asp-Asp-Asp-Asp-Asp-Arg, Glu-His-Arg-Arg,
Asp-Glue-His-Arg-Arg-Arg, Glu-His-His-His-Lys-Arg
, His-His-His-His-His-His, Asp-His-His-Arg, Asp
-Asp-Glu-His-His-His-Lys-Arg, Asp-Asp-Asp-Asp-Glu
-Glu-His-Arg-Arg-Arg-Arg-Arg, Asp-Asp-Asp-Glu-His
-His-Arg-Arg, Asp-Asp-Asp-Glu-Glu-Glu-His-Arg-Arg
And at least one selected from the group consisting of -Arg and Asp-Glu-Arg.

Further, the present invention provides an oligopeptide comprising at least Asn-Arg-Val-Tyr-Val-His-Pro-Phe, Asp-Arg-
Val-Tyr-Ile-His-Pro-Phe-His-Leu, Asp-Arg-Val-Tyr-
Ile-His-Pro-Phe, Gly-Arg-Gly-Asp, Tyr-His-Arg-Ar
g, Tyr-Tyr-His-Arg-Arg, Tyr-Tyr-His-Lys-Arg, Gl
u-His-Lys-Arg, Tyr-Tyr-His-His, Tyr-His-His, As
p-Glu-Tyr-His-Lys-Arg, Tyr-His, Asp-Asp-Asp-Asp-
Asp-Arg, Glu-His-Arg-Arg, Asp-Glue-His-Arg-Arg-A
rg, Glu-His-His-His-Lys-Arg, His-His-His-His-His-
His, Asp-His-His-Arg, Asp-Asp-Glu-His-His-His-Ly
s-Arg, Asp-Asp-Asp-Asp-Glu-Glu-His-Arg-Arg-Arg-Ar
g-Arg, Asp-Asp-Asp-Glu-His-His-Arg-Arg, Asp-Asp-
The present invention relates to a marker for fluorescence detection isoelectric focusing, comprising Asp-Glu-Glu-Glu-His-Arg-Arg-Arg and Asp-Glu-Arg.

[0033]

Embodiments of the present invention will be described below in detail.

(Oligopeptides) In the present invention, oligopeptides are used from various amphoteric substances usable as markers. In isoelectric focusing, most of the commonly used samples mainly contain proteins and the like, and therefore, in order to confirm the pI value of the sample, use an oligopeptide consisting of similar dissociation groups. Is preferred.

However, when the oligopeptide contains too many amino acids, the presence of a plurality of reaction sites and the mixing of by-products depending on the reaction conditions may occur when the group containing the fluorophore is bonded. It is possible that the resulting marker is not preferred because it does not give a single sharp peak.

Therefore, in the present invention, the number of amino acids forming the oligopeptide is not particularly limited, and may be any oligopeptide having an amino acid group sufficient to exhibit the desired pI.

Oligopeptides comprising amino acids as amphoteric compounds have various acidic and basic groups as acid-base dissociating groups, and N-terminal amino groups and C-terminal carboxyl groups. For example, as an amino acid group having an acidic group, aspartic acid, glutamic acid, tyrosine and the like, and as an amino acid group having a basic group, lysine,
Arginine, histidine and the like.

The oligopeptide exhibits a specific charge number at a certain pH due to these acid-base dissociation groups. Therefore, the number of charges of the oligopeptide as a whole can be calculated from the type and number of these acid-base dissociating groups.

For example, the charge number (Z) of the entire oligopeptide can be estimated by the following equation.

Here, an amino acid having a group having a negative charge by releasing a proton, wherein n _j amino acids having an acid dissociation constant of K _j of a group having a negative charge by releasing a proton, an amino acid having a group having a positive charge received, total number of charges oligopeptide containing a n _i number of amino acids to the acid dissociation constant of the group with a positive charge by receiving the proton and K _i (Z) _{is, Z = Σ i (n i} / (1 + K i / [H +])) - Σ j (n
_j / (1+ [H ⁺ ] / _Kj )) (Chemistry, 36 , 34-50).

Further, in this formula, -log ([H ⁺ ]) = pH at Z = 0 is the isoelectric point pI of the oligopeptide.

Therefore, by using this formula, it becomes possible to select an amino acid group necessary for an oligopeptide having a desired pI.

At this time, as a method of bonding a group containing a fluorophore, when the oligopeptide is bonded to the N-terminal amino group, basic dissociation by the N-terminal amino group is suppressed.

In order to make this estimation possible and to select a necessary amino acid group, the pK
As a, for example, α-carboxyl (C-terminal) (3.
6), β-carboxyl (Asp) (3.95), γ-
Carboxyl (Glu) (4.45), imidazole (His) (6.45), α-amino (N-terminal) (7.
6), thiol (Cys) (8.5), phenolic hydroxy (Tyr) (9.8), ε-amino (Lys)
(10.2), guanidium group (Arg) (12.5)
Etc. can be used.

For example, in an oligopeptide labeled at the N-terminus, aspartic acid or glutamic acid is selected to reduce pI, and arginine or lysine is selected to increase pI. Similarly, tyrosine or histidine may be selected to achieve a pI between them.

For example, when the pI is extremely small, the pI is close to 3 when at least five aspartic acids and one arginine are contained as an acid-base dissociating amino acid group.

Similarly, when at least two arginines and one lysine, one histidine and one tyrosine are included, the pI is 11 or more.

Similarly, by selecting various acid-base dissociable amino acid groups, a marker showing a desirable pI range can be constructed.

In the present invention, the range of pI is particularly preferably 2 or more and 13 or less.

In the present invention, the range of pI is particularly preferably 3 or more and 11 or less.

Further, in the present invention, the method for synthesizing an oligopeptide containing a desirably selected amino acid is not particularly limited. Synthesis by general synthesis methods (liquid phase method, solid phase method, etc. Basics and experiments of peptide synthesis, Nobuo Izumiya, Tetsuo Kato, Tohiko Aoyagi, Michinori Waki, Maruzen 1985
Year), or synthesis by an automatic synthesis method, etc. can be preferably used.

In the present invention, all or a part of natural product-derived oligopeptides can be suitably used as long as they have a necessary amino acid group and can control the reaction active site. It is.

For example, as an oligopeptide derived from a natural product that can be used in the present invention, angiotensin and its related substances (10 to 20 amino acids), bradykinin and its related substances, enkephalin and its related substances are preferably used. It is possible (Biochemical Dictionary (2nd edition), Tokyo Kagaku Dojin, 1990).

Further, it is possible to synthesize an oligopeptide having a desired amino acid group by chemically bonding amino acid groups necessary for the above-mentioned oligopeptide derived from a natural product.

(Fluorophore) The type of fluorophore usable in the present invention is not particularly limited as long as it contains a generally known fluorescent dye.

For example, rhodamine, fluorescein, cyanine, indocyanine, indocarbocyanine, pyronin, lucifer yellow, quinacrine, squaric acid,
Coumarin and fluoroanthenyl maleimide may be used.

In particular, rhodamine and cyanine dyes can be preferably used.

In the present invention, in particular, 5-carboxytetramethylrhodamine fluorophore (Handbook)
f Fluorescent Probes and
Research Chemicals, 5th Ed
ition MOLECULAR PROBES, IN
C. , 1992).

This rhodamine fluorophore has a maximum absorption at 546 nm (molar extinction coefficient 63,00) in methanol.
0) and has a fluorescence maximum at 579 nm.

Further, as a fluorophore which can be suitably used in the present invention, there is an aromatic heterocyclic compound or a polycyclic aromatic hydrocarbon (for example, fluorescent phosphorescence analysis, written by Taiji Nishikawa and Keizo Hiraki, Kyoritsu Shuppan). , 1989). For example, anthracene, naphthalene, phenanthrene, quinoline,
Pyrene, perylene and the like or derivatives thereof can be particularly preferably used. Providing these hydrocarbon fluorophores with an appropriate binding group enables them to be suitably bound to the oligopeptide.

(Binding Group) In the present invention, there is no particular limitation on the type of the binding group that binds the fluorophore group to the N-terminal of the oligopeptide. A group containing a fluorophore may be bonded to the N-terminal amino group without using a bonding group, or may be bonded via an appropriate bonding group.

In this case, it is preferable that the group containing the fluorophore is bonded to the N-terminal amino group by, for example, an amide bond, a thioamide bond, a sulfonamide bond, a urea bond, a thiourea bond, a urethane bond, or the like.

(Fluorophore dye group)-(bonding group) -N-terminal amino group The method for introducing the bonding group is not particularly limited. For example, in the case of an amide bond, the group containing the fluorophore dye preferably has an active ester group, an acid chloride, an acid anhydride, or the like.

For example, in the present invention, the rhodamine fluorophore group having an active ester group reacts with the N-terminal amino group of the oligopeptide to form an amide bond.

This reaction can be performed under mild conditions, and does not change other oligopeptide portions and fluorescent dye portions.

Further, when the oligopeptide has a plurality of reactive sites, only the N-terminal amino group can react, so that reaction conditions, reaction reagents and the like can be selected. In order to make this selection possible, general organic synthesis techniques can be suitably used.

For example, if necessary, protecting a reactive ε-amino acid (lysine) in advance is one of the preferable techniques.

In the present invention, the purity and storage stability of the obtained markers for fluorescence detection capillary isoelectric focusing can be confirmed by isoelectric focusing.

Further, if necessary, it can be easily purified using high performance liquid chromatography. In this case, efficient separation and purification can be achieved by using a reversed phase column, particularly, a packing material in which an octadecyl group is chemically bonded. 280 nm is a wavelength that can be suitably used for detection.

In the present invention, labeling is suitably carried out, for example, by bonding an amide bond to an angiotensin and an amino group of a peptide which is a derivative thereof to a rhodamine derivative.

(Measurement of pI) In the present invention, the pI of the marker for fluorescent isoelectric focusing is determined by performing isoelectric focusing.
It can be measured by comparing the mobility with a commercially available isoelectric focusing marker or by directly measuring the pH at the migration position of the labeled peptide.

For isoelectric focusing, slab (planar) gel isoelectric focusing using polyacrylamide as a support can be preferably used (Righetti, PG, Isoelectric Focu).
sing: Theory, Methodology and Applications, Elsevie
r, Amsterdam, 1983).

For detection by a staining method, a general method (for example, Coomassie brilliant blue-R250 staining) can be suitably used.

For detection by fluorescence analysis, a general method, for example, a method using a densitometer can be suitably used (for example, a Shimadzu dual wavelength flying spot scanning densitometer CS9300P manufactured by Shimadzu Corporation).
C).

(Capillary Isoelectric Focusing) There is no particular limitation on the capillary isoelectric focusing apparatus which can use the labeled oligopeptide according to the present invention as a marker.

In general, a peptide labeled with a fluorescent substance is concentrated by a capillary isoelectric focusing apparatus at a pH position formed on a carrier for electrophoresis in accordance with each pI and becomes stationary. Furthermore, these positions can be confirmed by detecting the fluorescence generated by the excitation light irradiation.

In the present invention, the excitation light source is not particularly limited, but it is particularly preferable to use a laser capable of stably irradiating light.

For example, in the present invention, a helium neon laser can be suitably used as excitation light for the rhodamine dye which is a fluorophore.

The pH gradient formed on the carrier for electrophoresis can be confirmed by a fluorescence detector provided at one end of the capillary.

As described above, the marker for fluorescence isoelectric focusing of the present invention is synthesized by binding a group having a fluorophore of an oligopeptide, and has a uniform pI and high stability. Having. Therefore, in fluorescence detection isoelectric focusing, it is a marker for high-sensitivity analysis when confirming the isoelectric point of the sample substance migrated based on the pH gradient formed on the carrier for electrophoresis.

Accordingly, the marker for isoelectric focusing on fluorescence detection of the present invention can be used in a capillary isoelectric focusing method.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples unless it exceeds the gist of the present invention.

[0083]

【Example】

(Synthesis reaction of fluorescent detection isoelectric focusing marker) Reaction of 5-carboxytetramethylrhodamine succinimidyl ester with oligopeptide The oligopeptide used in the reaction was purchased from Sigma, without further purification. It was used for the following reaction with 5-carboxytetramethylrhodamine succinimidyl ester. The oligopeptide used for the reaction was angiotensin I (aspartic acid-arginine-valine-tyrosine-isoleucine-histidine-proline-
Phenylalanine-histidine-leucine, Asp-Arg-Va
l-Tyr-Ile-His-Pro-Phe-His-Leu), angiotensin II (aspartic acid-arginine-valine-tyrosine-isoleucine-histidine-proline-phenylalanine, Asp-Arg-Val-Tyr-Ile-His-Pro -Phe), the derivative des-aspartate ¹ -angiotensin I
(Arginine-valine-tyrosine-isoleucine-histidine-proline-phenylalanine-histidine-leucine, Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu),
[Asparagine ¹ -valine ⁵ ] -angiotensin II
(Asparagine-arginine-valine-tyrosine-valine-histidine-proline-phenylalanine, Asn-Ar
g-Val-Tyr-Val-His-Pro-Phe), and a fibronectin fragment (glycine-arginine-glycine-aspartic acid, Gly-Arg-Gly-Asp).

Further, the following oligopeptides were obtained from Applie
d Synthesized based on the Boc method using an automatic peptide synthesizer manufactured by Biosystems (for example, see Chemistry Experiment Course 1, Protein VI, edited by The Biochemical Society of Japan, 1992), and the obtained oligopeptide was analyzed by high performance liquid chromatography. Purified. The conditions used for the high performance liquid chromatograph were YM
C-Pack ODS-AM (30mm inner diameter x 250m
length, YMC column, flow rate 20 ml /
The detection wavelength was 220 nm, and the mobile phase was acetonitrile gradient-0.1% trifluoroacetic acid. The gradient was set by the oligopeptide under the following conditions. Tyrosine-histidine-arginine-arginine (Tyr-His-Arg-Arg)
0.5% to 5%, 1% to 15% for tyrosine-tyrosine-histidine-arginine-arginine (Tyr-Tyr-His-Arg-Arg),
Tyrosine-tyrosine-histidine-lysine-arginine
(Tyr-Tyr-His-Lys-Arg) 1% to 15%, glutamic acid-
0.5% to 5% for histidine-lysine-arginine (Glu-His-Lys-Arg), 1% to 15% for tyrosine-tyrosine-histidine-histidine (Tyr-Tyr-His-His), tyrosine-histidine-histidine ( 1% to 10% for Tyr-His-His) and 1% to 1% for aspartic acid-glutamic acid-tyrosine-histidine-lysine-arginine (Asp-Glu-Tyr-His-Lys-Arg).
5%, 1% -15% for tyrosine-histidine (Tyr-His),
Aspartic acid-aspartic acid-aspartic acid-aspartic acid-aspartic acid-arginine (Asp-Asp-
0.1% to 15% for Asp-Asp-Asp-Arg), glutamate-histidine-arginine-arginine (Glu-His-Arg-Arg)
1% to 18%, aspartic acid-glutamic acid-histidine-arginine-arginine (Asp-Glue-His-Arg-Arg
-Arg) from 0% to 15%, glutamic acid-histidine-histidine-histidine-lysine-arginine (Glu-His-Hi
s-His-Lys-Arg) 0% to 15%, histidine-histidine-
Histidine-histidine-histidine (His-His-His-Hi
s-His-His) 0% to 10%, aspartic acid-histidine-
Histidine-arginine (Asp-His-His-Arg) 0% -5%,
Aspartic acid-aspartic acid-glutamic acid-histidine-histidine-histidine-lysine-arginine (Asp-Asp-Glu-His-His-His-Lys-Arg) 0% to 15%, aspartic acid-aspartic acid-aspartic acid-asparagine Acid-glutamic acid-glutamic acid-histidine-
Arginine-arginine-arginine-arginine-arginine (Asp-Asp-Asp-Asp-Glu-Glu-His-Arg-Arg-Arg-
Arg-Arg) 1% to 15%, aspartic acid-aspartic acid-aspartic acid-glutamic acid-histidine-histidine-histidine-arginine-arginine (Asp-Asp-
Asp-Glu-His-His-Arg-Arg) 0% to 15%, aspartic acid-aspartic acid-aspartic acid-glutamic acid-glutamic acid-glutamic acid-histidine-arginine-
Arginine-Arginine (Asp-Asp-Asp-Glu-Glu-Glu-Hi
s-Arg-Arg-Arg) 1% to 15%, and aspartic acid-glutamic acid-arginine (Asp-Glu-Arg) 0% to 4%.

The oligopeptide was prepared using 0.1M 2- (N-morpholino) ethanesulfonic acid (2- (N-Morpho)
lino) ethanesulfonic Acid,
(MES) -NaOH buffer (pH 6).

5-carboxytetramethylrhodamine
Succinimidyl ester (MOLECULAR PR
OBES) and dimethylformamide (dimet)
The solution was dissolved in H.sub.formamide to a concentration of 25 mM.

For 20 μl of the oligopeptide solution, 5
-Carboxytetramethyl rhodamine succinimidyl ester solution was added in 5 μl portions, and the reaction was completed at 27 ° C. overnight.

The reaction was stopped by adding 50 μl of 1 M Tris-HCl buffer (pH 8) to the reaction solution.

The labeled oligopeptide was separated and purified by high performance liquid chromatography under the following conditions.

Column: TSKgel ODS-80T _s
(4.6mm inner diameter x 25cm full length, manufactured by Tosoh Corporation) and TS
Kguardgel ODS-80T _s (3.2mm internal diameter × 1.5 cm total length, manufactured by Tosoh Corporation) Mobile phase: 5% to 55% acetonitrile gradient -0.1% trifluoroacetic acid flow rate; 1 ml / 1 minute Detection wavelength: obtained 280nm Chromatograms for the 14 types of fluorescent substance-labeled peptides and target peaks are shown in FIGS.

(P of 24 kinds of fluorescent substance labeled peptides)
I) The resulting 24 kinds of fluorescent substance-labeled oligopeptides were prepared using carrier ampholite (40% solution, pH 3.5).
-10, a polyacrylamide slab gel containing 2.5% of Pharmacia LKB (4.2% T, 4.8%)
C, 55 mm × 120 mm size, 0.5 mm thickness) and subjected to isoelectric focusing, using a commercially available isoelectric focusing marker (pI calibration kit 3-10, manufactured by Pharmacia Biotech). The mobilities were compared.

The electrophoresis conditions were as follows: after electrophoresis was started at a voltage of 50 mV, 100 V, 200 V, 400 V every 15 minutes.
V and 600V.

After completion of the electrophoresis, the fluorescent substance-labeled oligopeptide was irradiated with ultraviolet light through an orange filter, and photographed to confirm the electrophoretic position.

Thereafter, the gel was fixed with 20% trifluoroacetic acid for 20 minutes to determine the migration position of a commercially available isoelectric focusing marker, and then methanol 25%, acetic acid 1
Staining was performed for 20 minutes with a staining solution in which Selva Violet 49 (manufactured by Selva) was dissolved to a concentration of 0.05% in an aqueous solution containing 0%.

After staining, the migration position was confirmed by removing the background with an aqueous solution of methanol and acetic acid.

The migration of the obtained 24 kinds of fluorescent substance-labeled oligopeptides and commercially available markers for isoelectric focusing is shown in FIG.
7 are shown.

Table 1 summarizes the pI of each of the fluorophore-labeled oligopeptides obtained and the calculated value based on the formula (1). In this table, appropriately selected oligopeptides which will show a preferable various range of pI are also described for reference (the numbers are 25 or less).

(Table 1) pI measured and calculated values =================================== Number Structure Calculated value Measured value ------------------------------------------------------------------------------------ 1 Arg-Val-Tyr-Ile- His-Pro-Phe-His-Leu 8.28 8.20 2 Asn-Arg-Val-Tyr-Vyr-His-Pro-Phe 8.14 7.75 3 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu 6.46 6.65 4 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe 5.30 5.20 5 Gly-Arg-Gly-Asp 3.78 3.95 6 Tyr-His-Arg-Arg 11.00> 9.30 (Note 1) 7 Tyr-Tyr-His -Arg-Arg 9.80> 9.30 (Note 1) 8 Tyr-Tyr-His-Lys-Arg 9.62> 9.30 (Note 1) 9 Glu-His-Lys-Arg 8.34 7.65 10 Tyr-Tyr-His-His 6.46 6.70 11 Tyr -His-His 6.46 6.75 12 Asp-Glu-Tyr-His-Lys-Arg 5.56 5.55 13 Tyr-His 5.04 5.35 14 Asp-Asp-Asp-Asp-Asp-Arg 3.18 <3.50 (Note 1) 15 Glu-His- Arg-Arg 9.34 7.55 16 Asp-Glu-His-Arg-Arg-Arg 9.24 7.55 17 Glu-His-His-His-Lys-Arg 8.58 7.90 18 His-His-His-His-His-His 7.16 7 .10 19 Asp-His-His-Arg 6.46 6.55 20 Asp-Asp-Glu-His-His-His-Lys-Arg 6.18 4.70 21 Asp-Asp-Asp-Asp-Glu-Glu-His-Arg-Arg-Arg -Arg 4.88 4.90 22 Asp-Asp-Asp-Glu-His-His-Arg-Arg 4.62 4.55 23 Asp-Asp-Asp-Glu-Glu-Glu-His-Arg-Arg-Arg 4.26 4.05 24 Asp-Glu-Arg 3.66 <3.50 (Note 1) 25 Glu-His-Arg-Arg 25 Glu-His-Arg-Arg -Arg 12.20 ― 26 Tyr-His-Lys-Arg-Arg 11.28 ― 27 Glu-His-His-Lys-Arg-Arg 11.20 ― 28 Glu-Tyr-Tyr-His-His-Lys-Arg-Arg-Arg 10.24 ― 29 Glu-His-His-Lys-Lys-Arg 10.20 ― 30 Tyr-His-Lys-Arg 10.00 ― 31 Asp-Tyr-His-Lys-Arg-Arg 10.00 ― 32 Asp-Glu-Tyr-His-lys-Arg -Arg-Arg 10.00-33 Asp-Tyr-Tyr-His-Lys-Arg-Arg 9.62-34 Asp-Glu-Tyr-Tyr-His-His-Lys-Arg-Arg-Arg 9.60-35 Glu-His-His -Arg-Arg 9.48 ― 36 Asp-Glu-Tyr-Tyr-Tyr-His-His-Lys-Lys-Arg-Arg 9.34 ― 37 Glu-His-His-Lys-Arg 8.48 ― 38 Asp-Glu-Tyr-His -His-Arg-Arg-Arg 8.28 ― 39 Tyr-Tyr-His-His-His-Arg 8.22 ― 40 Asp-Glu-Tyr-His-Arg-Arg-Ar g 8.14 ― 41 Tyr-His-Arg 8.14 ― 42 Tyr-Tyr-Tyr-His-His-His-Arg 8.14 ― 43 Tyr-Tyr-His-His-Arg 8.14 ― 44 Glu-Tyr-His-Lys-Arg 8.06 ― 45 Asp-Asp-Tyr-His-Lys-Arg-Arg 8.06 ― 46 Asp-Tyr-His-Lys-Arg 8.06 ― 47 Tyr-His-Lys 8.06 ― 48 Glu-Tyr-His-Lys-Arg 8.05 ― 49 Tyr-Tyr-Tyr-His-His-Arg 8.04 ― 50 Tyr-Tyr-His-Lys-Arg 7.94 ― 52 Tyr-Tyr-Tyr-Tyr-His-Arg 7.90 ― 53 His- His-His-His-His 7.06 ― 54 Asp-His-His-His-His-Ar 6.94 ― 55 His-His-His-His 6.94 ― 56 Asp-His-His-His-Arg 6.76 ― 57 Tyr-Tyr- Arg 6.55 ― 58 Asp-Glu-His-His-Arg-Arg 6.48 ― 59 Asp-Asp-Glu-Tyr-His-His-Lys-Arg-Arg 6.48 ― 60 Tyr-Tyr-Tyr-Arg 6.48 ― 61 Asp- Asp-Glu-Tyr-His-Lys-Arg-Arg 5.60-62 Asp-His-Arg 5.30-63 Asp-Asp-Asp-Glu-Lys-Arg-Arg-Arg 4.64-64 Asp-Asp-Asp-Asp- His-His-Arg-Arg 4.50 ― 65 Asp-Glu-Arg-Arg 4.36 ― 66 Asp-Glu-Glu-Lys-Arg 4.12 ― 67 Asp-Asp-Glu-Arg 3.46 ― 68 Asp-Asp-Asp-Arg 3.38 ― 69 Asp-Asp-Asp-Asp-Arg 3.28 ― (Note 1: Fluorescently labeled oligo The measured position could not be obtained because the migration position of the peptide exceeded the mobility range (pI 9.30 to 3.50) of the commercially available isoelectric focusing marker used). =================================== (Fluorescence Detection Capillary Isoelectric Focusing) Obtained 14
The kinds of fluorescent substance-labeled oligopeptides were separated and detected by fluorescence detection capillary isoelectric focusing (Hj
erten, S.M. , Journal of Chrom
matography, 347, 191-198,
1985).

A fused silica capillary (manufactured by GL Sciences Co., Ltd.) having an inner wall coated with linear polyacrylamide and having an inner diameter of 75 μm, an outer diameter of 375 μm, and a total length of 6 cm was used.

The fluorescent substance-labeled peptide was dissolved in a solution containing 1% of ampholine and 0.1% of Tween 20, and then filled in a capillary.

The anolyte was methylcellulose (2% solution of 2%).
Viscosity at 5 ° C; 4,000 cP, manufactured by Sigma) 2%
And a catholyte 20 mM containing 2% of hydroxypropyl methylcellulose (viscosity of a 2% solution at 25 ° C .; 4,000 cP, manufactured by Sigma).
Using a NaOH solution, 2.4 kV, 10 μA, 2
The electrophoresis was performed for minutes.

After the electrophoresis, the fluorescent substance-labeled peptide focused in the capillary was detected by electrophoretically moving to the position of the detector.

A helium-neon laser (wavelength: 543.5 nm, output: 1 mW, model: 05-
Using a LGR-151-S (manufactured by Meles Griot), the generated fluorescence is condensed with a 40 × objective lens, and then a bandpass filter (590 nm, 30 nm bandwidth, model; DIF-BP-3, Nippon Vacuum Optical Co., Ltd.) Through a photomultiplier tube (model: R1387, manufactured by Hamamatsu Photonics), and the measurement results were analyzed using an integrator (model: CR4A, manufactured by Shimadzu Corporation).

FIG. 8 shows the migration time and fluorescence intensity of five of the 24 types of obtained fluorescent substance-labeled peptides when capillary isoelectric focusing was performed on a fluorescence detection capillary.

According to the above results, according to the present invention, a fluorescent substance-labeled oligopeptide can be obtained by including a fluorophore dye in the oligopeptide. At this time, since the oligopeptide and the fluorophore can be bound under mild conditions, the number and position of the fluorescent substance to be further bound to the oligopeptide can be specified without impairing the oligopeptide and the fluorescent substance, The resulting oligopeptide containing a fluorophore dye will have a highly stable and uniform single pI. Therefore, the fluorescence detection capillary isoelectric focusing marker obtained in the present invention can be used as a marker for estimating the isoelectric point of a sample substance.

[0106]

[Sequence list]

SEQ ID NO: 1 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 2 Sequence length: 8 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 3 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide SEQ ID NO: 4 Sequence length: 8 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 5 Sequence length: 4 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Gly Arg Gly Asp SEQ ID NO: 6 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr His Arg Arg SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 8 Sequence length: 5 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 9 Sequence length: 4 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Glu His Lys Arg SEQ ID NO: 10 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr Tyr His His SEQ ID NO: 11 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 12 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 13 Sequence length: 4 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Glu His Arg Arg SEQ ID NO: 14 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 15 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 16 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 17 Sequence length: 4 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp His His Arg SEQ ID NO: 18 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 19 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 20 Sequence length: 8 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 21 Sequence length: 10 Sequence type: amino acid Topology: Linear Sequence type: Peptide

[Brief description of the drawings]

FIG. 1 shows a chromatogram obtained by separating a peptide labeled with a fluorescent substance by high performance liquid chromatography after chemically binding a fluorescent substance to five kinds of peptides according to the present invention, and a target peak. It is a figure which shows (*).

FIG. 2 shows a chromatogram and a target peak when a peptide labeled with a fluorescent substance is separated by high performance liquid chromatography after chemically binding a fluorescent substance to nine kinds of peptides according to the present invention. It is a figure which shows (*).

FIG. 3 shows five peptides (Nos. 15 to 1) according to the present invention.
FIG. 9 is a diagram showing a chromatogram when a peptide labeled with a fluorescent substance is separated by high performance liquid chromatography after chemically binding the fluorescent substance to 9), and a target peak (*).

FIG. 4 shows five peptides (Nos. 20 to 2) according to the present invention.
FIG. 4C is a diagram showing a chromatogram when a peptide labeled with a fluorescent substance is separated by high performance liquid chromatography after chemically binding the fluorescent substance to 4), and a target peak (*).

FIG. 5 is a diagram showing electrophoretic images of five types of fluorescent substance-labeled peptides (Nos. 1 to 5) according to the present invention and a commercially available marker for isoelectric focusing when subjected to isoelectric focusing.

FIG. 6 is a view showing a migration image of the five kinds of fluorescent substance-labeled peptides according to the present invention and a commercially available marker for isoelectric focusing when isoelectric focusing is performed.

FIG. 7 shows an electrophoretic image of the five kinds of fluorescent substance-labeled peptides (Nos. 15 to 23) of the present invention and a commercially available marker for isoelectric focusing obtained by isoelectric focusing. FIG.

FIG. 8 shows five types of fluorescent substance-labeled peptides (numbers 1 to 5).
FIG. 5 is a diagram showing a moving time and a fluorescence intensity when performing fluorescence isoelectric focusing electrophoresis on a fluorescence detection capillary.

[Description of Signs] 1 ... des-aspartic acid ¹ -angiotensin I
(Arginine-valine-tyrosine-isoleucine-histidine-proline-phenylalanine-histidine-leucine, Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu), 2
... [asparagine ¹ -valine ⁵ ] -angiotensin I
I (asparagine-arginine-valine-tyrosine-valine-histidine-proline-phenylalanine, Asn-
Arg-Val-Tyr-Val-His-Pro-Phe), 3 ... Angiotensin I (aspartic acid-arginine-valine-tyrosine-isoleucine-histidine-proline-phenylalanine-histidine-leucine, Asp-Arg-Val-Tyr-Ile -Hi
s-Pro-Phe-His-Leu), 4 ... angiotensin II (aspartic acid-arginine-valine-tyrosine-isoleucine-histidine-proline-phenylalanine, As
p-Arg-Val-Tyr-Ile-His-Pro-Phe), 5 ... fibronectin fragment (glycine-arginine-glycine-aspartic acid (Gly-Arg-Gly-Asp), 6 ... tyrosine-histidine-arginine-arginine ( Tyr-His-Arg-Arg), 7 ... Tyrosine-Tyrosine-Histidine-Arginine-Arginine (Tyr-Tyr-His-Arg-Arg), 8 ... Tyrosine-Tyrosine-
Histidine-lysine-arginine (Tyr-Tyr-His-Lys-Ar
g), 9 ... Glutamic acid-histidine-lysine-arginine (Glu-His-Lys-Arg), 10 ... Tyrosine-tyrosine-
Histidine-histidine (Tyr-Tyr-His-His), 11 ... Tyrosine-histidine-histidine (Tyr-His-His), 12
... Aspartic acid-glutamic acid-tyrosine-histidine-lysine-arginine (Asp-Glu-Tyr-His-Lys-Arg),
13: tyrosine-histidine (Tyr-His), 14: aspartic acid-aspartic acid-aspartic acid-aspartic acid-aspartic acid-arginine (Asp-Asp-Asp-Asp)
-Asp-Arg), 15 ... Glutamic acid-histidine-arginine-arginine (Glu-His-Arg-Arg), 16 ... Aspartic acid-glutamic acid-histidine-arginine-arginine (Asp-Glue-His-Arg-Arg-Arg) , 17: Glytamate-histidine-histidine-histidine-lysine-arginine (Glu-His-His-His-Lys-Arg), 18: histidine-histidine-histidine-histidine-histidine (His-His-His-His-His-His) -His), 19 ... Aspartic acid-histidine-histidine-arginine (Asp-Hi)
s-His-Arg), 20 ... Aspartic acid-aspartic acid-glutamic acid-histidine-histidine-histidine-lysine-arginine (Asp-Asp-Glu-His-His-His-Lys-
Arg), 21 ... Aspartic acid-aspartic acid-aspartic acid-aspartic acid-glutamic acid-glutamic acid-histidine-arginine-arginine-arginine-arginine-arginine (Asp-Asp-Asp-Asp-Glu-Gl
u-His-Arg-Arg-Arg-Arg-Arg), 22 ... aspartic acid-aspartic acid-aspartic acid-glutamic acid-histidine-histidine-histidine-arginine-arginine (Asp-Asp-Asp-Glu-His-His- Arg-Arg), 23 ...
Aspartic acid-aspartic acid-aspartic acid-glutamic acid-glutamic acid-glutamic acid-histidine-arginine-arginine-arginine (Asp-Asp-Asp-
Glu-Glu-Glu-His-Arg-Arg-Arg), 24 ... Aspartic acid-glutamic acid-arginine (Asp-Glu-Arg)

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI C07K 5/113 C07K 5/113 7/06 ZNA 7/06 ZNA 7/14 7/14 G01N 27/447 G01N 33/68 33 / 68 27/26 301B (56) References JP-A-54-156385 (JP, A) JP-A-60-52758 (JP, A) JP-A-7-157717 (JP, A) JP-A-5-255390 ( JP, A) Special Table Hei 8-503775 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 33/48-33/52 G01N 33/58-33/98 G01N 27 / 447

Claims (10)

(57) [Claims] Claims: 1. A fluorescent detection isoelectric focusing marker used in a fluorescent isoelectric focusing method, wherein the marker is an oligopeptide containing a fluorophore dye. Marker. 2. The marker for fluorescence isoelectric focusing according to claim 1, wherein the fluorophore dye is an oligopeptide bonded to an N-terminal amino group via a bonding group. 3. The fluorescence detection and the like according to claim 2, wherein the binding group is at least selected from the group consisting of amide, thioamide, sulfonamide, urea, thiourea, and urethane binding groups. Marker for electrofocusing. Wherein said oligopeptide is at least, an amino acid having a group having a negative charge by releasing a proton, n _j of the acid dissociation constant of the groups with a negative charge by releasing proton and K _j number of amino acids and / or an amino acid having a group with a positive charge by receiving a proton, and a n _i number of amino acids to the acid dissociation constant of the group with a positive charge by receiving the proton and K _i, Z = Σ _i (n
_i / (1 + K _i / [H ⁺ ])) − Σ _j (n _j / (1+
[H ⁺ ] / K _j )), when −log ([H ⁺ ]) satisfying | Z | <0.01 is pI, 3 <
The marker for fluorescence detection isoelectric focusing according to claim 1, wherein pI <11. 5. The marker for fluorescence isoelectric focusing according to claim 1, wherein the oligopeptide is angiotensin or a derivative thereof. 6. The method according to claim 6, wherein the oligopeptide comprises at least Ar
g-Arg-Val-Tyr-Ile-His-Lys, Arg-Arg-Lys-His-Tyr, Ar
g-Arg-His-Tyr, Arg-Lys-His-Tyr, Arg-Arg-Val-Tyr-Il
e-Tyr, Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, Asn-
Arg-Val-Tyr-Val-His-Pro-Phe, Asp-Arg-Val-Tyr-Ile-
His-Pro-Phe-His-Leu, Asp-Arg-Val-Tyr-Ile-His-Pro-
6. The marker for fluorescence detection isoelectric focusing according to claim 5, wherein the marker is at least one oligopeptide selected from the group consisting of Phe and Gly-Arg-Gly-Asp. 7. The fluorophore dye is selected from the group consisting of rhodamine, fluorescein, cyanine, indocyanine, indocarbocyanine, pyronin, lucifer yellow, quinacrine, squaric acid, coumarin, fluoroanthenylmaleimide, and anthracene. The marker for fluorescence detection isoelectric focusing according to claim 1, which is a fluorophore dye. 8. The marker for fluorescence detection isoelectric focusing according to claim 1, wherein the fluorescence detection isoelectric focusing is a fluorescence detection capillary isoelectric focusing. 9. The method according to claim 9, wherein the oligopeptide comprises at least Ty.
r-His-Lys-Arg-Arg, Tyr-His-Lys-Arg, Asp-His-His-
Arg, Asp-His-Arg, Arg-Arg-Val-Tyr-Ile-His-Lys,
Arg-Arg-Lys-His-Tyr, Arg-Arg-His-Tyr, Arg-Lys-His-T
yr, Arg-Arg-Val-Tyr-Ile-Tyr, Arg-Val-Tyr-Ile-His-
Pro-Phe-His-Leu, Asn-Arg-Val-Tyr-Val-His-Pro-Phe
, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, Asp-A
rg-Val-Tyr-Ile-His-Pro-Phe, Gly-Arg-Gly-Asp, Tyr
-His-Arg-Arg, Tyr-Tyr-His-Arg-Arg, Tyr-Tyr-His-L
ys-Arg, Glu-His-Lys-Arg, Tyr-Tyr-His-His, Tyr-H
is-His, Asp-Glu-Tyr-His-Lys-Arg, Tyr-His, Asp-A
sp-Asp-Asp-Asp-Arg, Glu-His-Arg-Arg, Asp-Glue-Hi
s-Arg-Arg-Arg, Glu-His-His-His-Lys-Arg, His-His-H
is-His-His-His, Asp-His-His-Arg, Asp-Asp-Glu-His
-His-His-Lys-Arg, Asp-Asp-Asp-Asp-Glu-Glu-His-Arg
-Arg-Arg-Arg-Arg, Asp-Asp-Asp-Glu-His-His-Arg-Arg
, Asp-Asp-Asp-Glu-Glu-Glu-His-Arg-Arg-Arg, Asp-G
The fluorescent detection isoelectric focusing marker according to claim 1, wherein the marker is at least one oligopeptide selected from the group consisting of lu-Arg. 10. The oligopeptide, at least,
Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, Asn-Arg-Val-
Tyr-Val-His-Pro-Phe, Asp-Arg-Val-Tyr-Ile-His-Pro-
Phe-His-Leu, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Gl
y-Arg-Gly-Asp, Tyr-His-Arg-Arg, Tyr-Tyr-His-Arg-
Arg, Tyr-Tyr-His-Lys-Arg, Glu-His-Lys-Arg, Tyr-
Tyr-His-His, Tyr-His-His, Asp-Glu-Tyr-His-Lys-Ar
g, Tyr-His, Asp-Asp-Asp-Asp-Asp-Arg, Glu-His-Ar
g-Arg, Asp-Glue-His-Arg-Arg-Arg, Glu-His-His-His-
Lys-Arg, His-His-His-His-His-His, Asp-His-His-Ar
g, Asp-Asp-Glu-His-His-His-Lys-Arg, Asp-Asp-Asp-
Asp-Glu-Glu-His-Arg-Arg-Arg-Arg-Arg, Asp-Asp-Asp-
Glu-His-His-Arg-Arg, Asp-Asp-Asp-Glu-Glu-Glu-His-
2. The marker for fluorescence isoelectric focusing according to claim 1, comprising Arg-Arg-Arg and Asp-Glu-Arg.