JP3148153B2 - Ultraviolet absorption detection isoelectric focusing marker - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an isoelectric focusing marker for ultraviolet absorption detection which is used in an isoelectric focusing method for ultraviolet absorption detection.

[0002]

2. Description of the Related Art In 1966, electrophoresis made use of the difference in charge state at a specific pH.
terberg, O .; 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. .

[0003] The pH value at which the effective charge becomes zero is called the isoelectric point (pI) of the substance. In the isoelectric focusing method, a sample is concentrated at a position equal to its isoelectric point and stands still in a pH gradient formed on a carrier for electrophoresis. 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 mounting a detector at one end of the capillary and detecting the separated sample components.

[0005]

In the above-mentioned isoelectric focusing, the isoelectric point (pI) is estimated from the rest position of the separated sample in the electrophoresis carrier. It is necessary to clarify the pH gradient formed in, and a standard marker for this is required.

As a marker for isoelectric focusing used in the isoelectric focusing method, a method for detecting a change in absorption intensity due to a group that absorbs ultraviolet light or visible light in the marker (ultraviolet light) Absorption or visible light absorption detection), and the above-mentioned capillary isoelectric focusing method can also use the marker by this ultraviolet absorption detection method. For example, as the above-mentioned markers, conventionally known proteins having a clear pI (for example, trypsinogen (pI = 9.30), lentil lectin (pI = 7.80, 8.00, 8.20), human hemoglobin) C (pI = 7.50), human hemoglobin A
(PI = 7.10), equine myoglobin (pI = 7.10).
00), human carbonic anhydrase (pI =
6.50), bovine carbonic anhydrase (pI
= 6.00), β-lactoglobulin B (pI = 5.1)
0), phycocyanin (pI = 4.65), amyloglucosidase (pI = 3.50) and the like can be used.

[0007] A method of labeling the above-mentioned protein having a known pI with a substance having an appropriate high ultraviolet absorption intensity is also conceivable. However, selectivity of the labeling reaction, handling of the product, storage stability, etc. There is a problem in the point.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and have studied an oligopeptide containing an appropriate number of tryptophan groups as an ultraviolet absorbing group, wherein amino acids are selected so as to exhibit a wide range of pI. Show a sharp single characteristic pI and a wide range of pI values (3 <pI <1
The present invention was found to cover 1) and to be excellent in storage stability, and completed the present invention.

[0009]

SUMMARY OF THE INVENTION The present invention provides a method for isoelectric focusing by ultraviolet absorption detection, in which an appropriate amino acid residue is selected in order to accurately determine a pH gradient formed in a carrier for electrophoresis. By making the oligopeptide an oligopeptide having a structure containing an appropriate number of tryptophan, p is uniformly dispersed over a wide pH range.
The present invention relates to a marker for isoelectric focusing on ultraviolet absorption detection, which shows I and is superior in handling, storage stability and the like as compared with a conventional marker using a high molecular protein.

[0010] That is, the present invention relates to a marker for isoelectric focusing on ultraviolet absorption used in isoelectric focusing for ultraviolet absorption detection, wherein the marker is an oligopeptide containing at least one tryptophan. The present invention relates to a marker for isoelectric focusing on absorption detection.

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

Further, the oligopeptide is H-Trp-Tyr-
Lys-Arg-OH, H-Trp-Tyr-Lys-Lys-OH, H-Trp-Tyr-Tyr-Ly
s-Lys-OH, H-Trp-Tyr-Tyr-Tyr-Lys-Lys-OH, H-Trp-Tyr-
Lys-OH, H-Trp-Glu-Tyr-Tyr-Lys-Lys-OH, H-Trp-Glu-Hi
s-His-His-Arg-OH, H-Trp-Glu-His-Arg-OH, H-Trp-Glu-
His-His-OH, H-Trp-Glu-Arg-OH, H-Trp-Glu-His-OH, H-
Trp-Asp-Asp-His-His-OH, H-Trp-Glu-Glu-His-OH, H-Tr
p-Asp-Asp-Arg-OH, H-Trp-Glu-Glu-OH, H-Trp-Asp-Asp-
The present invention relates to the marker for isoelectric focusing on ultraviolet light detection described above, which is at least one selected from the group consisting of Asp-OH.

Further, the present invention provides the above-mentioned isoelectric focusing,
The present invention relates to the marker for ultraviolet absorption detection isoelectric focusing described above, which is a capillary isoelectric focusing electrophoresis for ultraviolet absorption detection.

Hereinafter, the present invention will be described with reference to embodiments.

[0015]

Embodiment

(Oligopeptide) In the present invention, an oligopeptide is used from various amphoteric substances that can be used 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.

Accordingly, 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 composed of amino acids as amphoteric compounds have various acidic groups and basic groups as acid-base dissociating groups, and N-terminal amino groups and C-terminal carboxyl groups. For example, amino acid groups having an acidic group include aspartic acid, glutamic acid, cysteine, tyrosine and the like, and amino acid groups having a basic group include lysine, arginine and histidine.

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 following equation makes it possible to estimate the number of charges (Z) as the entire oligopeptide.

Here, n _j amino acids having a group having a negative charge by releasing a proton, wherein the acid dissociation constant of the group having a negative charge by releasing a proton is K _j , 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 given by Z = Σ _i (n _i / (1 + K
_i / [H ⁺ ])) − Σ _j (n _j / (1+ [H ⁺ ] / K _j ))) (Regions of Chemistry, 36 , 470-486, 19)
82).

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. Further, regarding the convergence to the isoelectric point, it is necessary that the amount of change in charge with respect to the change in pH at the isoelectric point of the peptide, dZ / dpH, be large (Shimura, K., and Kasai, K., Electrophoresi
s, 16,1479-1484, 1995). In order to increase the dZ / dpH value, it is necessary that a dissociating group having a pKa value close to the isoelectric point value of the peptide be present.

At this time, the tryptophan group (Trp) does not significantly change the above-mentioned pI, and is an oligopeptide having a preferred number and position of Trp introduced therein, which can exhibit a preferable pI. Yes, basic dissociation by the N-terminal amino group is also exhibited, and the pKa of the N-terminal amino group is
As a result, a highly convergent labeled peptide having a pI around pH 7.6 can be obtained.

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, aspartic acid or glutamic acid is selected to reduce the pI, arginine or lysine is selected to increase the pI, and the α-amino group can be made free. It is.
Similarly, tyrosine or histidine may be selected to achieve a pI between them.

In the present invention, the range of pI is particularly preferably 3 or more and 11 or less. More particularly, the pI range is preferably 3 or more and 10 or less.

Further, in the present invention, the method for synthesizing an oligopeptide containing a desired amino acid is not particularly limited. Synthesis by general synthetic 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, an oligopeptide containing at least one tryptophan derived from a natural product is also provided.
All or some of them can be suitably used.

Further, the synthesis of the oligopeptide having a tryptophan group according to the present invention is not particularly limited, and can be performed by a method using ordinary chemical synthesis means. Also, a method using a commercially available automatic synthesizer can be preferably used.

In the present invention, the purity and storage stability of the obtained marker 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.

(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 the 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 ultraviolet absorption, a general ultraviolet absorption measuring device can be suitably used.

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

Generally, an oligopeptide containing tryptophan is converted by a capillary isoelectric focusing apparatus.
It is concentrated at the position of the pH formed on the carrier for electrophoresis according to each pI and stops. Furthermore, these positions can be confirmed by detecting ultraviolet absorption with an ultraviolet absorption measuring device. Alternatively, a pH gradient formed on the carrier for electrophoresis can be confirmed by an ultraviolet absorption measurement device provided at one end of the capillary.

As described above, the isoelectric focusing marker for ultraviolet absorption detection of the present invention is an oligopeptide having a sufficient number of tryptophan residues,
It exhibits a moderately dispersed pI over a wide pH range and has high stability. Therefore, in isoelectric focusing on ultraviolet absorption detection, it becomes 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. Furthermore, the marker for isoelectric focusing on ultraviolet absorption detection of the present invention can be used for capillary isoelectric focusing.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples as long as the gist of the present invention is not exceeded.

[0038]

【Example】

(Synthesis of isoelectric focusing marker for UV absorption detection) (1) Peptide; H-Trp-Tyr-Lys-Arg-OH, H-Trp-Tyr-
Lys-Lys-OH, H-Trp-Tyr-Tyr-Lys-Lys-OH, H-Trp-Tyr-Ty
r-Tyr-Lys-Lys-OH, H-Trp-Tyr-Lys-OH, H-Trp-Glu-Tyr-
Tyr-Lys-Lys-OH, H-Trp-Glu-His-His-His-Arg-OH, H-Tr
p-Glu-His-Arg-OH, H-Trp-Glu-His-His-OH, H-Trp-Glu-
Arg-OH, H-Trp-Glu-His-OH, H-Trp-Asp-Asp-His-His-O
H, H-Trp-Glu-Glu-His-OH, H-Trp-Asp-Asp-Arg-OH, HT
rp-Glu-Glu-OH and H-Trp-Asp-Asp-Asp-OH are available from Applied Bio
It was synthesized based on the Boc method by an automatic peptide synthesizer manufactured by systems Co., Ltd. (for example, see Shinsei Kagaku Kenkyusho 1, Protein VI, edited by The Japanese Biochemical Society, 1992). The obtained oligopeptide was separated and purified by high performance liquid chromatography under the following conditions. The conditions used for the high-performance liquid chromatograph were columns S-5, 1
20A ODS (30 mm inner diameter x 250 mm full length) was used, the flow rate was 20 ml / min, the detection wavelength was 220 nm, and the mobile phase was an acetonitrile gradient-0.1% trifluoroacetic acid. did.

For H-Trp-Tyr-Lys-Arg-OH, 10.8% to 30.8%
/ 60 min; for H-Trp-Tyr-Lys-Lys-OH, 2% to 22% / 60 min; for H-Trp-Tyr-Tyr-Lys-Lys-OH, 4% to 24% / 60 min. Min; for H-Trp-Tyr-Tyr-Tyr-Lys-Lys-OH, 6% to 26% / 60
Min; in H-Trp-Tyr-Lys-OH, 6% to 22.6% / 60 min; HT
rp-Glu-Tyr-Tyr-Lys-Lys-OH: 3.9% to 23.9% / 60 min; H-Trp-Glu-His-His-His-Arg-OH: 2.2% to 22.2%
/ 60 min; for H-Trp-Glu-His-Arg-OH, 2% to 22% / 60 min; for H-Trp-Glu-His-His-OH, 1.7% to 21.7% / 60 min. For H-Trp-Glu-Arg-OH, 3.6% to 23.6% / 60 min; HT
For rp-Glu-His-OH, 3% to 23% / 60 min; H-Trp-Asp-Asp-
For His-His-OH, 3.1% to 23.1% / 60 min; H-Trp-Glu-Glu
For -His-OH, 2.5% to 22.5% / 60 min; H-Trp-Asp-Asp-Ar
For g-OH, 2.4% to 22.4% / 60 min; for H-Trp-Glu-Glu-OH, 3.7% to 23.7% / 60 min; for H-Trp-Asp-Asp-Asp-Asp-OH, 3.2 % To 23.2% / 60 minutes.

(PI) The oligopeptide obtained above is
The pI was measured by the following method, and the pI of the obtained measured value of each oligopeptide was summarized in Table 1 together with the calculated value.

Table 1 No. Synthetic peptide pI marker structure pI calculated value pI measured value ================================ 28 H-Trp-Tyr-Lys-Arg-OH 10.02 10.24 29 H-Trp-Tyr-Lys-Lys-OH 9.76 10.05 30 H-Trp-Tyr-Tyr-Lys-Lys-OH 9.52 9.77 31 H-Trp-Tyr- Tyr-Tyr-Lys-Lys-OH 9.36 9.57 32 H-Trp-Tyr-Lys-OH 8.62 8.52 33 H-Trp-Glu-Tyr-Tyr-Lys-Lys-OH 8.47 8.52 34 H-Trp-Glu-His- His-His-Arg-OH 7.34 7.38 35 H-Trp-Glu-His-Arg-OH 7.04 7.15 36 H-Trp-Glu-His-His-OH 6.42 6.75 37 H-Trp-Glu-Arg-OH 6.06 6.10 38 H-Trp-Glu-His-OH 5.48 5.70 39 H-Trp-Asp-Asp-His-His-OH 5.24 5.58 40 H-Trp-Glu-Glu-His-OH 4.54 4.38 41 H-Trp-Asp-Asp- Arg-OH 4.16 4.13 42 H-Trp-Glu-Glu-OH 3.82 3.86 43 H-Trp-Asp-Asp-Asp-OH 3.38 3.46 ==================== =========== The measured value is the isoelectric point by slab gel electrophoresis according to the following method. It was carried out by constant.

That is, on a 1 × 65 × 125 mm synthetic silica plate, a 1 × 50 × 115 mm polyacrylamide gel (4.2%) containing Pharmalyte manufactured by Pharmacia Biotech Inc. as a final concentration at a 1/16 dilution of a stock solution as an amphoteric carrier for forming a pH gradient.
T, 4.8% C). Basic side peptide number 28-3
3 for Pharmalyte8-10.5, and for peptide numbers 34-39
Pharmalyte 5-8, Pha No.
rmalyte2.5-5 was used. The silica plate on which the gel was placed was placed on a horizontal cooling plate in which constant temperature water was circulated, and electrophoresis was performed in a glove box filled with nitrogen gas in order to avoid the influence of carbon dioxide in the air. An electrode was placed along the strip, and about 50 nmol of each peptide was added as a sample to the gel surface and finally focused at a voltage of about 100 V / cm (about 2 V).
time). After the focusing is completed, adjust the temperature of the water circulating through the gel cooling plate so that the gel temperature is 25 ° C with the voltage applied, and adjust the pH of the gel surface at 1 cm intervals along the pH gradient. Was measured. For the measurement of gel temperature and pH, a METHOXY pH meter (HM-17MX type) manufactured by Toa Denpa Kogyo KK was used. The focus of the peptide was determined by scanning the absorbance at 280 nm of the entire gel with a densitometer (Densitometer, Model CS-9300PC manufactured by Shimadzu Corporation). The pH was plotted against the position of the gel to determine the pH gradient in the gel, and the isoelectric point of the peptide was determined from the focused position of the peptide.

Further, capillary electrophoresis was carried out in a capillary having an inner wall coated with polyacrylamide and having an inner diameter of 50 μm, using Pharmalyte 3-10 as an amphoteric carrier. Apply a voltage of 500 V / cm, hold the same voltage after 2 minutes, apply pressure to the anode end, move the pH gradient to the cathode side, and detect the peptide by absorption at 280 nm at a position 7 cm from the cathode end did.

The results obtained are shown in FIG. 1. When these peptides were added from the cathode side, the peptides formed once on the anode side of the detection point after passing the detection point once at the beginning of electrophoresis. Converged during the pH gradient (FIGS. 1, 5 min). Thereafter, sharp peaks were sequentially detected from the basic peptide with the movement of the pH gradient (FIG. 1, up to about 13 minutes). Similar separation results were obtained for the converged peptide, except that no peptide peak before convergence was observed when the peptide was added to the anode side. Under these conditions, one of the peptides was observed overlapping without being separated from the other peaks. The most acidic peptide was detected after 30 minutes.

According to the above results, according to the present invention, by including an appropriate number of tryptophan amino acid residues in an oligopeptide, an oligopeptide as a pI marker by ultraviolet absorption can be obtained.

[0046]

According to the present invention, the marker for isoelectric focusing on ultraviolet absorption detection is an oligopeptide containing an appropriate number of tryptophan for ultraviolet absorption detection and containing an amino acid residue having a desirable pI. It is characterized by.
Therefore, the obtained marker for isoelectric focusing according to the present invention has a sharp separation, a large ultraviolet absorption intensity, a wide range of pI, and easy handling, as compared with a marker based on ordinary macromolecular proteins. Becomes

Therefore, not only normal isoelectric focusing but also
It can also be used for capillary isoelectric focusing. Detection can be easily performed by a usual ultraviolet absorption measuring device.

[0048]

[Sequence list]

SEQ ID NO: 1 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Tyr Lys Arg SEQ ID NO: 2 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Tyr Lys Lys SEQ ID NO: 3 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 4 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 5 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 6 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 7 Sequence length: 4 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Trp Glu His Arg SEQ ID NO: 8 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: peptide Sequence Trp Glu His His SEQ ID NO: 9 Sequence length: 5 Sequence type: amino acid Topology: linear Sequence type: peptide SEQ ID NO: 10 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Glu Glu His SEQ ID NO: 11 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Asp Asp Arg SEQ ID NO: 12 Sequence length: 5 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Trp Asp Asp Asp

[Brief description of the drawings]

FIG. 1 is a diagram showing the migration time and ultraviolet absorption intensity of capillary isoelectric focusing electrophoresis markers for ultraviolet absorption detection isoelectric focusing (numbers 28 to 43) according to the present invention.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-52-92590 (JP, A) JP-A-9-171020 (JP, A) JP-A-7-157717 (JP, A) JP-A 54-925 156385 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/447 C07K 1/28 C07K 5/097 C07K 5/117 ZNA C07K 7/06 JICST file (JOIS)

Claims (4)

(57) [Claims] 1. An ultraviolet absorption isoelectric focusing marker used in ultraviolet absorption detection isoelectric focusing, which is an oligopeptide containing at least one tryptophan. Point electrophoresis marker. 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 )), −Z | <0.01 is satisfied.
When log ([H ⁺ ]) is pI, 3 <pI <11
The isoelectric focusing marker for ultraviolet absorption detection according to claim 1, wherein: 3. The method according to claim 2, wherein the oligopeptide is H-Trp-Tyr-Lys-
Arg-OH, H-Trp-Tyr-Lys-Lys-OH, H-Trp-Tyr-Tyr-Lys-Ly
s-OH, H-Trp-Tyr-Tyr-Tyr-Lys-Lys-OH, H-Trp-Tyr-Lys-
OH, H-Trp-Glu-Tyr-Tyr-Lys-Lys-OH, H-Trp-Glu-His-Hi
s-His-Arg-OH, H-Trp-Glu-His-Arg-OH, H-Trp-Glu-His-
His-OH, H-Trp-Glu-Arg-OH, H-Trp-Glu-His-OH, H-Trp-
Asp-Asp-His-His-OH, H-Trp-Glu-Glu-His-OH, H-Trp-As
p-Asp-Arg-OH, H-Trp-Glu-Glu-OH, H-Trp-Asp-Asp-Asp-
The marker for isoelectric focusing on ultraviolet absorption detection according to claim 1, wherein the marker is at least one selected from the group consisting of OH. 4. The isoelectric focusing according to any one of claims 1 to 3, wherein the isoelectric focusing is capillary isoelectric focusing with ultraviolet absorption detection. Marker.