Isoelectric point marker

A group of compounds having the following general formula can be obtained by reacting chromoprotein and organic acid: ##STR1## wherein X is a chromophore, P is a protein, NH.sub.2 is mainly .epsilon.-amino group of lysine, n is a positive integer of 1 to 18, and m is a positive integer in the relation of m.ltoreq.n. This compound group has one to eighteen organic acids bonded thereto, and shows various isoelectric points in accordance with the number of the organic acids, but the respective isoelectric points are maintained constant. If such compounds each having an individual isoelectric point, are used as isoelectric point markers, it is possible to recognize accurately the position of isoelectric point only by a visual operation.

FIELD OF THE INVENTION 
The present invntion relates to a novel isoelectric point marker and a 
novel method of isoelectric point determination, and more particularly to 
an isoelectric point marker composed of chromoprotein treated with organic 
acid anhydride. 
BACKGROUND OF THE INVENTION 
Recently, in the fields of biochemistry and clinical inspection, attention 
has been paid to gel isoelectric separation (also referred to as gel 
isoelectric focusing or gel isoelectric fractionation) as a means of 
separation, purification and analysis of protein. This method has been 
thought of as a simplified version of the density gradient isoelectric 
separation developed by Svenseon et al. Namely, when a mixture of special 
amphoteric electrolytes having an isoelectric point at various pH values 
is enclosed in a gel, such as acrylamide or agar, etc., and electrodes are 
provided at both ends of the enclosure, and electricity is passed 
therebetween, a pH-gradient derived from the amphoteric electrolytes is 
produced between both electrodes. The present invention is based on the 
principle that protein can be focused to the position of the isoelectric 
point in the pH-gradient produced by use of such an amphoteric electrolyte 
mixture. According to the method (C. W. Wringley: "Methods in Enzymology", 
Vol. 22, p559-564, Academic Press), it is, of couse, possible to isolate 
and purify proteins by the difference of their isoelectric points and also 
to obtain the isoelectric point of protein by measurement of pH-gradient 
in a gel, and therefore it has also a large value of utilization for a 
method of analyzing protein. 
However, this method as the drawback that in order to measure the 
pH-gradient formed in a gel, there is required a complicated process 
comprising taking out the gel immediately after the electrophoresis, 
cutting it into as thin slices as possible at uniform intervals, 
extracting the amphoteric electrolyte existing in the respective gel 
slices by use of decarboxylated pure water, and then measuring pH of the 
respective extract. However, this method for pH measurement has 
difficulties in practical use such that it takes a long time to extract 
the amphoteric electrolyte and the cutting of the gel is difficult and 
poor in accuracy and, as well, pH values may fluctuate in accordance with 
the extraction conditions. Various ways have been tried heretofore, such 
as a method in which, in isoelectric separation, with respect to coloring 
matter of amphoteric electrolyte, by utilizing it as pH-indicator, 
electrophoresis is made together with a sample (A. Conway-Jacobs and L. M. 
Lewin: Analytical Biochemistry, vol. 43, p394-400, 1971), and a method in 
which in a similar object phenanthroline and iron complex are used (E. T. 
NAKHLEH et al.: Analytical Biochemistry, vol. 49, p218-224, 1972). 
However, these substances have drawbacks such that since these substances 
are low molecular weight compounds, diffusion takes place rapidly and also 
since the dissociation constants of dissociation groups at both ends of 
the isoelectric point values are far apart from each other, the separation 
layer spreads. 
SUMMARY OF THE INVENTION 
The inventors have sought a superior isoelectric point marker, namely an 
isoelectric point marking material, in order to eliminate the difficulties 
of pH measurement in such a gel isoelectric separation, thus resulting in 
completion of the present invention. 
The present invention is an isoelectric point marker composed of 
chromoprotein treated by organic acid anhydride and having one of the 
following general formulas. 
(1) An isoelectric point marker composed of a compound shown in the 
following general formula: 
##STR2## 
wherein X is a chromophore, 
P is a protein, 
NH.sub.2 is .epsilon.-amino group mainly derived from lysine, 
n is a positive integer of 1-18, and 
m is a positive integer in the relation of m.ltoreq.n. 
(2) An isoelectric point marker composed of a compound having the following 
general formula: 
##STR3## 
which is obtained by acetylation of the compound shown in the above 
general formula (1). 
(3) An isoelectric point marker composed of a compound having the following 
general formula: 
##STR4## 
which is obtained by succinylation of the compound shown in the above 
general formula (1). 
(4) An isoelectric point marker composed of a compound having the following 
general formula: 
##STR5## 
which is obtained by acetylation and succinylation of the compound shown 
in the above general formula (1). 
Within each general formula the compound with each individual value of m 
(or m' and m") have different fixed isoelectric points. A plurality of 
such compounds with fixed isoelectric points may then be combined to 
produce an isoelectric point marker having a known number of fixed known 
isoelectric points. When added to a gel and subjected to gel isoelectric 
separation, colored bands are formed in the gel at each of the specific 
isoelectric point positions in the gel corresponding to the known 
isoelectric points in the marker. It is thus possible to easily determine 
the pH-gradient in the gel from a perusal of the position of the known 
isoelectric points.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
A raw material for producing the isoelectric point marker of the present 
invention is a chromoprotein, and such substances are exemplified by the 
following, derived from various animals and vegetables or microorganisms: 
cytochrome c group, myoglobin, hemoglobin, flavin protein, copper protein 
and so forth. 
Table 1 shows representative examples of these chromoproteins. 
TABLE 1 
______________________________________ 
Kind, Type of Isoelectric 
Protein Origin Point at 0-2.degree. C. 
______________________________________ 
Cytochrome c of 
Horse 10.6 
oxidized type 
Cytochrome c.sub.2 of 
Rhodopseudomonas 
10.0 
reduced type palustris 
Myoglobin Sperm whale 8.1 
Hemoglobin Rat 7.5 
Myoglobin Horse 7.3 
Cytochrome c.sub.2 of 
Rhodospirillum 
6.2 
reduced type rubrum 
Cytochrome c.sub.2 of 
Rhodopseudomonas 
5.8 
reduced type spheroides 
Cytochrome c' of 
Rhodospirillum 
5.6 
oxidized type rubrum 
Cytochrome c' of 
Rhodopseudomonas 
5.0 
oxidized type spheroides 
Cytochrome c-551 
Pseudomonas 4.7 
of oxidized type 
aeruginosa 
______________________________________ 
These chromoproteins are treated by use of organic acid anhydride. As 
examples of organic acid anhydrides which may be used there are acetic 
anhydride, propionic anhydride, butyric anhydride, succinic anhydride, 
malonic anhydride, etc. 
The reaction is initiated by adding a certain amount of the organic acid 
anhydride to an aqueous solution of a certain concentration of the 
chromoprotein and gently stirring the solution. As the reaction proceeds, 
free organic acid and hydrogen ion are generated, and therefore the 
solution is neutralized by an alkali such as caustic soda and so forth, to 
permit the reaction to be sufficiently carried out under the neutral 
condition. The reaction is completed after 5 to 30 min. and the solution 
obtained from the above reaction is then separated and purified by means 
of an ion exchanger, thereby obtaining a plurality of isoelectric point 
markers having various specific isoelectric points. 
The isoelectric point marker composed of a mixture of a plurality of the 
thus obtained organic acid anhydride treated chromoproteins according to 
the present invention, each of the plurality having a known isoelectric 
point, forms a colored band at each of the specific isoelectric point 
positions in the gel when subjected to gel isoelectric separation together 
with a sample according to the known prior art method. The position of 
these colored bands can be determined at a glance without conducting 
complicated operations, and thus it is possible to easily determine a 
pH-gradient in the gel from both the respective known isoelectric points 
and their position. 
The analysis of protein by use of such an isoelectric point marker is 
carried out as follows. In the case that the isoelectric point of a test 
protein is known, the position of the intended protein can immediately be 
determined from the pH-gradient in the gel previously obtained by use of 
an isoelectric point marker of the present invention, and therefore a 
portion at the determined position may be taken out and the necessary 
measurements carried out on said portion. Also, in the case of measurement 
of an isoelectric point of a protein whose isoelectric point is unknown, a 
pH-gradient of the gel is obtained by use of an isoelectric point marker 
in accordance with the present invention, and on the other hand the gel is 
taken out and color is formed by use of a color former or the gel is 
sliced at certain intervals and the activity of protein in each sliced 
section is measured and so forth, thereby determining the position of the 
protein, and the thus determined position is applied to the pH-gradient 
obtained by an isoelectric point marker. 
In the case that gels of the same composition are subjected to isoelectric 
separation under the same conditions, since it is thought that the 
pH-gradient is produced in a similar way in any of the gels, it is not 
always necessary for the isoelectric point marker to flow together with 
the sample, and it may also be allowed that isoelectric point marker alone 
is separately subjected to electrophoresis in an identical gel. This is 
particularly convenient when a number of samples are desired to be 
measured simultaneously. 
As explained above, if the isoelectric separation of protein is carried out 
by use of the isoelectric point marker of the present invention, the 
measurement of the isoelectric point is remarkably simplified. This gives 
an advantage that many samples can be treated in a short time as well or 
decreasing the possibility of denaturation of protein during 
pH-measurement, so that the protein activity can be measured more 
accurately, resulting in a further propagation of the gel isoelectric 
separation in many fields. 
PRODUCTION EXAMPLE 1 
A 2 mM aqueous solution of cytochrome c derived from a horse heart was 
prepared, and acetic anhydride was added so that the final concentration 
thereof was from 2 mM to 360 mM. Each reaction medium containing 2 mM, 10 
mM, 41 mM, 81 mM, 204 mM and 360 mM of acetic anhydride was respectively 
allowed to react at room temperature for 15 min. as it was stirred gently. 
The reactions were carried out under neutral condition as the media were 
neutralized with 1 N NaOH during the reaction. The reaction proceeds 
according to the following reaction scheme: 
##STR6## 
wherein X-P denotes cytochrome c. 
One molecule of cytochrome c has eighteen lysine NH.sub.2 groups, which can 
react with acetic anhydride. In the reaction, the number of lysine 
NH.sub.2 groups which are reacted is initiated in accordance with the 
concentration of the acetic anhydride, and when the concentration of 
acetic anhydride becomes 360 mM, all the lysine NH.sub.2 groups are 
acetylated. The acetylation number differs according to each concentration 
of acetic anhydride, and also even by using an identical concentration, 
several kinds of cytochrome c having different acetylation numbers can be 
obtained. 
For example, in the case of using 2 mM cytochrome c, when acetic anhydride 
of the final concentration of 2 mM was added thereto, cytochrome c with 
one of the amino groups from lysine having been acetylated, and cytochrome 
c with two thereof having been acetylated, and cytochrome c with three 
thereof having been acetylated were respectively produced, and in the case 
of addition of 360 mM acetic anhyride, six, seven and twelve of amino 
groups of lysine had respectively been acetylated. 
Each reaction medium was purified by ion-exchange chromatography and 
sucrose density gradient isoelectric separation, and thereby nine separate 
kinds of acetylated cytochrome c, respectively having isoelectric points 
of 3.9, 4.1, 4.9, 5.6, 6.5, 8.1, 9.1, and 10.2, were obtained. 
PRODUCTION EXAMPLE 2 
A 2 mM aqueous solution of cytochrome c originating from a horse heart was 
prepared, and succinic anhydride was added, so that the final 
concentration thereof was from 2 mM to 360 mM. Each reaction medium 
containing 2 mM, 90 mM, 180 mM, 270 mM and 360 mM of succinic anhydride 
was respectively allowed to react at room temperature for 15 min. as it 
was stirred gently. The reactions were carried out under neutral condition 
as the media were neutralized with 1 N NaOH during the reaction. The 
reaction proceeds according to the following reaction scheme. 
##STR7## 
wherein X-P denotes cytochrome c. 
One molecule of cytochrome c has eighteen lysine NH.sub.2 groups, and in 
the reaction with succinic anhydride, the number of NH.sub.2 groups which 
are reacted is initiated in accordance with the concentration of succinic 
anhydride, and when the concentration of succinic anhydride becomes 360 
mM, all eighteen of the lysine NH.sub.2 groups are entirely succinylated. 
The succinylation number differs according to each concentration of 
succinic anhydride, and also even by using an identical concentration, 
several kinds of succinylated cytochrome c having different succinylation 
number can be obtained. 
For example, in the case of 2 mM cytochrome c, when succinic anhydride of 
the final concentration of 2 mM was added thereto, ones having isoelectric 
point of 10.2 and 9.4 were respectively obtained, and in the case of 
addition of 6 mM succinic anhydride, ones having isoelectric point of 
10.2, 9.4 and 7.5 were respectively obtained, and in the case of addition 
of 18 mM succinic anhydride, ones having isoelectric point of 10.2, 9.4 
and 7.5 were respectively obtained, and in the case of addition of 30 mM 
succinic anhyride, ones having isoelectric point of 9.4, 7.5 and 6.6 were 
respectively obtained, and in the case of addition of 64 mM succinic 
anhydride, ones having isoelectric point of 9.4, 7.5, 6.6 and 5.4 were 
respectively obtained, and in the case of addition of 360 mM succinic 
anhydride, ones having isoelectric point of 5.4 and 4.1 were respectively 
obtained. 
Each reaction medium was purified by ion-exchange chromatography and 
sucrose density gradient isoelectric separation, and thereby nine separate 
kinds of succinyl cytochrome c respectively having isoelectric point of 
3.9, 4.2, 4.5, 5.0, 5.6, 6.0, 7.8, 9.5 and 10.2 were obtained. 
PRACTICAL EXAMPLE 1 
1.2 g of acrylamide, 60 mg of N,N'-methylenebisacrylamide, 0.012 mg of 
riboflavin, 0.135 ml of N,N,N',N'-tetramethylethylene diamine, 5 mg of 
ammonium persulfate and 1.2 ml of 40% Ampholine (pH 3.5-10) (Trade name, 
made by LKB Co., Sweden) was dissolved in pure water and brought to 24 ml. 
This solution was sufficiently mixed together in a flask, and after the 
deairing thereof in a suction desiccator, it was charged in a glass column 
(5.times.100 mm) up to about 70 mm, and photopolymerization was carried 
out and thus a gel for isoelectric separation was prepared. 
On the other hand, as isoelectric point markers, a combination of five of 
the separate kinds of acetylated cytochrome c having isoelectric points 
respectively of 4.9, 5.6, 6.5, 8.1 and 9.5 obtained by Production Example 
1 was provided. 
EDTA, mercaptoethyl alcohol and Tris-HCl buffer of pH 7.5 were added to the 
liver of a rat, and the mixture was ground and centrifuged at 
105,000.times.g thereby obtaining a supernatant liquid. 14 ml of said 
supernatant liquid (corresponding to 7 mg of the liver) was used as a 
sample, to which was added 10 ml of the combined solution containing 20 mg 
of each said isoelectric point marker acetylated cytochome c and 24 ml of 
60% glycerine solution (Ampholine concentration 4% of Ampholine (pH 
3.5-10), and the resulting mixture was quietly poured onto said gel 
thereby preparing a sample layer. On this sample layer 50 ml of a 
protective layer composed of 15% glycerine and 2% Ampholine (pH 3-6) was 
overlaid. 
The lower end of the sample gel thus obtained was dipped in a catholyte 
composed of 1 M NaOH aqueous solution, and the upper end thereof was 
dipped in an anolyte composed of 0.02 M phosphoric acid aqueous solution, 
and electricity was passed between the lower and upper ends at a constant 
voltage of 200 v for five hours. During this passage of electricity, the 
temperature of the column was maintained at a temperature of 
0.degree.-1.degree. C. by means of cooling water. 
After stopping the pass of electricity, the gel was taken out of the 
column, the position of isoelectric point marker was detected by the naked 
eye, and on the other hand, the gel was sliced to obtain pieces of 5 mm in 
breadth, and Ampholine was extracted by use of 0.5 ml of water, and pH was 
respectively measured, and a pH-gradient diagram was made from each said 
position and isoelectric point. 
The result thereof is shown in FIG. 1 from which it is clear that the 
pH-gradient formed by use of the prior art 5 mm-slicing method and the 
pH-gradient formed by the visual observation by use of the isoelectric 
point marker of the present invention coincide well. 
Moreover, each of the pieces sliced into fourteen equal pieces at intervals 
of 5 mm was added with 0.6 ml of 0.1 M Tris-HCl buffer containing 10 mM 
EDTA and 20 mM 6-mercaptoethyl alcohol and then homogenized. This 
homogenized liquid was centrifuged at 3000.times.g for 15 min., and the 
pyruvate-kinase activity of the resulting supernatant liquid was measured. 
As shown in FIG. 2 showing the result, the pyruvate-kinase of rat liver was 
separated into three isoelectric point isoenzymes and the isoelectric 
points were obtained from positions showing each active peak, with the 
result that the respective value of 1 (pI=4.9), 2 (pI=6.2) and 3 (pI=7.8), 
placing in order from the lower one, were obtained. 
PRACTICAL EXAMPLE 2 
Quite like Practical Example 1, pI isoenzyme pattern of pyruvate kinase of 
normal rat and tumor-bearing rat (inoculated with Rhodamine-sarcoma) were 
inspected. The cutting at the time of measurement was roughly performed 
centering around the positions of pH 4.9=L-type, pH 6.2=L-type, pI 
7.8=S-type according to the pH-gradient obtained from the pI marker, and 
thus the measurement was carried out. 
The result is shown in FIG. 3, wherein c denotes the pH-gradient of the pI 
marker, IV denotes the detection amount of L-type pyruvate kinase, V 
denotes that of L-type pyruvate kinase, and VI denotes that of S-type 
pyruvate kinase. The solid line shows those of the normal rat, and the 
broken line shows those of the tumor-bearing rat. 
From this drawing it is apparent that by use of the pI marker of the 
present invention it is possible to easily detect the fact that in the 
case of the tumor-bearing rat S-type pyruvate kinase was increased 
abnormally. 
It will be obvious to those skilled in the art that various changes may be 
made without departing from the scope of the invention and the invention 
is not to be considered limited to what is described in the specification.