Separation and purification of biomembrane proteins

A method for separating and purifying a biomembrane protein from biomembrane by subjecting the biomembrane to gel electrophoresis in the presence of at least one anionic surfactant having the general formula (I): EQU RO--XO).sub.m (YO).sub.n SO.sub.3 M (I) wherein R represents an alkyl group having 6 to 22 carbon atoms or an alkylphenyl group having 6 to 22 carbon atoms, X and Y independently represent a hydrocarbon residue having 1 to 4 carbon atoms, m and n independently represent a number of from zero to 40 provided that m+n is 4 to 40, and M represents an alkali metal, an alkaline earth metal, an amine, or ammonium. Thus, the desired biomembrane protein can be separated and purified with a high purity without denaturing the protein and also without impairing the biological function thereof.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method for separating and purifying a 
biomembrane protein from a biomembrane. A biomembrane protein separated 
and purified at a high purity is important not only for studying the 
function and structure of a biomembrane protein, but also because it has 
become useful as a product in the fields of pharmacology, medicine, and 
engineering. According to the present invention, the desired biomembrane 
protein can be separated and purified from a biomembrane, with a high 
purity and without impairing the biological function thereof, by 
subjecting the biomembrane to gel electrophoresis in the presence of a 
specified anionic surfactant. 
2. Description of the Related Art 
A biomembrane is composed mainly of polar lipids and membrane proteins. The 
membrane proteins maintaining their biological functions are inserted into 
a bilayer membrane composed of the polar lipid, especially phospholipids. 
Most of the biomembrane proteins such as porin of the Escherichia coli 
outer membrane, cytochrome b.sub.5, (Na.sup.+, K.sup.+)ATPase, 
(Ca.sup.++)ATPase, and (H.sup.+)ATPase are only slightly soluble in water 
and, therefore, when these proteins are separated from the biomembrane, 
the desired biomembrane protein should be solubilized in the first step of 
the separation and purification operation, unlike water-soluble globular 
proteins. To solubilize membrane proteins, media having an environment or 
situation similar to that of the lipid bilayer are required. Various 
organic solvents and surfactants have been used for the above-mentioned 
purpose. Typical examples of such organic solvents being acetone, butanol, 
ethanol, pyridine, and so on and typical examples of such surfactants 
being anionic surfactants represented by sodium dodecylsulfate, cationic 
surfactants represented by trimethyldodecyl ammonium chloride, and 
nonionic surfactants represented by polyoxyethylene dodecyl ether. 
However, since most organic solvents act as a strong denaturing agent 
against proteins, it is usually difficult to separate and purify the 
desired biomembrane protein from the biomembrane without impairing the 
biological function thereof. Furthermore, since sodium dodecylsulfate, 
(i.e., "SDS") conventionally used as a typical anionic surfactant in 
biochemical fields acts as a strong protein denaturing agent, it is 
usually difficult to separate and purify the desired biomembrane protein 
without impairing the biological function thereof. Various attempts have 
been made to solve the above-mentioned difficulties by using, as a medium 
for solubilizing biomembrane proteins, nonionic surfactants having a low 
protein denaturation power. However, the critical micelle concentrations 
of most nonionic surfactants are so low that it becomes difficult to 
remove the surfactant molecules bound to the protein by dialysis after the 
separation and purification of the desired biomembrane protein. 
Bile acid salts may be used, as an anionic surfactant having a low protein 
denaturation power, for solubilizing biomembrane protein. However, the 
bile acid salts or similar natural surface active substances are 
practically useless in that they are not available in large amounts for 
commercial or industrial use. On the other hand, cationic surfactants are 
commonly used as a germicide, since they are strongly bound to lipids 
constituting the biomembrane when compared with the other surfactants, and 
since the denaturation power of cationic surfactants against protein is 
not weak. There are few (or substantially no) cases in which the 
separation and purification of the biomembrane proteins can be 
successfully carried out by using cationic surfactants. Thus, these 
cationic surfactants are not widely used in the separation of the 
biomembrane proteins. 
Various separation and purification methods utilizing the physical or 
chemical characteristics of proteins have been proposed, such as thermal 
or pH treating methods, fractional precipitation methods, absorption and 
desorption methods, chromatographic methods utilizing ion exchanging, 
isoelectric fractionation methods, density gradient centrifugation 
methods, electrophoresis methods, affinity chromatographic methods, 
molecular sieve methods, two phase partition methods, and crystallization 
methods. These methods have both merits and demerits. 
SUMMARY OF THE INVENTION 
Accordingly, the objects of the present invention are to eliminate the 
above-mentioned disadvantages and to provide a method for separating and 
purifying a biomembrane protein from a biomembrane, by utilizing gel 
electrophoresis in the presence of a specified anionic surfactant, with a 
high purity and without causing any denaturation of the protein or 
impairing the biological function thereof. 
Other objects and advantages of the present invention will be apparent from 
the description set forth hereinbelow. 
In accordance with the present invention, there is provided a method for 
separating and purifying a biomembrane protein from a biomembrane by 
subjecting the biomembrane to gel electrophoresis in the presence of at 
least one anionic surfactant having the general formula (I): 
EQU RO--XO).sub.m (YO).sub.n SO.sub.3 M (I) 
wherein R represents an alkyl group having 6 to 22 carbon atoms or an alkyl 
phenyl group having 6 to 22 carbon atoms, X and Y independently represent 
a hydrocarbon residue having 1 to 4 carbon atoms, m and n independently 
represent a number of from zero to 40 provided that m+n is 4 to 40, and M 
represents an alkali metal, an alkaline earth metal, an amine, or 
ammonium. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
(1) Surfactants 
The anionic surfactants usable in the present invention for solubilizing a 
biomembrane protein without denaturing the protein are those having the 
above-mentioned general formula (I). In the general formula (I), R 
represents an alkyl group having 6 to 22 carbon atoms or an alkylphenyl 
group having 6 to 22 carbon atoms. The alkyl group may be linear or 
branched and R may have unsaturated hydrocarbon atoms. X and Y 
independently represent a hydrocarbon residue having 1 to 4 carbon atoms 
such as --CH.sub.2 --, --CH.sub.2 CH.sub.2 --, --CH.sub.2 CH(CH.sub.3)--, 
and --CH.sub.2 CH(C.sub.2 H.sub.5)-- and these may be either a 
homopolymer, block copolymer, or random copolymer. The references m and n 
independently represent a number from zero to 40, provided that m+n is 4 
to 40, which represent the addition mole number or the average addition 
mole number of aklylene oxides. M represents an alkali metal such as 
sodium, lithium, potassium, an alkaline metal such as magnesium, calcium, 
an amine such as triethanolamine, diethanolamine, monoethanolamine, 
monoisopropanolamine, diisopropanolamine, triisopropanolamine, pyridine, 
morpholine, or ammonium. When the carbon atom number of R in the formula 
(I) is less than 6, the biomembrane protein cannot be effectively 
solubilized due to a decrease in the surface activity of the surfactant. 
Contrary to this, when the carbon atom number of R in the formula (I) is 
more than 22, it becomes difficult to remove the surfactant molecules by 
dialysis from the membrane protein-surfactant complexes after the 
separation of the protein. 
Preferably, group R is a linear alkyl group having 8 to 16 carbon atoms or 
a branched alkylphenyl group having 6 to 14 carbon atoms and m+n is 4 to 
20. Especially, anionic surfactants having the general formula (I) in 
which R is a linear alkyl group having 10 to 14 carbon atoms or a branched 
alkylphenyl group having 8 to 12 carbon atoms, X and Y are --CH.sub.2 
CH.sub.2 --, and m+n is 4 to 15. In the general formula (I), when m+n is 3 
or less, the kinds of biomembrane proteins separated without impairing 
their biological functions are disadvantageously limited, since the 
denaturation power of the surfactant against the protein is increased. 
Contrary to this, when m+n is 21 or more, the surfactant exhibits nonionic 
surfactant-like phenomena in spite of being anionic surfactants and, 
therefore, it becomes difficult to remove the surfactant molecules by 
dialysis from the membrane protein-surfactant complexes after the 
separation of the desired biomembrane protein. 
Examples of the anionic surfactants having the general formula (I) are as 
follows: 
Polyoxyethylene (ave. 5 mol) octyl ether sulfates; polyoxyethylene (ave. 5 
mol) decyl ether sulfates; polyoxyethylene (ave. 5 mol) lauryl ether 
sulfates; polyoxyethylene (ave. 8 mol) lauryl ether sulfates; 
polyoxyethylene (ave. 20 mol) lauryl ether sulfates; hexaoxyethylene 
dodecyl ether sulfates; octaoxyethylene dodecyl ether sulfates; poly 
(oxypropylene (ave. 2 mol)-oxyethylene (ave. 5 mol)) lauryl ether 
sulfates; poly (oxybutylene (ave. 1 mol)-oxyethylene (ave. 5 mol)) lauryl 
ether sulfates; polyoxyethylene (ave. 7 mol) cetyl ether sulfates; 
polyoxyethylene (ave. 10 mol) oleyl ether sulfates; polyoxyethylene (ave. 
6 mol) sec-lauryl ether sulfates; polyoxyethylene (ave. 7 mol) octylphenyl 
ether sulfates; polyoxyethylene (ave. 7 mol) nonylphenyl ether sulfates; 
polyoxyethylene (ave. 12 mol) nonylphenyl ether sulfates. 
Preferable examples of the anionic surfactants usable in the present 
invention are sodium polyoxyethylene (ave. 8 mol) lauryl ether sulfate, 
sodium octaoxyethylene dodecyl ether sulfate, sodium polyoxyethylene (ave. 
6.9 mol) branched nonylphenyl ether sulfate, ammonium polyoxyethylene 
(ave. 7 mol) lauryl ether sulfate, triethanolammonium polyoxyethylene 
(ave. 6 mol) dodecyl ether sulfate, sodium poly (oxypropylene (ave. 2 
mol)-oxyethylene (ave. 5 mol)) lauryl ether sulfate. 
(2) Gel Electrophoresis 
According to the present invention, the solubilized biomembrane protein is 
subjected to gel electrophoresis. The supporting media usable in the gel 
electrophoresis according to the present invention include, for example, 
cellulose acetate, Sephadex.RTM. (Sepharose available from Pharmacia), 
vinyl chloridevinyl acetate copolymer, polyvinyl chloride, starch powder, 
starch gel, agarose gel, and polyacrylamide gel. Of these media, the use 
of polyacrylamide gel is especially preferable. This is because the 
polyacrylamide gel has various advantages; namely, it is chemically 
stable, the gel concentration and crosslink proportion can be freely 
controlled (i.e., the average pore size can be readily varied depending 
upon the intended use), it has substantially no electroendosmosis, it is 
not substantially affected by a change in the ambient pH and temperature, 
it can be formed or molded in any desired form or shape, and the 
reproducibility in the gel electrophoresis is very high. 
A typical example of the electrophoresis operation according to the present 
invention will be now specifically explained. Although polyacrylamide gel 
is used as a supporting medium in the following explanation, it should be 
noted that the supporting medium usable in the present invention is by no 
means limited to polyacrylamide. 
(i) Apparatus and Devices 
These consist of a cylindrical tube supporting the polyacrylamide gel and 
vessels containing a buffers solutions provided at both sides of the tube. 
The dimensions and volumetric capacities of the cylindrical tube and 
vessels can be optionally decided or selected depending upon, for example, 
the kinds and amount of protein to be separated and purified. 
(ii) Electric Power 
The use of direct current generating apparatus, especially constant current 
or constant voltage generating apparatus, is preferable in that the 
capability and reproducibility of the method in the present invention is 
thereby increased, although any conventional electric power generation 
apparatus can be used in the practice of the present invention. 
(iii) Preparation of Polyacrylamide Gel for Electrophoresis 
A 1% to 30% by weight amount of acrylamide, 0.05% to 10% by weight, based 
on the amount of the acrylamide, of N,N'-methylene bisacrylamide, and 
0.01% to 1% by weight of an anionic surfactant having the above-mentioned 
general formula (I) are dissolved in water. The resultant aqueous solution 
is polymerized to cause gelation. The preferable gel concentration is such 
that the total amount of the acrylamide and the 
N,N'-methylenebisacrylamide in the gel is 3% to 15% by weight, and the 
amount of the N,N'-methylenebisacrylamide is 1% to 5% by weight based on 
the amount of the acrylamide. Although there is no specific limitation to 
the concentration of the surfactant, the preferable concentration of the 
surfactant in the gel is 0.01 to 1% by weight, especially 0.05 to 0.2% by 
weight. It should be noted that any conventional additives such as 
polymerization catalysts, polymerization accelerators, pH buffers, and 
preservatives can be used in the gelation. For instance, photoinitiators 
such as riboflavin or radical polymerization initiators such as ammonium 
persulfate may be used in any conventional amount, preferably 0.04% to 
0.12% by weight based on the weight of the polymerization mixture. Any 
conventional polymerization accelerators such as N,N,N',N'-tetramethyl 
ethylenediamine may also be used in any appropriate concentration, 
preferably 0.1% to 0.5% by weight. 
Since the dissolved oxygen inhibits the gelation, it is sometimes 
preferable if the dissolved oxygen in the aqueous polymerization solution 
is degassed under reduce pressure and that the upper layer portion of the 
aqueous polymerization solution is overlayered with distilled water to 
prevent direct contact with air. The pH buffers optionally usable in the 
practice of the gelation include, for example, sodium dibydrogen 
phosphate-disodium hydrogen phosphate system, sodium carbonate-sodium 
hydrogen carbonate system. Thus the desired gel electrophoresis can be 
effected at an intended pH condition. Although there is no critical 
limitation to the concentration of the pH buffers, the concentration of 
the pH buffers is generally adjusted in the range of 10 to 500 mM, 
preferably 50 to 200 mM, more preferably about 100 mM. 
(iv) Aqueous Solution in Buffer Vessels 
Both ends of the polyacrylamide gel for the electrophoresis should be 
filled with a buffer solution. The buffer solution generally comprises a 
buffer which is used in the preparation of the aqueous solution, the 
surfactant, and water. The concentrations of the buffer and the surfactant 
are preferably the same as those used in the aqueous solution for the 
preparation of the above-mentioned polyacrylamide gel. 
(v) Aqueous Solution Containing a Biomembrane Protein to be Separated 
An aqueous solution containing biomembrane protein to be separated 
generally contains, in addition to the protein, 0.1% to 5% by weight, 
preferably 0.2% to 3% by weight, more preferably 0.5% to 2% by weight of 
the surfactant. This aqueous solution optionally contains 1% to 30% by 
weight, preferably 10% to 20% by weight, of viscous liquid such as 
glycerine to improve the desired separation and purification efficiency. 
The above-mentioned aqueous solution may contain the buffer in the gel and 
the buffer solution. The concentration of the buffer in the aqueous 
solution is, preferably, 500 mM or less and is the same as or less than 
that of the above-mentioned buffer solution. The most preferable 
concentration of the buffer in the aqueous solution is 1/2 to 1/20 that of 
the above-mentioned buffer solution. 
Furthermore, the above-mentioned aqueous solution may contain water-soluble 
anionic dyes such as bromphenol blue for showing the relative mobility, 
and micelle-soluble but water-insoluble coloring pigments or dyes such as 
oil-soluble Yellow OB dye for showing the mobility of the surfactant 
micelle. The concentration of these dyes and pigments can be appropriately 
selected but, preferably, the concentration of the water-soluble dyes is 
about 0.001% to 0.05% by weight and that of the water-insoluble pigments 
or dyes is 0.01% to 0.5% by weight. 
The desired electrophoresis is started by placing the above-mentioned 
aqueous solution containing the biomembrane protein on the upper end 
portion of the polyacrylamide gel. The biomembrane protein is separated 
and purified in the form of a disc in the gel. After a dye staining is 
accomplished by immersing the gel in a dye solution, excess back ground 
stain is removed by repeated washing of the gel in 7% by weight acetic 
acid. The separated biomembrane protein in the form of a disc can be 
observed as a colored band. The dyes usable in this operation include, for 
example, amido black and Coomassie Brilliant Blue. The separation and 
purification of the designed biomembrane protein can be confirmed by any 
conventional method (e.g., see Katsuya Hayashi, "Experimental Method of 
Biochemistry-Electrical Properties of Protein" ed. by Ikuzo Uritani, 
Kensuke Shimura, Michinori Nakamura, and Katsuji Funazu, pages 30 to 39, 
published in 1971 by Gakkai Shuppan Center, Japan). 
(3) Substance to be Separated and Purified According to the Present 
Invention 
The substances to be separated and purified according to the present method 
are all biomembrane proteins extracted and solubilized from, for example, 
animal organs, cultured cells, microorganism cells, and plant cells. 
(4) Separation and Purification 
Supernatant solutions containing all of the solubilized biomembrane protein 
can be directly separated and purified by the method according to the 
present invention. Furthermore, solutions containing the membrane proteins 
obtained by treatment of any conventional nucleic acid removal, or 
solutions obtained by previous treatment conventional initial purification 
methods according to with their intended purpose, such as fractional 
precipitation methods and density gradient centrifugation methods, can be 
further highly purified. 
Note that, when the above-mentioned rough pre-treatment is carried out 
prior to the practice of the separation and purification of biomembrane 
protein according to the present invention, it is preferable to use a 
method such that the biological function of the desired biomembrane 
protein is not impaired or that, even if the denaturing of the desired 
protein occurs, the desired activity can be reversibly recovered when 
factors causing the denaturation are eliminated. As mentioned above, the 
separation and purification method of a biomembrane protein according to 
the present invention can be advantageously applied to any kind of 
biomembrane proteins and to any separation and purification step of a 
biomembrane protein. 
Although there are no critical limitations to the temperature and pH of the 
system during the separation and purification according to the present 
invention, the preferable temperature is 0.degree. C. to 40.degree. C., 
more preferably 0.degree. C. to 20.degree. C., and the preferable pH is 4 
to 9, especially about 7. A temperature of less then 0.degree. C. 
sometimes tends to allow the water in the gel to be frozen and, therefore, 
electrophoresis becomes impossible. Contrary to this, a temperature of 
more than 40.degree. C. sometime tends to cause thermal denaturation of 
the desired biomembrane protein, whereby the biological function of the 
biomembrane protein is impaired. On the other hand, when the pH of the 
system is less than 4 or more than 9, an unpreferable acid or alkali 
denaturation of the protein sometimes occurs. 
As mentioned hereinabove, according to the present invention, the following 
advantageous characteristic are obtained. 
(a) The anionic surfactants having the general formula (I) have low protein 
denaturing properties and, unlike bile acid salts, they are readily 
synthesized industrially at a low cost. 
(b) The protein denaturing properties of the anionic surfactants (I) are 
extremely low and similar to those of nonionic surfactants. Since the 
amount of the anionic surfactants (I) bound to the solubilized biomembrane 
proteins differs from one another, each protein-surfactant complex has a 
different electric charge. Therefore, these complexes move through 
electrophoresis supporting media at inherent velocities toward an anode. 
During this movement, molecular sieving effects of the supporting media 
can be utilized and, therefore, the separation and purification efficiency 
is extremely increased when compared with a conventional gel filtration 
method of a biomembrane protein solubilized with nonionic surfactants. 
(c) The second defect of the conventional gel filtration methods of a 
biomembrane protein solubilized with nonionic surfactants is that the use 
of a large amount of a solvent is required. In the practice of the 
separation and purification method according to the present invention, the 
use of such a solvent is advantageously not required. 
(d) It is often difficult to remove nonionic surfactants bound to a 
biomembrane protein by dialysis, because the critical micellar 
concentrations of nonionic surfactants are quite low. Contrary to this, 
since the critical micellar concentration of the anionic surfactants (I) 
used in the present invention is high when compared with nonionic 
surfactants, the anionic surfactants (I) can be readily removed by 
dialysis or electrodialysis. 
(e) The anionic surfactants (I) usable in the present invention not only 
solubilize biomembrane proteins without denaturing, but also remarkably 
suppress the denaturation of a biomembrane protein in a medium utilized in 
the present invention.

EXAMPLE 
The present invention will now be further illustrated by, but is by no 
means limited to, the following Examples, Reference Examples, and 
Comparative Examples, in which (Na.sup.+, K.sup.+) ATPase of canine renal 
outer medulla was separated and purified. 
(1) The ATPase activity after the separation and purification was 
determined as follows. 
An appropriate amount of phospholipids derived from soybean is added to a 
30 mM imidazol/30 mM glycidyl glycine buffer solution (pH 7.2, 20.degree. 
C.) containing 4 mM ATP, 100 mM NaCl, 25 mM KCl, 3.9 mM MgCl.sub.2, and 
0.2 mM EDTA. The mixture was treated at a temperature of 37.degree. C. for 
2.5 to 4 minutes. Then, a concentrated aqueous SDS solution was added to 
terminate the reaction. Thereafter, the formation or non-formation of 
inorganic phosphoric acid was determined according to the methods of 
Hegyvary et al (see Anal. Biochem., 94, 397-401(1979)). 
(2) The degree of the separation and purification of biomembrane protein 
was evaluated as follows. 
After the polyacrylamide gel electrophoresis according to the present 
invention was completed, the gel was taken out and, then, a conventional 
SDS-polyacrylamide gel electrophoresis of it was performed using a slab 
type two-dimensional electrophoresis device. It is reported by Y. Hayashi 
et al., (B.B.A., 748, 153-167(1983)) that (Na.sup.+, K.sup.+) ATPase 
comprises two kinds of subunits, .alpha. and .beta., each having different 
molecular weights, and that a few or several subunits are gathered to form 
oligomers, such as .alpha..beta., (.alpha..beta.).sub.2 and 
(.alpha..beta.).sub.n type. When pure (Na.sup.+, K.sup.+) ATPase is 
subjected to SDS-polyacrylamide gel electrophoresis, only two bands 
corresponding to .alpha.- and .beta.-subunits are obtained and no other 
bands are observed. Accordingly, the purity of (Na.sup.+, K.sup.+) ATPase 
can be evaluated by the presence or absence of the third band after the 
electrophoresis. 
REFERENCE EXAMPLE 
Polyacrylamide gel electrophoresis of (Na.sup.+, K.sup.+) ATPase purified 
by a Jorgensen method disclosed in B.B.A., 356, 36-52(1974) was carried 
out at room temperature in the presence of C.sub.12 H.sub.25 O(CH.sub.2 
CH.sub.2 O).sub.8 SO.sub.3 Na. 
The compositions of gel and buffer solutions used in the one dimensional 
and two-dimensional polyacrylamide gel electrophoresis were as shown in 
Tables 1 and 2, respectively. 
TABLE 1 
______________________________________ 
Gel Composition 
______________________________________ 
Acrylamide 5 wt. % 
N,N'--methylenebisacrylamide 
2.7 wt. % 
(based on the 
amount of the 
acrylamide) 
Ammonium persulfate 0.07 wt. % 
N,N,N',N'--tetramethylene diamine 
0.15 wt. % 
Phosphate buffer (pH = 7) 
100 mM 
Surfactant 0.1 wt. % 
Water Balance 
______________________________________ 
TABLE 2 
______________________________________ 
Buffer Composition 
______________________________________ 
Phosphate buffer (pH = 7) 
100 mM 
Surfactant 0.1 wt. % 
Water Balance 
______________________________________ 
The gel electrophoresis was carried out according to a conventional 
SDS-polyacrylamide gel electrophoresis method disclosed in, for example, 
J. V. Maizel, Jr., "Methods in Virology", Academic Press. (1971), p179 and 
Toshio Takagi and Jun Miyake "Shin Jikken Kagaku Kouza Vol. 20, Seibutsu 
Kagaku I (Edited by Nippon Kagaku Kai)" Maruzen (1978), p109, except that 
no SS linkage dissociating agents were used and that SDS was substituted 
for the anionic surfactant, i.e., C.sub.12 H.sub.25 O--(CH.sub.2 CH.sub.2 
O).sub.8 SO.sub.3 Na. 
In summary, the polyacrylamide gel electrophoresis was carried out as 
follows: 
Polyacrylamide gel electrophoresis was carried out in gels (0.5.times.8.0 
cm) having a composition illustrated in Table 1. Bromophenol Blue was used 
as a marker dye. About 20 .mu.l of a sample solution containing about 10 
.mu.g of (Na.sup.+, K.sup.+) ATPase were applied per gel. Electrophoresis 
was performed at 10 mA per gel for 3 hr. Protein bands in the gel were 
stained by Amido Black, followed by destaining in acetic acid solution in 
order to remove excess stain from the background. 
As a result of the above mentioned electrophoresis, two clear bands were 
obtained. After migrating protein bands out of the gel, of course the gel 
is another one performed at the same time, the presence or absence of 
ATPase activity was determined. The result was positive. In addition, 
after removing the gel from the tube, the gel was subjected to a 
conventional SDS-polyacrylamide gel electrophoresis using a slab-type two 
dimensional electrophoresis apparatus. As a result, two spots 
corresponding to .alpha.- or .beta.-subunit, respectively, were obtained 
from two bands. No other spots were observed. It should be noted, however, 
that a difference between the mobility of the bands of the one-dimensional 
and two-dimensional gel electrophoresis was observed. It is believed that 
this exhibits the difference between the effect of the use of the anionic 
surfactant according to the present invention and SDS on the hydrodynamic 
volume of the .alpha.- and .beta.-subunits. That is, the two bands 
obtained by the polyacrylamide gel electrophoresis according to the 
present invention are .alpha.- and .beta.-subunits respectively and these 
subunits can retain the enzymatic activity by the reconstitution. That is, 
according to the present invention, the desired biomembrane protein can be 
separated and purified from a biomembrane, with a high purity and without 
impairing its biological function. 
EXAMPLE 1 
Microsome obtained from canine kidney was mixed with a 1% by weight aqueous 
solution of C.sub.12 H.sub.25 O (CH.sub.2 CH.sub.2 O).sub.8 SO.sub.3 Na. 
The mixture was subjected to ultrasound treatment and, then, was 
centrifugally separated. The supernatant was collected. The separation and 
purification of this supernatant was carried out under the same conditions 
as in the Reference Example. 
The gel portions exhibiting mobility corresponding to that of the two bands 
obtained in the Reference Example were cut off and the membrane protein 
contained in these portions was recovered. The ATPase activity of the 
recovered protein was positive. When the degree of the separation and 
purification was confirmed in SDS-polyacrylamide gel electrophoresis using 
a slab-type two-dimensional gel electrophoresis apparatus, clear spots 
were observed corresponding to .alpha.- and .beta.-subunits from the two 
bands and no other spots were observed. Thus, the degree of separation and 
purification of the membrane protein was extremely high. Furthermore, the 
difference in the mobility of each band between in the one-dimensional gel 
electrophoresis and two-dimensional gel electrophoresis was observed. 
EXAMPLE 2 
The gel electrophoresis separation and purification of the biomembrane 
protein was carried out in the same manner as in Example 1, except that 
the anionic surfactant of Example 1 was replaced by sodium polyoxyethylene 
(ave, 7 mol) laurylether sulfate. However, since the migration velocity of 
(Na.sup.+, K.sup.+) ATPase in the polyacrylamide gel electrophoresis 
according to the present invention is different depending upon the anionic 
surfactant used, the preliminary test set forth in the Reference Example 
was carried out by substituting the above-mentioned surfactant for the 
surfactant in the Reference Example, whereby the migrated position of the 
desired membrane protein was previously determined. 
As a result, the ATPase activity was positive and the purity was very high. 
EXAMPLE 3 
The gel electrophoresis separation and purification of the biomembrane 
protein was carried out in the same manner as in Examples 1 and 2, except 
that sodium polyoxyethylene (ave, 10 mol) nonylphenyl ether sulfate was 
used as the anionic surfactant. 
The ATPase activity was positive and the purity was extremely high. 
COMATIVE EXAMPLE 1 
The gel electrophoresis separation and purification of the Reference 
Example was carried out by using, as a surfactant, SDS. As a result, two 
clear bands were obtained. The two clear band portions were cut off, and 
then the two band portions were reconstituted. When the ATPase activity 
was determined the result was negative. Furthermore, when the purity was 
determined by using a slab-type two dimensional gel electrophoresis 
method, one of the two clear bands having the larger mobility yielded a 
single spot also having a large mobility in the two-dimensional 
electrophoresis. On the other hand, the other clear band having a 
relatively small mobility also yielded a single spot having a relatively 
small mobility. There was no substantial difference in the mobility of 
each hand between the two dimensional electrophoresis and the first 
one-dimensional electrophoresis. 
As is clear from the above-mentioned results, when SDS is used, the 
hydrodynamic volumes of the .alpha.- and .beta.-subunits of (Na.sup.+, 
K.sup.+) ATPase were changed so that recovery of the enzymatic activity 
was impossible. 
EXAMPLE 4 
The gel electrophoresis separation and purification of the biomembrane 
protein was carried out in the same manner as in Example 3, except that 
sodium poly(oxypropylene (ave. 2 mol)-oxyethylene (ave. 3 mol.) lauryl 
ether sulfate was used as the anionic surfactant. 
As a result, the ATPase activity was positive and the purity was extremely 
high. 
EXAMPLE 5 
Microsome fractionatedly obtained from canine kidney was mixed with a 1% by 
weight aqueous solution of C.sub.12 H.sub.25 O (CH.sub.2 CH.sub.2 O).sub.8 
SO.sub.3 Na. The mixture was subjected to ultrasound treatment and, then, 
was centrifugally separated. The supernatant was collected. The separation 
and purification of this supernatant was carried out under the same 
conditions as in the Reference Example, except that the gel was cooled 
with water having a temperature of 0.degree. C. during the 
electrophoresis. 
As a result, one clear band was observed. The portion was recovered from 
the gel, in which electrophoresis was performed in parallel and not 
stained, by cutting off the gel portion corresponding to the clear band. 
The ATPase activity of this was positive. In addition, the degree of 
separation and purification was determined by a conventional 
SDS-polyacrylamide gel electrophoresis using a slab-type two-dimensional 
electrophoresis apparatus. As a result, spots corresponding to .alpha.- 
and .beta.-subunits, respectively, were obtained. No other spots were 
observed. 
Accordingly, it is clear that, when the gel electrophoresis was carried out 
around 0.degree. C., the recovery of the enzymatic activity is 
advantageously effected by the reconstitution since the splitting of the 
protein into .alpha.- and .beta.-subunits was suppressed. 
EXAMPLE 6 
The gel electrophoresis separation and purification of the biomembrane 
protein was carried out in the same manner as in Example 5, except that 
sodium polyoxyethylene (ave. 7 mol) lauryl ether sulfate was used as the 
anionic surfactant. 
As a result, the ATPase activity was positive and the purity was extremely 
high. 
EXAMPLE 7 
The gel electrophoresis separation and purification of the biomembrane 
protein was carried out in the same manner as in Example 5, except that 
sodium poly(oxypropylene (ave. 2 mol)-oxyethylene (ave. 5 mol) lauryl 
ether sulfate was used as the anionic surfactant. 
As a result, the ATPase activity was positive and the purity was extremely 
high.