- Email: [email protected]
Effect of nonionic detergent on fractionation of proteins by isoelectric focusing
ANALYTICAL
Effect
BIOCHEMISTRY
of
Nonionic
41,
149-157
(1971)
Detergent
on Fractionation
by Isoelectric A. D. FRIESEN, Department
of
Chemistry,
Received
of
Proteins
Focusing
J. C. JAMIESON, University
of
Manitoba,
October
.4XD
F. E. ASHTON
Winrkpeg
19, Manitoba,
Canada
28. 1970
The technique of isoelectric fractionation or isoelectric focusing involves the fractionation of large molecular weight ampholytes, such as proteins, according to their isoelectric points by exposure to an applied voltage in a natural pH gradient. Although the technique has been known to be theoretically possible for some time (1) it did not become a practical reality until 1966, when Vesterberg and Svensson (2) successfully synthesized a series of ampholytes capable of producing a natural equilibrated pH gradient. Since that time isoelectric focusing has been used to prepare a variety of proteins and other large molecular weight ampholytes (e.g., 3-6). One disadvantage of the t’echnique, when applied to the separation of proteins, is that there is a tendency for precipitation to occur when proteins attain their isoelectric points, thus resulting in wide bands and poor fractionation. The present work describes a technique which essentially eliminates precipit’ation of proteins during isoelectric focusing. This was accomplished by including the nonionic detergent Brij 35 in the pH gradient. Proteins were recovered by chromatographic and electrophoretic techniques and were examined by electrophoresis on starch gel. Infrared spectroscopy was employed to determine whether any detergent was bound to proteins recovered after isoelectric focusing in presence of Brij 35. MATERIALS
Chemicals were obtained as follows: Bovine serum albumin icrystallized and lyophilized), bovine hemoglobin, type 1, human albumin, grade III, and bovine thyroglohulin, type 1, Sigma Chemical Company, St. T,oui?;, MO. Brij 35 (polyoxyethylene lauryl ether), Nutritional Biochemicals Corp., Cleveland, Ohio. Starch, hpdrolyzcd, Connaught Medical Research I,nboratorie$, Toronto, Ontario. 149
150
Extinctions at 280 mp were measured with a Beckman model DB ~l)cctrol)hotonletep. Extinctions in the visible region of the spectrum were measured with a Enicam SP 600 spectrophotometer. Measurements of pH were made with a radiometer model 28 b pH-meter. Infrared spectra were obtained on a Perkin-Elmer 337 spectrophotometer; KBr discs were l)repared from a mixture of 1 mg protein and 6 mg KBr. Isoelectric
Focusing
The isoelectric focusing column (LKB 8100-10, Volume 110 ml) gradient mixing device (LKB 8121) and pH 3-10 ampholine carrier ampholytes were obtained from LKB Producter AB, Stockholm-Bromma 1, Sweden. ,4 dense electrode solution containing 0.2 ml concentrated sulfuric acid, 12 gm sucrose and 14 ml water was added to the anode at the hottom of the column. A sucrose gradient cont’aining carrier ampholytes was slowly introduced into the column using the LKB gradient device. The dense gradient solution contained 28 gm sucrose, 2 ml (40% w,/v) carrier ampholytcs, and 42 ml water. The light gradient solution contained 0.5 ml carrier ampholytes in 60 ml water. The final concentration of carrier ampholytc,s in the gradient was 1% (w/v). After about 50 ml of the gradient had entered the column, t,hc sample (10 mg protein, WC “Results”), dissolved in 1 ml wat.er, was added to the light gradient soIution. When the column had filled, the light electrode solution, consisting of 10 ml NaOH (1% w/v), was added to the cathode at the top of the column. About 3 hr was required to fill the column. When fractionations wcrc carried out in presence of detergent 1.0 ml of Brij 35 (30% w/v) was added to each gradient solution. A potential of 300V was applird to the column for 72 hr, after which time the current dropped from 10 to 1 mA. ,411 operations wrre performed at 2” and the column n-as maintaiucd at 2” during fractionation by circulating refrigerant through the cooling system of the column. When isoelectric focusing was cornplctcd the valrc at the bottom of the column was closed and the column was cmpticd by the lower exit at a flow rate of about 1-2 ml/min. Fractions of 1 ml were collected by hand. The pH and extinction at 280 rnp of each sample was determined. Recovery
of Proteins after
Isoelectric Focusing
After isoelectric focusing, tubes containing protein were pooled and concentrated to about 5 ml by ultrafiltration with concurrent dialysis against water as described by Sober, Gutter, Wycoff, and Pet’erson (7).
Samples were finally freeze-dried. Detergent, when present, was removed in two ways: (1) 11 cm x 3 cm columns of DEAE-cellulose were prc1~~1 and equilibratccl with 0.02 M phosphate buffer, pH 8.0, as describetl l)y A&ton, ,Jamiebon, and Friesen (8). The protein sample was dissolved in I ml phosphate buffer, applied to the top of the column, and elutetl with buffer at a flow rate of 2 ml/min ; 3 ml fractions wcare collected 1,) hand. The detergent, which was neutral, passed through the column unabsorbed and was detected by measuring extinctions at, 250 mp. When no morn E,,,,-positive material eluted from the column 0.02 31 phosphate buffer, pH 5.0, containing 0.3 M NaCl, leas passed through to remove the prot’ein. Tubes containing protein were poolecl, concentrated, and freezeclricd ati described above. In a few instances protein was recovered by prcl)arativc elcrtrophoresis on Cellogcl strips or blocks as described by Ashton et al. (8). In most cases prot’eins migrated rapidly and could easily be separated from the neutral detergent, which did not migrate significantly. Electrophoretic
Methods
Starch gel electrophoresis was carried out by a method based on that described by Simkin, Skinner, and Scshadri (9). The troughs measured 18.5 cm X 5 cm X 0.6 cm and the horizontal procedure of Smithies (10) was followed; sampleswere applied on Rhatman No. 3 paper. The buffer used to prepare the gel contained 76 mM Tris and 6 mM citric acid, pH 9.1; the electrode and bridge compartments of the tank contained 300
IWfect
of Presence Proteins
TABLE 1 of 0.5y0 Brij 35 on Precipitation dlwiug Isoelectric F’oc.lwillg
Bovine albumill Hllman albllmin Bovine hemoglobin Bovine thyrogloblllin Rat serum Rat ~7,-glob11lin fmcl ion I:tit ~z-glot~~dilk frnc*tion
-
1 race
of
+ + + + if +++ ++
n 10 mg protein was used in all experiments except in the case of rat serllm when (ca. 18 mg proteill) were llsed. bThe degree of precipitation is itldicatetl by plus siglls: :t lighl precipitate, moderate precipit at,e, + + ; and a heavy prccipitatc + + +.
0.3 ml +;
a
152
FRIDSEN,
JAMIESON,
AND
ASHTON
mM boric acid and 50 mM NaOH, pH 8.2. A potential of 170V was applied for 6 hr at 2”. Gels were stained for protein with naphthalene black 10B (10). RESULTS
Isoelectric
Focusing
of Proteins
Samples of bovine and human albumin, bovine hemoglobin and thyroglobulin, rat serum and aI- and a,-globulin-containing fractions isolated from rat serum (the CQ- and a,-globulin fractions were fractions 5 and 4, respectively, as described by Ashton et al. (8)) were subjected to isoelectric focusing in presence and absence of 0.5% Brij 35. Table 1 shows that, in most cases, precipitation of protein that occurred during isoelectric fractionation was eliminated in presence of detergent. Typical elution profiles obtained following fractionation of bovine albumin and i.O-
.lO
Detergent
present
0.6.
-6 PH
20
Volume FIG.
0.5%
1. Isoelectric Brij 35: (0)
60
40
80
(ml)
focusing of 10 mg bovine optical density at 280 m&,
albumin (A) pH.
in absence
and
presence
of
ISOELECTRIC
FOCUSIKG
Detergent
WITH
Present
-2
N
: ;;
153
DETERGENT
vo-
m .o a 0
-10 Detergent
Absent
0.6 -
-6 PH
0.2
20
40
60
FIG. 2. Isoelectric focusing of 10 mg bovine thyroglobulin of 0.5% Brij 35: (0) optical density at 280 ~JL, (A) pH.
80
in absence and presence
bovine thyroglobulin in presence and absence of detergent are shown in Figs. 1 and 2. The elution profiles obtained following fractionation in presence of detergent were generally sharper t’han those obtained in it,s absence (Figs. 1 and 2). Furthermore, there did not appear to be any change in isoelectric points of the proteins examined as a result of the presence of detergent during isoelectric focusing. Examination
of Proteins after Isoelectric Focusing
Detergent was removed from proteins after isoelectric focusing by preparative electrophoresis or by stepwise elution from columns of DEAE-cellulose. Figure 3 shows a typical elution profile of bovine albumin following chromatography on DEAE-cellulose; similar profiles were obtained with the other proteins examined. The technique of isoelectric focusing in presence of detergent will only be useful for fractioning proteins that tend to precipitate if proteins can
154
FIG.
cellulose 35. The densities position protein;
FRIE.C;EN,
JAMIESOP;,
ATW
SSH’WN
3. Stepwise elution chromatography on a 11 cm X 3 cm column of DEAEof bovine albumin recovered after isoelectric focusing in presence of Brij column was cluted initially with 0.02 M phosphate buffer, pH S.0; optical at 250 mp were determined to detect Brij 35. The arrow indicates the at which 0.02111 phosphate buffer, 0.3 M NaCI, pH 5.0, was added to elutc optical densities at 280 ~JL were determined to detect protein.
be removed in an unaltered form without having detergent bound to them. In order to test for the presence of det,ergent, protcii>s were examined by infrared spectroscopy. Brij 35 has two strong absorption bands at 1120 cm1 and 2950 cm-l (Fig. 4a) and was detected by infrared spectroscopy in quantities as low as 20 pg in presence of 1 mg protein (cf. Figs. 4b and 4~). Therefore, proteins recovered after isoelectric focusing in presence and absence of Brij 35 were examined by infrared spectroscopy. A typical infrared red spectrum obtained from bovine albumin isolated following isoelectric focusing in presence of detergent is shown in Fig. 4~1; the spectrum shown in Fig. 4d was identical t’o that of unfractionated bovine albumin (Fig. 4~). With all the proteins used in the present work (Table I) there was no difference between the infrared spectra of proteins recovered after fractionation in presence of detergent when compared with corresponding proteins recovered aft,er fractionation in absence of detergent. In none of t’he samples examined was there any indication t,hat t’here was contamination of protein by residual Brij 35. In order to determine if there was any change in physical characteristics of prot’eins as a result of isoelectric focusing, recovered proteins were compared with samples of original unfractionated prot#eins by electrophoresis on starch gel. Typical electrophoresis pat,terns of some of the proteins examined are shown in Fig. 5. There was no apparent. difference in electrophoret,ic mohilities of proteins isolated following isoelectric focusing in presence and absence of detergent when compared with corresponding unfrartionatcd proteins. Moreover, in most cases, the prot,eins recovered after isoelectric focusing were devoid of minor contaminating proteins that were present in the unfractionated material.
ISOELECTRIC
FOCUSING
WITH
DETERGENT
156
FRIESEN,
JAMIESON,
AND
ASHTON
thyroglobulin I.” ._“.
.
i
._
a
_. -. . .“.“& ---““.-b c
a
b
c
FIG. 5. Electrophoresis on starch gel of bovine albumin and bovine thyroglobulin: (a) protein recoveredafter isoelectricfocusingin absenceof Brij 35, (b) unfractionated protein, (c) protein recoveredafter isoelectricfocusingin presenceof Brij 35.
DISCUSSION
The procedure of separating biologically interesting proteins by isoelectric focusing has, in many instances, proved disappointing because of the tendency for precipitation to occur. In order to avoid or minimize precipitation workers have resorted to a variety of techniques such as increasing ampholyte concentration (11)) decreasing the concentration of material to be fractioned, adding urea (12)) or focusing proteins in a region of the column containing a high sucrose concentration (5). These techniques either require large amounts of expensive ampholytes or are otherwise undesirable. In the present work it was found that the addition of 0.576 of the nonionic detergent, Brij 35, was sufficient to keep proteins in solution during isoelectric focusing. The use of detergent had been previously described by Godson (13) for the isoelectric focusing of hemo-
globin, but no attempt
was made to reisolate and characterize the protein
examined. In the present studies proteins isolated by isoelectric focusing in presence of detergent were characterized by electrophoresis on starch gel and found to be identical with corresponding proteins recovered after isoelectric focusing in absence of detergent and with unfractionated proteins. Furthermore, the isoelectric points of the proteins examined did not appear to change when detergent was present during isoelectric focusing.
The detergent was easily removed by chromatography
or electrophoresis
and proteins were recovered free of detergent as determined by infrared spectroscopy. The technicme of adding detergent during isoelectric focusing of proteins has the effect of increasing resolution of proteins in cases in which precipitation of proteins resulted in wick bands. The technique
ISOELECTRIC
FOCUSING
WITH
DETERGENT
157
has the further advantage of allowing larger alnounts of protein to be focused into a single band. The technique of isoelectric focusing in presc‘nce of detergent has been used rout’inely in this laboratory and appears to be desirable when there is a chance of precipitation of protein occurring. SUMMARY
The nonionic detergent Brij 35 was found to eliminate precipitation of protein during isoelectric focusing. Proteins were recovered after isoelectric focusing by chromatographic or electrophoretic methods. Recovered proteins were identical with unfractionated proteins and with corresponding proteins recovered after isoelectric focusing in absence of detergent when examined by electrophoresis on starch gel. Examination of proteins by infrared spectroscopy did not reveal the presence of any protein-bound detergent. The technique of isoelectric focusing in presence of Brij 35 appears to be useful for fractionating proteins when there is a possibility of precipit,ation occurring. AICKNOWLEDGMENTS This work was supported by the National Research Council of Canada (grant A5394) nnd the rrsenrch fund of the Faculty of Graduate Studies, University of Manitoba. REFERENCES 1. 2. 3.
H.. Acta Chem. &and. 15, 325 (1961). 0.. AND SYENSSON, H., Acta Chem. Stand. 20, 820 (1966). QUAST. R.. ASD VESTEREIERG, O., Acta Chem. &and. 22, 1499 (1968). 4. WENN, R. V.. AND WILLIAMS, J., Biochem. J. 108, 69 (1968). 5. GORDON, A. H.. AND LOUIS, L. N., Biochem. J. 113, 481 (1969). 6. IA, Y.-T.. AND J,I. S.-C., J. Biol. Chem. 245, 825 (1970). 7. SOBER. H. A., GUTTER, F. J., WYCOFF, M. M., AND PETERSON, E. A.! J. Am. Chem. Sot. 78, 756 (1956). 8. ASHTON. F. E.. J.~MIESON, J. C., AND FRIESEN. A. D., Can. J. B&hem. 48, 841 (1970). 9. SIMKIN, J. I,., SKINNER, E. R., AND SESH.~DRI, H. S., Biochem. J. 90, 316 (1964). 10. SMITHIES. 0.. Biochem. J. 61, 629 (1955). 11. PECHET. L.. AND SMITH. J. A., Biochim. Biophys. Acta 200, 475 (1970). 12. HAGLUND. H.. Sci. Tools (published by LKB-Produkter AB. Stockholm-Bromma, Sweden) 14, 17 (1967). 13. GODSON, G. IT., Awl. Biochem. 35, 66 (1970). SVENSSON. VESTERBERC.
Effect
BIOCHEMISTRY
of
Nonionic
41,
149-157
(1971)
Detergent
on Fractionation
by Isoelectric A. D. FRIESEN, Department
of
Chemistry,
Received
of
Proteins
Focusing
J. C. JAMIESON, University
of
Manitoba,
October
.4XD
F. E. ASHTON
Winrkpeg
19, Manitoba,
Canada
28. 1970
The technique of isoelectric fractionation or isoelectric focusing involves the fractionation of large molecular weight ampholytes, such as proteins, according to their isoelectric points by exposure to an applied voltage in a natural pH gradient. Although the technique has been known to be theoretically possible for some time (1) it did not become a practical reality until 1966, when Vesterberg and Svensson (2) successfully synthesized a series of ampholytes capable of producing a natural equilibrated pH gradient. Since that time isoelectric focusing has been used to prepare a variety of proteins and other large molecular weight ampholytes (e.g., 3-6). One disadvantage of the t’echnique, when applied to the separation of proteins, is that there is a tendency for precipitation to occur when proteins attain their isoelectric points, thus resulting in wide bands and poor fractionation. The present work describes a technique which essentially eliminates precipit’ation of proteins during isoelectric focusing. This was accomplished by including the nonionic detergent Brij 35 in the pH gradient. Proteins were recovered by chromatographic and electrophoretic techniques and were examined by electrophoresis on starch gel. Infrared spectroscopy was employed to determine whether any detergent was bound to proteins recovered after isoelectric focusing in presence of Brij 35. MATERIALS
Chemicals were obtained as follows: Bovine serum albumin icrystallized and lyophilized), bovine hemoglobin, type 1, human albumin, grade III, and bovine thyroglohulin, type 1, Sigma Chemical Company, St. T,oui?;, MO. Brij 35 (polyoxyethylene lauryl ether), Nutritional Biochemicals Corp., Cleveland, Ohio. Starch, hpdrolyzcd, Connaught Medical Research I,nboratorie$, Toronto, Ontario. 149
150
Extinctions at 280 mp were measured with a Beckman model DB ~l)cctrol)hotonletep. Extinctions in the visible region of the spectrum were measured with a Enicam SP 600 spectrophotometer. Measurements of pH were made with a radiometer model 28 b pH-meter. Infrared spectra were obtained on a Perkin-Elmer 337 spectrophotometer; KBr discs were l)repared from a mixture of 1 mg protein and 6 mg KBr. Isoelectric
Focusing
The isoelectric focusing column (LKB 8100-10, Volume 110 ml) gradient mixing device (LKB 8121) and pH 3-10 ampholine carrier ampholytes were obtained from LKB Producter AB, Stockholm-Bromma 1, Sweden. ,4 dense electrode solution containing 0.2 ml concentrated sulfuric acid, 12 gm sucrose and 14 ml water was added to the anode at the hottom of the column. A sucrose gradient cont’aining carrier ampholytes was slowly introduced into the column using the LKB gradient device. The dense gradient solution contained 28 gm sucrose, 2 ml (40% w,/v) carrier ampholytcs, and 42 ml water. The light gradient solution contained 0.5 ml carrier ampholytes in 60 ml water. The final concentration of carrier ampholytc,s in the gradient was 1% (w/v). After about 50 ml of the gradient had entered the column, t,hc sample (10 mg protein, WC “Results”), dissolved in 1 ml wat.er, was added to the light gradient soIution. When the column had filled, the light electrode solution, consisting of 10 ml NaOH (1% w/v), was added to the cathode at the top of the column. About 3 hr was required to fill the column. When fractionations wcrc carried out in presence of detergent 1.0 ml of Brij 35 (30% w/v) was added to each gradient solution. A potential of 300V was applird to the column for 72 hr, after which time the current dropped from 10 to 1 mA. ,411 operations wrre performed at 2” and the column n-as maintaiucd at 2” during fractionation by circulating refrigerant through the cooling system of the column. When isoelectric focusing was cornplctcd the valrc at the bottom of the column was closed and the column was cmpticd by the lower exit at a flow rate of about 1-2 ml/min. Fractions of 1 ml were collected by hand. The pH and extinction at 280 rnp of each sample was determined. Recovery
of Proteins after
Isoelectric Focusing
After isoelectric focusing, tubes containing protein were pooled and concentrated to about 5 ml by ultrafiltration with concurrent dialysis against water as described by Sober, Gutter, Wycoff, and Pet’erson (7).
Samples were finally freeze-dried. Detergent, when present, was removed in two ways: (1) 11 cm x 3 cm columns of DEAE-cellulose were prc1~~1 and equilibratccl with 0.02 M phosphate buffer, pH 8.0, as describetl l)y A&ton, ,Jamiebon, and Friesen (8). The protein sample was dissolved in I ml phosphate buffer, applied to the top of the column, and elutetl with buffer at a flow rate of 2 ml/min ; 3 ml fractions wcare collected 1,) hand. The detergent, which was neutral, passed through the column unabsorbed and was detected by measuring extinctions at, 250 mp. When no morn E,,,,-positive material eluted from the column 0.02 31 phosphate buffer, pH 5.0, containing 0.3 M NaCl, leas passed through to remove the prot’ein. Tubes containing protein were poolecl, concentrated, and freezeclricd ati described above. In a few instances protein was recovered by prcl)arativc elcrtrophoresis on Cellogcl strips or blocks as described by Ashton et al. (8). In most cases prot’eins migrated rapidly and could easily be separated from the neutral detergent, which did not migrate significantly. Electrophoretic
Methods
Starch gel electrophoresis was carried out by a method based on that described by Simkin, Skinner, and Scshadri (9). The troughs measured 18.5 cm X 5 cm X 0.6 cm and the horizontal procedure of Smithies (10) was followed; sampleswere applied on Rhatman No. 3 paper. The buffer used to prepare the gel contained 76 mM Tris and 6 mM citric acid, pH 9.1; the electrode and bridge compartments of the tank contained 300
IWfect
of Presence Proteins
TABLE 1 of 0.5y0 Brij 35 on Precipitation dlwiug Isoelectric F’oc.lwillg
Bovine albumill Hllman albllmin Bovine hemoglobin Bovine thyrogloblllin Rat serum Rat ~7,-glob11lin fmcl ion I:tit ~z-glot~~dilk frnc*tion
-
1 race
of
+ + + + if +++ ++
n 10 mg protein was used in all experiments except in the case of rat serllm when (ca. 18 mg proteill) were llsed. bThe degree of precipitation is itldicatetl by plus siglls: :t lighl precipitate, moderate precipit at,e, + + ; and a heavy prccipitatc + + +.
0.3 ml +;
a
152
FRIDSEN,
JAMIESON,
AND
ASHTON
mM boric acid and 50 mM NaOH, pH 8.2. A potential of 170V was applied for 6 hr at 2”. Gels were stained for protein with naphthalene black 10B (10). RESULTS
Isoelectric
Focusing
of Proteins
Samples of bovine and human albumin, bovine hemoglobin and thyroglobulin, rat serum and aI- and a,-globulin-containing fractions isolated from rat serum (the CQ- and a,-globulin fractions were fractions 5 and 4, respectively, as described by Ashton et al. (8)) were subjected to isoelectric focusing in presence and absence of 0.5% Brij 35. Table 1 shows that, in most cases, precipitation of protein that occurred during isoelectric fractionation was eliminated in presence of detergent. Typical elution profiles obtained following fractionation of bovine albumin and i.O-
.lO
Detergent
present
0.6.
-6 PH
20
Volume FIG.
0.5%
1. Isoelectric Brij 35: (0)
60
40
80
(ml)
focusing of 10 mg bovine optical density at 280 m&,
albumin (A) pH.
in absence
and
presence
of
ISOELECTRIC
FOCUSIKG
Detergent
WITH
Present
-2
N
: ;;
153
DETERGENT
vo-
m .o a 0
-10 Detergent
Absent
0.6 -
-6 PH
0.2
20
40
60
FIG. 2. Isoelectric focusing of 10 mg bovine thyroglobulin of 0.5% Brij 35: (0) optical density at 280 ~JL, (A) pH.
80
in absence and presence
bovine thyroglobulin in presence and absence of detergent are shown in Figs. 1 and 2. The elution profiles obtained following fractionation in presence of detergent were generally sharper t’han those obtained in it,s absence (Figs. 1 and 2). Furthermore, there did not appear to be any change in isoelectric points of the proteins examined as a result of the presence of detergent during isoelectric focusing. Examination
of Proteins after Isoelectric Focusing
Detergent was removed from proteins after isoelectric focusing by preparative electrophoresis or by stepwise elution from columns of DEAE-cellulose. Figure 3 shows a typical elution profile of bovine albumin following chromatography on DEAE-cellulose; similar profiles were obtained with the other proteins examined. The technique of isoelectric focusing in presence of detergent will only be useful for fractioning proteins that tend to precipitate if proteins can
154
FIG.
cellulose 35. The densities position protein;
FRIE.C;EN,
JAMIESOP;,
ATW
SSH’WN
3. Stepwise elution chromatography on a 11 cm X 3 cm column of DEAEof bovine albumin recovered after isoelectric focusing in presence of Brij column was cluted initially with 0.02 M phosphate buffer, pH S.0; optical at 250 mp were determined to detect Brij 35. The arrow indicates the at which 0.02111 phosphate buffer, 0.3 M NaCI, pH 5.0, was added to elutc optical densities at 280 ~JL were determined to detect protein.
be removed in an unaltered form without having detergent bound to them. In order to test for the presence of det,ergent, protcii>s were examined by infrared spectroscopy. Brij 35 has two strong absorption bands at 1120 cm1 and 2950 cm-l (Fig. 4a) and was detected by infrared spectroscopy in quantities as low as 20 pg in presence of 1 mg protein (cf. Figs. 4b and 4~). Therefore, proteins recovered after isoelectric focusing in presence and absence of Brij 35 were examined by infrared spectroscopy. A typical infrared red spectrum obtained from bovine albumin isolated following isoelectric focusing in presence of detergent is shown in Fig. 4~1; the spectrum shown in Fig. 4d was identical t’o that of unfractionated bovine albumin (Fig. 4~). With all the proteins used in the present work (Table I) there was no difference between the infrared spectra of proteins recovered after fractionation in presence of detergent when compared with corresponding proteins recovered aft,er fractionation in absence of detergent. In none of t’he samples examined was there any indication t,hat t’here was contamination of protein by residual Brij 35. In order to determine if there was any change in physical characteristics of prot’eins as a result of isoelectric focusing, recovered proteins were compared with samples of original unfractionated prot#eins by electrophoresis on starch gel. Typical electrophoresis pat,terns of some of the proteins examined are shown in Fig. 5. There was no apparent. difference in electrophoret,ic mohilities of proteins isolated following isoelectric focusing in presence and absence of detergent when compared with corresponding unfrartionatcd proteins. Moreover, in most cases, the prot,eins recovered after isoelectric focusing were devoid of minor contaminating proteins that were present in the unfractionated material.
ISOELECTRIC
FOCUSING
WITH
DETERGENT
156
FRIESEN,
JAMIESON,
AND
ASHTON
thyroglobulin I.” ._“.
.
i
._
a
_. -. . .“.“& ---““.-b c
a
b
c
FIG. 5. Electrophoresis on starch gel of bovine albumin and bovine thyroglobulin: (a) protein recoveredafter isoelectricfocusingin absenceof Brij 35, (b) unfractionated protein, (c) protein recoveredafter isoelectricfocusingin presenceof Brij 35.
DISCUSSION
The procedure of separating biologically interesting proteins by isoelectric focusing has, in many instances, proved disappointing because of the tendency for precipitation to occur. In order to avoid or minimize precipitation workers have resorted to a variety of techniques such as increasing ampholyte concentration (11)) decreasing the concentration of material to be fractioned, adding urea (12)) or focusing proteins in a region of the column containing a high sucrose concentration (5). These techniques either require large amounts of expensive ampholytes or are otherwise undesirable. In the present work it was found that the addition of 0.576 of the nonionic detergent, Brij 35, was sufficient to keep proteins in solution during isoelectric focusing. The use of detergent had been previously described by Godson (13) for the isoelectric focusing of hemo-
globin, but no attempt
was made to reisolate and characterize the protein
examined. In the present studies proteins isolated by isoelectric focusing in presence of detergent were characterized by electrophoresis on starch gel and found to be identical with corresponding proteins recovered after isoelectric focusing in absence of detergent and with unfractionated proteins. Furthermore, the isoelectric points of the proteins examined did not appear to change when detergent was present during isoelectric focusing.
The detergent was easily removed by chromatography
or electrophoresis
and proteins were recovered free of detergent as determined by infrared spectroscopy. The technicme of adding detergent during isoelectric focusing of proteins has the effect of increasing resolution of proteins in cases in which precipitation of proteins resulted in wick bands. The technique
ISOELECTRIC
FOCUSING
WITH
DETERGENT
157
has the further advantage of allowing larger alnounts of protein to be focused into a single band. The technique of isoelectric focusing in presc‘nce of detergent has been used rout’inely in this laboratory and appears to be desirable when there is a chance of precipitation of protein occurring. SUMMARY
The nonionic detergent Brij 35 was found to eliminate precipitation of protein during isoelectric focusing. Proteins were recovered after isoelectric focusing by chromatographic or electrophoretic methods. Recovered proteins were identical with unfractionated proteins and with corresponding proteins recovered after isoelectric focusing in absence of detergent when examined by electrophoresis on starch gel. Examination of proteins by infrared spectroscopy did not reveal the presence of any protein-bound detergent. The technique of isoelectric focusing in presence of Brij 35 appears to be useful for fractionating proteins when there is a possibility of precipit,ation occurring. AICKNOWLEDGMENTS This work was supported by the National Research Council of Canada (grant A5394) nnd the rrsenrch fund of the Faculty of Graduate Studies, University of Manitoba. REFERENCES 1. 2. 3.
H.. Acta Chem. &and. 15, 325 (1961). 0.. AND SYENSSON, H., Acta Chem. Stand. 20, 820 (1966). QUAST. R.. ASD VESTEREIERG, O., Acta Chem. &and. 22, 1499 (1968). 4. WENN, R. V.. AND WILLIAMS, J., Biochem. J. 108, 69 (1968). 5. GORDON, A. H.. AND LOUIS, L. N., Biochem. J. 113, 481 (1969). 6. IA, Y.-T.. AND J,I. S.-C., J. Biol. Chem. 245, 825 (1970). 7. SOBER. H. A., GUTTER, F. J., WYCOFF, M. M., AND PETERSON, E. A.! J. Am. Chem. Sot. 78, 756 (1956). 8. ASHTON. F. E.. J.~MIESON, J. C., AND FRIESEN. A. D., Can. J. B&hem. 48, 841 (1970). 9. SIMKIN, J. I,., SKINNER, E. R., AND SESH.~DRI, H. S., Biochem. J. 90, 316 (1964). 10. SMITHIES. 0.. Biochem. J. 61, 629 (1955). 11. PECHET. L.. AND SMITH. J. A., Biochim. Biophys. Acta 200, 475 (1970). 12. HAGLUND. H.. Sci. Tools (published by LKB-Produkter AB. Stockholm-Bromma, Sweden) 14, 17 (1967). 13. GODSON, G. IT., Awl. Biochem. 35, 66 (1970). SVENSSON. VESTERBERC.