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A method for electrophoretic characterization on polyacrylamide gel of circulating insulin immunoreactive substances
ASALYTICAL
40, 241-246 (1971)
BIOCHEMISTRY
A Method for Electrophoretic Characterization Polyacrylamide Gel of Circulating Insulin lmmunoreactive Substances NORMAN Diabetes
R. LAZARUS, AND LILLIAN
Research Laboratory,
RAUL A. GUTMAN, RECANT
VA Hospital,
Received
July
on
Washington,
D. C. 20422
10, 1970
Since the discovery of proinsulin (l), a biosynthetic precursor of insulin, the number of insulin immunoreactive substances that could possibly be present in the circulation has proliferated. These substances include proinsulin, insulin, various intermediates of proinsulin, as well a8 the mono- and di-arginyl derivatives of insulin (2). The nearly intact connecting peptide of proinsulin may also be present in the serum (3). A method was necessary, therefore, that would permit characterization of insulin immunoreactive substances which exist in the circulation at concentrations of the order of lo-lo M. The present method utilizing a modification of the polyacrylamide gel procedure originally described by Cain and Pitney (4) (to determine the relative specific activity of radioactive proteins) accomplishes this purpose by the use of insulin immunoassay of the gel segments. METHODS
All buffer8 and acrylamide solutions are previously described (4)) except that a 15% running gel was routinely used. Electrophoresis was carried out on a Canalco apparatus utilizing tubes 7.5 cm X 0.5 cm i.d. The procedure of Davis was followed (5). Running gel volume was 0.75 ml, stacking gel volume 0.15 ml. Samples were dissolved in equal volumes of buffer and 40% sucrose in buffer. Up to 0.4 ml of sample could be placed on the column. Serum samples were extracted in acid alcohol by a modification (6) of the Davoren procedure (7) prior to electrophoresis. All electrophoretic runs were performed at 4” with 5 mA/tube. Electrophoreses were terminated when the tracking dye reached 1 mm from the end of the running gel. Utilizing purified proinsulin, proinsulin intermediates, or insulin materials, el&,rophore& took up to 1 hr. The acid alcohol extracts of serum required considerably 241 0 1971 by Academic
Press, Inc.
242
LAZARUS,
GUTMAN,
AND
RECANT
longer electrophoresing time, up to 3 hr. The increased time of electrophoresis is probably due to the presence of proteins and salts that are extracted together with insulin. After removal, gels were sliced into 3 mm sections and each section extracted overnight at 4” in 1.5 ml 0.1 N NH,OH that had previously been cooled to 0”. After overnight extraction, the samples were vigorously stirred with a vortex stirrer and then freeze dried for 36 hr. After freeze drying, 4% albumin-Verona1 buffer, pH 8.6, was added to the tubes. The dried gels floated on the buffer and could be easily removed with forceps. After removal of the gel, the amount of insulin immunoreactivity (IRI) in each sample solution was determined by radioimmunoassay (8). RESULTS
AND
DISCUSSION
Figure 1 shows the migration pattern of porcine proinsulin intermediates. Porcine proinsulin is a single chain molecule
and three which has
IRI PAlTERNS ON 15% POLYACRYLAMIDE GELS Proinsulin
5678910 3mm SLICES FIG.
1. Polyacrylamide
gels of porcine
proinsulin
and intermediates
of proinsulin
ACRYLAMIDE
GELS
OF
SERUM
243
INSULINS
SERUM ACID-ALCOHOL EXTRACTS OF PORCINE PROINSULIN AND INSULIN
f
fINSULIN +
1
I
I
I
PROINSULIN
Fia
I
I-
PROINSULIN
INSULIN 40
30 < 2 20 3 a 10
0i
SLICE No. FIG.
2. IRI
patterns
of
acid
alcohol
extracts
of
porcine
insulin
and proinsulin.
been definitively characterized as a porcine insulin molecule with a 33 amino acid connecting peptide from the carboxyl terminal end of the B chain to the amino terminus of the A chain (9). The gel cannot differentiate between intact proinsulin and the intermediate of proinsulin IMMUNOREACTIVE
MATERIAL
OBTAINED FROM BEEF INSULIN
15% polyacrylamide
Sephadex G-50 superfine 1 x 40 cm. 1 M Acetic Acid
1.6 2 s '.2 d 0.8 d 0.4
”
0 12345678910 3mm
18 Sections
20
22 24 26 28 Fraction (1.5 ml.1
M
32
Fm. 3. At right, elution profile of commercial beef insulin after chromatography on Sephadex; tube 22, the peak of the crude proinsulin fraction, was electrophoresed on polyacrylamide. At left, stained protein bands obtained after polyacrylamide electrophoresis of tube 22; shown below is immunoreactive pattern obtained using same material.
244
LAZARUS,
GUTMAN,
AND
RECANT
with cleavage between residues 54 and 55. There is clear differentiation between desnonapeptide proinsulin (missing 55-63) and desdipeptide proinsulin (missing 62-63). The proinsulin which was recovered from the gel retains its biological activity when tested in vitro on rat hemidiaphragm muscle. Re-electrophoresis of this proinsulin on polyacrylamide reveals an unaltered IRI migration pattern. Because this electrophoretic method is to be used on acid alcohol extracts of serum it was necessary to test the effect of the extraction procedure on the electrophoretic mobilities of insulin and proinsulin. Figure 2 shows the migration pattern of an acid alcohol extract and of serum to which known amounts of porcine proinsulin and insulin were added. The migration pattern of both these species is unchanged from that shown in Fig. 1. The recovery of both species was in the order of 70~0. To test the differentiating power of the present method when presented with a heterogenous mixture of materials, 15 mg beef insulin (Eli Lilly & Co.) were chromatographed on Sephadex G-50 Superfine, ,
IRI
PATTERNS
15% polyacrylamide Acid alcohol extract of serum
Sephadex G-M superfine 1 x 40 cm. A-V Buffer pH8.6 2cc serum
1 2 3 4 5 6 7 8 9 10 +
+ 18
M
22 24 26 28 Fraction L5ml. i
30
32
1 2 3 4 5 6 7 8 9 10 3mm Sections
FIG. 4. At right, immunoreactive elution Rrofile obtained after chromatography of 2 ml serum obtained 15 min after I. V.. Tolbutamide (80 @ IRI/ml) ; the immunoreactive peak migrates in a position consistent with insulin. At left, polyacrylamide electrophoretic patterns obtahed, respectively, from acid alcohol extracts of 2 ml and 1 ml of same serum.
ACRYLAMIDE
GELS
OF
SERUM
245
IKSULINS
1 M acetic acid (Fig. 3). After freeze drying, the fraction eluting at the proinsulin peak position was divided into two parts and each electrophoresed. One sample was stained with amido black and the other subjected to immunoassay (Fig. 3). The following points are noteworthy. The protein bands on the stained gel correspond in number and mobility to the previously described species present in commercial beef insulin (10). Beef proinsulin migrates in section 7 while porcine proinsulin migrates in section 8 (Fig. 1). The immunoreactive pattern corresponds to the stained pattern. To determine whether the method could be applied to serum, 2 ml and 1 ml of serum (containing 86 pIJ IRIJml) obtained from a normal subject 15 min after I. V. Tolbutamide, were extracted with acid alcohol and then electrophoresed. Recovery of IRI after acid alcohol extraction was 80%. The electrophoretic immunoreactive patterns of the acid alcohol extracts were compared to the immunoreactive pattern obtained by chromatographing 2 ml of unextracted serum on Sephadex G-50 Superfine, 5% albumin-Verona1 buffer, pH 8.6 (Fig. 4). As can be seen, approximately 80% of the immunoreactive material present in the acid alcohol extracts of serum has been recovered on the polyacrylamide gel. The 2 ml and 1 ml extracts show similar profiles. The immunoreactive material eluting as a single peak at the insulin position on Sephadex is shown on polyacrylamide to consist almost entirely of a single species. SUMMARY
A method is described for determining the electrophoretic behavior of circulating insulin immunoreactive materials. A polyacrylamide immunoreactive pattern of serum can be determined on as little as 100 PIJ of insulin immunoreactive material. It is suggested that the utility of the method would be increased if it were coupled to a prior chromatographic separation of proinsulin like components from insulin by means of gel filtration. There is also considerable potential for use of this method as a preparative procedure for study of human circulating insulin components. ACKNOWLEDGMENT Dr. Ronald Chance, Eli Lilly Research Laboratories, is thanked for his generous gifts of the porcine proinsulin intermediates which were isolated, purified, and characterized by him. REFERENCES 1. STEINER, D. F., DUNNINGHAM, D., SPIGELMAN, 697 (1967). 2. CHANCE, R. E., personal communication.
L.,
AND
ATEN,
B.,
fJ&~e
157,
246
LAZARUS,
GUTMAN,
AND
RECANT
3. CLARK, J. L., CHO, S., RTJBENSTEIN, A. H., AND STEINER, D. F., B&hem. Biophys. Res. Commun. 35, 456 (1969). 4. CAIN, D. F., AND PITNEY, R. E., Anal. Biochem. 22, 11 (196-S). 5. DAVIS, B. J., Ann. ,N. Y. Acad. Sci. 121, 404 (1964). 6. MELANI, F., RUBENSTEIN, A. H., AND STEINER, D. F., J. Clin. Invest. 49, 497 (1970). 7. DAVOREN, D. R., B&him. Biophys. Acta 63, 150 (1962). 8. MORGAN, C. R., AND LAZAROW, A., Diabetes 12, 115 (1963). 9. CHANCE, R. E., ELLIS, R. M., AND BROMER, W. W., Science 161, 165 (1968). 10. STEINER, D. F., HALLUND, O., RUBENSTEIN, A., CHO, S., AND BA~LISS, C., Diabetes 17, 725 (1968).
40, 241-246 (1971)
BIOCHEMISTRY
A Method for Electrophoretic Characterization Polyacrylamide Gel of Circulating Insulin lmmunoreactive Substances NORMAN Diabetes
R. LAZARUS, AND LILLIAN
Research Laboratory,
RAUL A. GUTMAN, RECANT
VA Hospital,
Received
July
on
Washington,
D. C. 20422
10, 1970
Since the discovery of proinsulin (l), a biosynthetic precursor of insulin, the number of insulin immunoreactive substances that could possibly be present in the circulation has proliferated. These substances include proinsulin, insulin, various intermediates of proinsulin, as well a8 the mono- and di-arginyl derivatives of insulin (2). The nearly intact connecting peptide of proinsulin may also be present in the serum (3). A method was necessary, therefore, that would permit characterization of insulin immunoreactive substances which exist in the circulation at concentrations of the order of lo-lo M. The present method utilizing a modification of the polyacrylamide gel procedure originally described by Cain and Pitney (4) (to determine the relative specific activity of radioactive proteins) accomplishes this purpose by the use of insulin immunoassay of the gel segments. METHODS
All buffer8 and acrylamide solutions are previously described (4)) except that a 15% running gel was routinely used. Electrophoresis was carried out on a Canalco apparatus utilizing tubes 7.5 cm X 0.5 cm i.d. The procedure of Davis was followed (5). Running gel volume was 0.75 ml, stacking gel volume 0.15 ml. Samples were dissolved in equal volumes of buffer and 40% sucrose in buffer. Up to 0.4 ml of sample could be placed on the column. Serum samples were extracted in acid alcohol by a modification (6) of the Davoren procedure (7) prior to electrophoresis. All electrophoretic runs were performed at 4” with 5 mA/tube. Electrophoreses were terminated when the tracking dye reached 1 mm from the end of the running gel. Utilizing purified proinsulin, proinsulin intermediates, or insulin materials, el&,rophore& took up to 1 hr. The acid alcohol extracts of serum required considerably 241 0 1971 by Academic
Press, Inc.
242
LAZARUS,
GUTMAN,
AND
RECANT
longer electrophoresing time, up to 3 hr. The increased time of electrophoresis is probably due to the presence of proteins and salts that are extracted together with insulin. After removal, gels were sliced into 3 mm sections and each section extracted overnight at 4” in 1.5 ml 0.1 N NH,OH that had previously been cooled to 0”. After overnight extraction, the samples were vigorously stirred with a vortex stirrer and then freeze dried for 36 hr. After freeze drying, 4% albumin-Verona1 buffer, pH 8.6, was added to the tubes. The dried gels floated on the buffer and could be easily removed with forceps. After removal of the gel, the amount of insulin immunoreactivity (IRI) in each sample solution was determined by radioimmunoassay (8). RESULTS
AND
DISCUSSION
Figure 1 shows the migration pattern of porcine proinsulin intermediates. Porcine proinsulin is a single chain molecule
and three which has
IRI PAlTERNS ON 15% POLYACRYLAMIDE GELS Proinsulin
5678910 3mm SLICES FIG.
1. Polyacrylamide
gels of porcine
proinsulin
and intermediates
of proinsulin
ACRYLAMIDE
GELS
OF
SERUM
243
INSULINS
SERUM ACID-ALCOHOL EXTRACTS OF PORCINE PROINSULIN AND INSULIN
f
fINSULIN +
1
I
I
I
PROINSULIN
Fia
I
I-
PROINSULIN
INSULIN 40
30 < 2 20 3 a 10
0i
SLICE No. FIG.
2. IRI
patterns
of
acid
alcohol
extracts
of
porcine
insulin
and proinsulin.
been definitively characterized as a porcine insulin molecule with a 33 amino acid connecting peptide from the carboxyl terminal end of the B chain to the amino terminus of the A chain (9). The gel cannot differentiate between intact proinsulin and the intermediate of proinsulin IMMUNOREACTIVE
MATERIAL
OBTAINED FROM BEEF INSULIN
15% polyacrylamide
Sephadex G-50 superfine 1 x 40 cm. 1 M Acetic Acid
1.6 2 s '.2 d 0.8 d 0.4
”
0 12345678910 3mm
18 Sections
20
22 24 26 28 Fraction (1.5 ml.1
M
32
Fm. 3. At right, elution profile of commercial beef insulin after chromatography on Sephadex; tube 22, the peak of the crude proinsulin fraction, was electrophoresed on polyacrylamide. At left, stained protein bands obtained after polyacrylamide electrophoresis of tube 22; shown below is immunoreactive pattern obtained using same material.
244
LAZARUS,
GUTMAN,
AND
RECANT
with cleavage between residues 54 and 55. There is clear differentiation between desnonapeptide proinsulin (missing 55-63) and desdipeptide proinsulin (missing 62-63). The proinsulin which was recovered from the gel retains its biological activity when tested in vitro on rat hemidiaphragm muscle. Re-electrophoresis of this proinsulin on polyacrylamide reveals an unaltered IRI migration pattern. Because this electrophoretic method is to be used on acid alcohol extracts of serum it was necessary to test the effect of the extraction procedure on the electrophoretic mobilities of insulin and proinsulin. Figure 2 shows the migration pattern of an acid alcohol extract and of serum to which known amounts of porcine proinsulin and insulin were added. The migration pattern of both these species is unchanged from that shown in Fig. 1. The recovery of both species was in the order of 70~0. To test the differentiating power of the present method when presented with a heterogenous mixture of materials, 15 mg beef insulin (Eli Lilly & Co.) were chromatographed on Sephadex G-50 Superfine, ,
IRI
PATTERNS
15% polyacrylamide Acid alcohol extract of serum
Sephadex G-M superfine 1 x 40 cm. A-V Buffer pH8.6 2cc serum
1 2 3 4 5 6 7 8 9 10 +
+ 18
M
22 24 26 28 Fraction L5ml. i
30
32
1 2 3 4 5 6 7 8 9 10 3mm Sections
FIG. 4. At right, immunoreactive elution Rrofile obtained after chromatography of 2 ml serum obtained 15 min after I. V.. Tolbutamide (80 @ IRI/ml) ; the immunoreactive peak migrates in a position consistent with insulin. At left, polyacrylamide electrophoretic patterns obtahed, respectively, from acid alcohol extracts of 2 ml and 1 ml of same serum.
ACRYLAMIDE
GELS
OF
SERUM
245
IKSULINS
1 M acetic acid (Fig. 3). After freeze drying, the fraction eluting at the proinsulin peak position was divided into two parts and each electrophoresed. One sample was stained with amido black and the other subjected to immunoassay (Fig. 3). The following points are noteworthy. The protein bands on the stained gel correspond in number and mobility to the previously described species present in commercial beef insulin (10). Beef proinsulin migrates in section 7 while porcine proinsulin migrates in section 8 (Fig. 1). The immunoreactive pattern corresponds to the stained pattern. To determine whether the method could be applied to serum, 2 ml and 1 ml of serum (containing 86 pIJ IRIJml) obtained from a normal subject 15 min after I. V. Tolbutamide, were extracted with acid alcohol and then electrophoresed. Recovery of IRI after acid alcohol extraction was 80%. The electrophoretic immunoreactive patterns of the acid alcohol extracts were compared to the immunoreactive pattern obtained by chromatographing 2 ml of unextracted serum on Sephadex G-50 Superfine, 5% albumin-Verona1 buffer, pH 8.6 (Fig. 4). As can be seen, approximately 80% of the immunoreactive material present in the acid alcohol extracts of serum has been recovered on the polyacrylamide gel. The 2 ml and 1 ml extracts show similar profiles. The immunoreactive material eluting as a single peak at the insulin position on Sephadex is shown on polyacrylamide to consist almost entirely of a single species. SUMMARY
A method is described for determining the electrophoretic behavior of circulating insulin immunoreactive materials. A polyacrylamide immunoreactive pattern of serum can be determined on as little as 100 PIJ of insulin immunoreactive material. It is suggested that the utility of the method would be increased if it were coupled to a prior chromatographic separation of proinsulin like components from insulin by means of gel filtration. There is also considerable potential for use of this method as a preparative procedure for study of human circulating insulin components. ACKNOWLEDGMENT Dr. Ronald Chance, Eli Lilly Research Laboratories, is thanked for his generous gifts of the porcine proinsulin intermediates which were isolated, purified, and characterized by him. REFERENCES 1. STEINER, D. F., DUNNINGHAM, D., SPIGELMAN, 697 (1967). 2. CHANCE, R. E., personal communication.
L.,
AND
ATEN,
B.,
fJ&~e
157,
246
LAZARUS,
GUTMAN,
AND
RECANT
3. CLARK, J. L., CHO, S., RTJBENSTEIN, A. H., AND STEINER, D. F., B&hem. Biophys. Res. Commun. 35, 456 (1969). 4. CAIN, D. F., AND PITNEY, R. E., Anal. Biochem. 22, 11 (196-S). 5. DAVIS, B. J., Ann. ,N. Y. Acad. Sci. 121, 404 (1964). 6. MELANI, F., RUBENSTEIN, A. H., AND STEINER, D. F., J. Clin. Invest. 49, 497 (1970). 7. DAVOREN, D. R., B&him. Biophys. Acta 63, 150 (1962). 8. MORGAN, C. R., AND LAZAROW, A., Diabetes 12, 115 (1963). 9. CHANCE, R. E., ELLIS, R. M., AND BROMER, W. W., Science 161, 165 (1968). 10. STEINER, D. F., HALLUND, O., RUBENSTEIN, A., CHO, S., AND BA~LISS, C., Diabetes 17, 725 (1968).