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Zone electrophoresis on starch of preparations of follicle-stimulating hormone from sheep pituitary glands
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
77,
138-146 (1%8)
Zone Electrophoresis on Starch of Preparations of FollicleStimulating Hormone from Sheep Pituitary Glands I. D. Raacke,l Ardis J. Lostroh2 and Choh Hao Li3 From
the Hormone
Research
Laboratory
University of California,
and the Department Berkeley, California
of Biochemistry,
Received January 3, 1958
Ample demonstration is available in the literature to the effect that the appearance of a single peak in the course of moving-boundary electrophoresis or ultracentrifugation is in itself insufficient to establish the homogeneity of a protein preparation (1, 2). This is particularly true in the case of proteins like the pituitary hormones (3, 4), which possess high biological activity and yet may occur in a complex mixture of other proteins that, although closely associated with the active component and very similar in physicochemical behavior, are themselves biologically inert. Some years ago, a method was described for the isolation from sheep pituitaries of a follicle-stimulating hormone (FSH) preparation (5) which behaved as a homogenous protein both in moving-boundary electrophoresis and in the ultracentrifuge (6). In view of the fact that zone electrophoresis on starch has been used successfully in studying the homogeneity of biologically active proteins (7), it seemed desirable to reinvestigate the electrophoretic behavior of similar FSH preparations by this method. EXPERIMENTAL Preparations of FSH pituitary glands by the 1 Present address: 2 Present address: celin Berthelot, Paris 3 Reprint requests to C. H. Li.
employed in this investigation were obtained from sheep method previously described (5). Zone electrophoresis on
Virus Laboratory, University of California, Berkeley, Calif. Laboratoire d’Embryologie, College de France, Place MarVe, France. and inquiries concerning this paper should be addressed
138
ZONE
ELECTROPHORESIS
ON
STARCH
OF
139
FSH
starch (8) was performed according to the procedure previously described (9), but with reversible silver-silver chloride electrodes. The dimensions of the troughs were 4 cm. in diameter and 40 or 60 cm. in length. Unless otherwise stated, borate buffer of pH 8.5 (0.05 M H3B03-0.05 M KC1-0.012 N NaOH) was used. The amount of material in each experiment varied from 10 to 300 mg. ; runs were usually made at 185 v. for 48 or 72 hr. The starch in each trough was cut into l-cm. sections, which were eluted with water; the protein was determined by the method of Lowry et al. (10). Prior to biological assay, the protein concentration was ascertained by determining Kjeldahl nitrogen in pooled samples; in those instances where individual tubes were to be assayed, the protein concentrations were estimated from the Folin color (10) as read from a standard curve. The isoelectric point was determined according to the procedure previously employed for this determination in the cases of cY-corticotropin (11) and of human chorionic gonadotropin (HCG) (12). The same FSH preparation (L2037B) was run at four different pH’s in univalent buffers of 0.1 ionic strength (containing 0.06 IIf NaCI); the mobilities of FSH activity were estimated from the position of the center of the active zone. The assay method of Steelman and Pohley (13), based on the augmentation of ovarian weight obtained with FSH and HCG in intact rats, was used for estimating the follicle-stimulating potency of the preparations. Immature female rats of the Long-Evans strain, 21 and 22 days of age, were injected, once daily for 3 days, intraperitoneally with HCG and subcutaneously with the FSH preparation to be tested. The dose of HCG4 was adjusted to give control ovaries weighing 3&35 mg. Both hormones were usually dissolved in distilled water, although the FSH solutions obtained from the various procedures were sometimes diluted directly to the proper concentration without prior lyophilization. Autopsy was performed 24 hr. aft,er the last inject.ion. The biological activity of the unknown preparation was estimated in terms of a slope unit, given by the following equation: Slope unit
=
test ovaries
(mg.) -
control
ovaries
(mg.)
dose (mg.)
Further biological characterization was carried out in hypophysectomized animals. For assessing the minimal effective dose (M.E.D.) required to stimulate follicular development with begnining antrum formation in the ovaries of hypophysectomized animals (14), female rats of the Long-Evans strain were operated on at 28 days of age and injected subcutaneously, once daily, beginning at 35 days of age. Twenty-four hours after the last of three such injections, the animals were sacrificed; the ovaries and uteri were dissected free from the surrounding tissue, weighed on a Roller-Smith torsion balance, and fixed in neutral formalin. Subsequently, histological sections were prepared using the celloidin method with the hematoxylin-eosin stain. An estimation of the interstitial-cell stimulating (ICSH) activity in the preparations was obtained in immature male rats hypophysectomized at 24 days of age. 4 We wish to thank Leo Pharmaceutical of a highly purified HCG preparation.
Products,
Copenhagen,
for
the gift
140
RAACKE, LOSTROH AND LI
Intraperitoneal injections were begun 3 days after the operation and continued daily for 4 days. On the fifth day, the animals were autopsied, and the weights of the ventral prostates were obtained. RESULTS
For the sake of convenient identification, the electrophoretic patterns have been divided into seven peaks, labeled from A to G (Fig. 2), whose mobilities in borate buffer are given in Table I. Even though these peaks may not represent single components, they could be identified in a large number of different preparations. Figures 1 and 2 show the pattern obtained by means of zone electrophoresis on starch of a preparation (L2037B) which appeared reasonably homogeneous in free electrophoresis (5, 6). It can be seen from Table II that fraction 131aD, obtained from the D region (shaded area in Fig. 2), had an FSH activit,y averaging 900 slope units/mg. in contrast to the starting material which had an activity of only 80-100 slope units. It should be noted that the main peak, comprising segments 27-35 in Fig. 1, had very low FSH activity (see Table II), with the highest activity being found in segment 22. The active material in the D zone (Fig. 2) was isolated and rerun either twice or three times, depending on the purity of the starting material. TABLE
I
Mobilities on Starch of Several Components Commonly Encountered in Crude FSH PreparaGons
Electrophoretic
u x 106sq. cm./v./sec.b Pear
Uncorrecled A B c D E F G
-3.0 -2.5
-1.8 -1.5 -0.5 +0.4 +0.9
Corrected -6.5 -6.0 -5.3
-4.9 -4.0 -3.2 -2.6
0 See Fig. 2. b All mobilities were measured in 0.1 M borate buffer, pH 8.5 (pH 7.9 in the trough). Calculations were made according to the formula fi = dZ/Vt, where d is the observed distance of migrat,ion, 1 the length of the trough, V the voltage across the ends of the trough and t the time in seconds. The corrections for electroosmotic flow were made by assuming an electroosmotic flow of 3.5 X 10-b sq. cm./v./sec.
ZONE
0
BLECTROPHORESIS
OIi
STARCH
OF
Segment Number (cm.)
141
FSH
0
FIG. 1. Pattern obtained after zone electrophoresis on starch in borate buffer, pH 8.5 (7.36 in a 60-cm. trough, 150 v. for 72 hr.) of FSH preparation L2037B.
FIQ. 2. Pattern obtained after zone electrophoresis on starch in borate buffer, pH 8.5 (7.9 in a 60-cm. trough, 190 v. for 72 hr.) of FSH preparation L2037B.
142
RAACKE,
LOSTROH
TABLE Estimation Purified
AND
LI
II
by the Augmentation Assay Method of the Potency of FSH Obtained from Zone Electrophoresis on Starch Ovarian weights
Preparation
Total dose’=
No. of rats
w.
L2037Bb R20B”
ME-500(8)” ME-500(18) ME-500 (20) ME-500 (22) ME-509 (24) ME-500(27) ME-500(30) ME-500(35) 131aDd
0.10 0.15 0.50 0.15 0.30 0.50 0.50 0.50 0.15 0.15 0.15 0.50 0.50 0.50 0.048
4 3 3 8 4 8 2 3 3 3 3 5 6 3 12
Test w.
HCG control w.
40.8 52.0 85.9 55.2 58.5 93.2 28.6 91.8 80.8 87.7 57.9 58.5 40.8 30.5 79.5
32.4 37.1 37.1 45.7 34.8 45.7 37.1 37.1 37.1 37.1 37.1 37.1 37.1 37.1 36.3
Activity slope units
85 100 100 65 80 95 0 110 290 335 140 45 10 0 900
a The total dose for preparations L2037B and R2OB (starting material) was estimated on a dry-weight basis; the doses for preparation ME-500 were estimated from the Folin color, and the dose for preparation 13laD was estimated on a nitrogen basis, assuming 16% N for the FSH protein. b Starting materials for zone electrophoresis. = The numbers in parentheses correspond to the segments (in cm.) of the starch electrophoresis experiment depicted in Fig. 1. d See Fig. 3.
In this way, highly active material could be obtained, material which appears as a reasonably homogeneous peak but which probably does not yet represent a pure substance (see Fig. 3), despite the fact that considerable purification has been achieved. It was found that a total dose of 0.025 mg. when injected into young female hypophysectomized rats caused follicular development with beginning antrum formation (Table III) in contrast to a total dose of 0.3 mg. of the starting material required to achieve the same effect. It was apparent that the purified fraction (131aD) was not completely free from ICSH, as evidenced by the uterine development in hypophysectomized females and by the growth of the ventral prostate in hypophysectomized males (Table III). It can be seen from Fig. 4, which presents a plot of electrophoretic mobility versus pH, that the isoelectric point of the active component
4,0-
c----a
h
Protein Concentrohon Biological Activity
c
c aI -5 a .- o- 2 2
b‘-_ !!a??%
4 6
w
IO
14
Segment
18
22
Number
30
26
34
38 _
(cm.)
FIG. 3. Pattern obtained with an FSH preparation after purification by means of zone electrophoresis on starch (borate buffer, pH 7.9 in a 40-cm. trough, 206 v., 72 hr.).
Biological
TABLE III Characterization in Hypophysectomized FSH Obtained from Zone Electrophoresis Organ
Preparation
Total dos.5’
lo. oitrats
Rats of Purified on Starch
weights
Female
uterus
Ovaries
Prostate
.I-
Saline 131aD” 131aD 131aD 131aD 131aD Saline
9.075 0.100 0.150 0.300 0 0.010 0.025 0.038 0.040 1.30 0
m&z.
+w.
m&-.
R2OB
*5 5 5 4 7 4 4 3 3 3 5
9.8
f
0.76
11.7 10.9 14.2 9.5 7.4 10.6 15.5 17.2 26.9 7.6
& f f f f f f f f f
1.1 0.5 2.3~ 0.7 0.5 O.gc 0.1 2.8 4.7 0.8
15.4 14.7 14.9 14.1 ,66.0 12.7
f * f f f f
mg.
0.8 1.2 0.6 2.3 11.0 0.8
6.3 f 4.2 f
0.3 0.5
13.9 f 7.9 f
1.1 0.5
a The total dose for the R-preparation (representing starting material) was estimated on a dry-weight basis; the dose for the purified preparations (D-fractions) was estimated on a nitrogen basis, assuming 16% N for the FSH protein. h Mean f standard error. c Total dose producing follicular development with beginning antrum formation (M.E.D.). d See Fig. 3. 143
144
RAACKE,
4.0
LOSTROH
5.0
AND LI
6.0PH7.0
8.0
9.0
-1.0
FIQ. 4. Plot of zone electrophoretic mobilities as a function of pH of several commonly encountered components in FSH preparations; the active component is D.
(D fraction in Fig. 2) is 4.6. This is very close to the previously published isoelectric point for FSH of 4.5 (6), a coincidence which might be explained by the fact that the mobility curves of all the commonly encountered contaminants in the FSH preparations investigated converge at 4.6. DISCUSSION
It was evident throughout the present investigation that the FSH activity was not associated with the main protein component in any of the preparations studied, as evidenced by the fact that the main component (segments 27-35 in Fig. 1, A zone in Fig. 2) was found to be comparatively inactive, at the same time that a highly active material (segment 22 in Fig. 1, D zone in Fig. 2) could be located. Although none of the preparations described in previous publications (5, 6) were available when the present study was undertaken, preparations obtained by the same method of isolation and possessing comparable activity show mainly one peak (Figs. 1 and 2). The previous erroneous identification of the main protein component with the activity can easily be understood
ZONE ELECTROPHORESIS ON STARCH OF FSH
145
on the basis of certain characteristics of the material as well as certain factors inherent in the previous methods of analysis. When examined in free solution by means of optical devices (moving-boundary electrophoresis and ultracentrifugation), these preparations tend to appear nearly homogeneous, not only because the A peak (Fig. 2) represents the greater bulk of the material, but also because the rest of the material (including the active substance) is not detectable as a separate component, but, rather, spreads out in such a way as to raise the base line more or less uniformly. Furthermore, because of the coincidence that all the components have an isoelectric point in the neighborhood of pH 4.5 (see Fig. 4), repeated fractionation with ammonium sulfate at this pH produces only an insignificant shift in the relative proportions of all the components; this was verified by the almost identical electrophoretic patterns given by all the fractions obtained from such fractional precipitation experiments. Because of this, the ratio of biological activity to total protein remains constant, within the limits of error of the assay, so that the illusion of homogeneity is again given. From an inspection of Figs. 1 and 2, it is evident that zone electrophoresis on starch accomplishes a very substantial purification of the starting material. On the basis of the yields obtained for the pooled fraction 131aD, approximately a tenfold purification could be expected, and the activity of the isolated material indicates that such a purification was actually attained. In normal rats, the activity of the purified fraction was 900 slope units as compared with SO-100 units for the starting material (Table II); and, in hypophysectomized animals, the M.E.D. for the former was 0.025 mg. as compared with 0.300 mg. for the latter (Table III). It should be pointed out again that, despite this considerable purification, even our best preparations of ovine FSH obtained by zone electrophoresis on starch are not yet pure. From the work of Steelman et al. (15, 16), the same appears to be true for FSH of porcine origin. All that can be said with any certainty at present is that the behavior of the active principle throughout has been consistent with that of an acid protein with an isoelectric point in the neighborhood of pH 4.5. ACKNOWLEDGMENTS This work was supported in part by grants from the National Institutes of Health, United States Public Health Service (G-4097). We wish to acknowledge the able technical assistance of Charles W. Jordan, Jr. and Elizabeth Hageman.
146
RAACKE, LOSTROH AND LI SUMMARY
Preparations of follicle-stimulating hormone from sheep pituitary glands have been examined by means of zone electrophoresis on starch. It was found that the activity was not associated with the main protein component, and that the active component as well as the contaminating components had an isoelectric point at pH 4.6. Approximately a tenfold purification could be achieved by the electrophoretic procedure, REFERENCES 1. LI, C. H., in “Amino Acids and Proteins” (D. M. Greenberg, ed.), p. 487.
C. C Thomas, Springfield, Mass., 1951. 2. COLVIN, J. R., SMITH, D. B., AND COOK, W. H., Chem. Revs. 64, 687 (1954). 3. LI, C. H., Acta Endocrinol. 10, 255 (1952). 4. ACHER, R., AND FROMAGEOT,C., Ergeb. Physiol. biol. Chem. u. exptl. Pharmakol. 48, 287 (1955).
5. LI, C.H.,SIMPSON, M.E., AND EVANS, H.M.,Science 109, 445 (1949). 6. LI, C. H., AND PEDERSEN, K. O., J. Gen. Physiol. 36,629 (1952). 7. TISELIUS, A., AND FLODIN, O., Advances in Protein Chem. 8, 461 (1954). 8. KUNKEL, H. G., AND SLATER, R. J., Proc. Sot. Exptl. Biol. Med. 80, 42 (1952). 9. F~NSS-BECH, P., AND LI, C. H., J. BioZ. Chem. 207, 175 (1954). 10. LOWRY,O.H.,ROSEBROUGH,N. J., FARR, A.L., AND RANDALL, R.J.,J. Biol. Chem. 193, 265 (1951). 11. RAACKE, I. D., AND Lr, C. H., J. Biol. Chem. 216, 277 (1955). 17, 366 (1954). 12. RAACKE, I. D., LI, C. H., AND LOSTROH, A., Acta Endocrinol. 13. STEELMAN, S. L., AND POHLEY, F. M., Endocrinology 63, 604 (1953). 14. EVANS, H.M., SIMPSON,M.E., TOLKSDORF, S., AND JENSEN, H., Endocrinology 26, 529 (1939); SIMPSON, M. E., VAN WAGENEN, G., AND CARTER, F., Proc. Sot. Exptl. Biol. Med. 91, 6 (1956). 15. STEELMAN, S. L., LAMONT, W. A., AND BALTES, B. J., Endocrinology 66, 216 (1965). 16. STEELMAN, S. L., LAMONT, W. A., AND BALTES, B. J., Acta Endocrinol. 22, 186 (1966).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
77,
138-146 (1%8)
Zone Electrophoresis on Starch of Preparations of FollicleStimulating Hormone from Sheep Pituitary Glands I. D. Raacke,l Ardis J. Lostroh2 and Choh Hao Li3 From
the Hormone
Research
Laboratory
University of California,
and the Department Berkeley, California
of Biochemistry,
Received January 3, 1958
Ample demonstration is available in the literature to the effect that the appearance of a single peak in the course of moving-boundary electrophoresis or ultracentrifugation is in itself insufficient to establish the homogeneity of a protein preparation (1, 2). This is particularly true in the case of proteins like the pituitary hormones (3, 4), which possess high biological activity and yet may occur in a complex mixture of other proteins that, although closely associated with the active component and very similar in physicochemical behavior, are themselves biologically inert. Some years ago, a method was described for the isolation from sheep pituitaries of a follicle-stimulating hormone (FSH) preparation (5) which behaved as a homogenous protein both in moving-boundary electrophoresis and in the ultracentrifuge (6). In view of the fact that zone electrophoresis on starch has been used successfully in studying the homogeneity of biologically active proteins (7), it seemed desirable to reinvestigate the electrophoretic behavior of similar FSH preparations by this method. EXPERIMENTAL Preparations of FSH pituitary glands by the 1 Present address: 2 Present address: celin Berthelot, Paris 3 Reprint requests to C. H. Li.
employed in this investigation were obtained from sheep method previously described (5). Zone electrophoresis on
Virus Laboratory, University of California, Berkeley, Calif. Laboratoire d’Embryologie, College de France, Place MarVe, France. and inquiries concerning this paper should be addressed
138
ZONE
ELECTROPHORESIS
ON
STARCH
OF
139
FSH
starch (8) was performed according to the procedure previously described (9), but with reversible silver-silver chloride electrodes. The dimensions of the troughs were 4 cm. in diameter and 40 or 60 cm. in length. Unless otherwise stated, borate buffer of pH 8.5 (0.05 M H3B03-0.05 M KC1-0.012 N NaOH) was used. The amount of material in each experiment varied from 10 to 300 mg. ; runs were usually made at 185 v. for 48 or 72 hr. The starch in each trough was cut into l-cm. sections, which were eluted with water; the protein was determined by the method of Lowry et al. (10). Prior to biological assay, the protein concentration was ascertained by determining Kjeldahl nitrogen in pooled samples; in those instances where individual tubes were to be assayed, the protein concentrations were estimated from the Folin color (10) as read from a standard curve. The isoelectric point was determined according to the procedure previously employed for this determination in the cases of cY-corticotropin (11) and of human chorionic gonadotropin (HCG) (12). The same FSH preparation (L2037B) was run at four different pH’s in univalent buffers of 0.1 ionic strength (containing 0.06 IIf NaCI); the mobilities of FSH activity were estimated from the position of the center of the active zone. The assay method of Steelman and Pohley (13), based on the augmentation of ovarian weight obtained with FSH and HCG in intact rats, was used for estimating the follicle-stimulating potency of the preparations. Immature female rats of the Long-Evans strain, 21 and 22 days of age, were injected, once daily for 3 days, intraperitoneally with HCG and subcutaneously with the FSH preparation to be tested. The dose of HCG4 was adjusted to give control ovaries weighing 3&35 mg. Both hormones were usually dissolved in distilled water, although the FSH solutions obtained from the various procedures were sometimes diluted directly to the proper concentration without prior lyophilization. Autopsy was performed 24 hr. aft,er the last inject.ion. The biological activity of the unknown preparation was estimated in terms of a slope unit, given by the following equation: Slope unit
=
test ovaries
(mg.) -
control
ovaries
(mg.)
dose (mg.)
Further biological characterization was carried out in hypophysectomized animals. For assessing the minimal effective dose (M.E.D.) required to stimulate follicular development with begnining antrum formation in the ovaries of hypophysectomized animals (14), female rats of the Long-Evans strain were operated on at 28 days of age and injected subcutaneously, once daily, beginning at 35 days of age. Twenty-four hours after the last of three such injections, the animals were sacrificed; the ovaries and uteri were dissected free from the surrounding tissue, weighed on a Roller-Smith torsion balance, and fixed in neutral formalin. Subsequently, histological sections were prepared using the celloidin method with the hematoxylin-eosin stain. An estimation of the interstitial-cell stimulating (ICSH) activity in the preparations was obtained in immature male rats hypophysectomized at 24 days of age. 4 We wish to thank Leo Pharmaceutical of a highly purified HCG preparation.
Products,
Copenhagen,
for
the gift
140
RAACKE, LOSTROH AND LI
Intraperitoneal injections were begun 3 days after the operation and continued daily for 4 days. On the fifth day, the animals were autopsied, and the weights of the ventral prostates were obtained. RESULTS
For the sake of convenient identification, the electrophoretic patterns have been divided into seven peaks, labeled from A to G (Fig. 2), whose mobilities in borate buffer are given in Table I. Even though these peaks may not represent single components, they could be identified in a large number of different preparations. Figures 1 and 2 show the pattern obtained by means of zone electrophoresis on starch of a preparation (L2037B) which appeared reasonably homogeneous in free electrophoresis (5, 6). It can be seen from Table II that fraction 131aD, obtained from the D region (shaded area in Fig. 2), had an FSH activit,y averaging 900 slope units/mg. in contrast to the starting material which had an activity of only 80-100 slope units. It should be noted that the main peak, comprising segments 27-35 in Fig. 1, had very low FSH activity (see Table II), with the highest activity being found in segment 22. The active material in the D zone (Fig. 2) was isolated and rerun either twice or three times, depending on the purity of the starting material. TABLE
I
Mobilities on Starch of Several Components Commonly Encountered in Crude FSH PreparaGons
Electrophoretic
u x 106sq. cm./v./sec.b Pear
Uncorrecled A B c D E F G
-3.0 -2.5
-1.8 -1.5 -0.5 +0.4 +0.9
Corrected -6.5 -6.0 -5.3
-4.9 -4.0 -3.2 -2.6
0 See Fig. 2. b All mobilities were measured in 0.1 M borate buffer, pH 8.5 (pH 7.9 in the trough). Calculations were made according to the formula fi = dZ/Vt, where d is the observed distance of migrat,ion, 1 the length of the trough, V the voltage across the ends of the trough and t the time in seconds. The corrections for electroosmotic flow were made by assuming an electroosmotic flow of 3.5 X 10-b sq. cm./v./sec.
ZONE
0
BLECTROPHORESIS
OIi
STARCH
OF
Segment Number (cm.)
141
FSH
0
FIG. 1. Pattern obtained after zone electrophoresis on starch in borate buffer, pH 8.5 (7.36 in a 60-cm. trough, 150 v. for 72 hr.) of FSH preparation L2037B.
FIQ. 2. Pattern obtained after zone electrophoresis on starch in borate buffer, pH 8.5 (7.9 in a 60-cm. trough, 190 v. for 72 hr.) of FSH preparation L2037B.
142
RAACKE,
LOSTROH
TABLE Estimation Purified
AND
LI
II
by the Augmentation Assay Method of the Potency of FSH Obtained from Zone Electrophoresis on Starch Ovarian weights
Preparation
Total dose’=
No. of rats
w.
L2037Bb R20B”
ME-500(8)” ME-500(18) ME-500 (20) ME-500 (22) ME-509 (24) ME-500(27) ME-500(30) ME-500(35) 131aDd
0.10 0.15 0.50 0.15 0.30 0.50 0.50 0.50 0.15 0.15 0.15 0.50 0.50 0.50 0.048
4 3 3 8 4 8 2 3 3 3 3 5 6 3 12
Test w.
HCG control w.
40.8 52.0 85.9 55.2 58.5 93.2 28.6 91.8 80.8 87.7 57.9 58.5 40.8 30.5 79.5
32.4 37.1 37.1 45.7 34.8 45.7 37.1 37.1 37.1 37.1 37.1 37.1 37.1 37.1 36.3
Activity slope units
85 100 100 65 80 95 0 110 290 335 140 45 10 0 900
a The total dose for preparations L2037B and R2OB (starting material) was estimated on a dry-weight basis; the doses for preparation ME-500 were estimated from the Folin color, and the dose for preparation 13laD was estimated on a nitrogen basis, assuming 16% N for the FSH protein. b Starting materials for zone electrophoresis. = The numbers in parentheses correspond to the segments (in cm.) of the starch electrophoresis experiment depicted in Fig. 1. d See Fig. 3.
In this way, highly active material could be obtained, material which appears as a reasonably homogeneous peak but which probably does not yet represent a pure substance (see Fig. 3), despite the fact that considerable purification has been achieved. It was found that a total dose of 0.025 mg. when injected into young female hypophysectomized rats caused follicular development with beginning antrum formation (Table III) in contrast to a total dose of 0.3 mg. of the starting material required to achieve the same effect. It was apparent that the purified fraction (131aD) was not completely free from ICSH, as evidenced by the uterine development in hypophysectomized females and by the growth of the ventral prostate in hypophysectomized males (Table III). It can be seen from Fig. 4, which presents a plot of electrophoretic mobility versus pH, that the isoelectric point of the active component
4,0-
c----a
h
Protein Concentrohon Biological Activity
c
c aI -5 a .- o- 2 2
b‘-_ !!a??%
4 6
w
IO
14
Segment
18
22
Number
30
26
34
38 _
(cm.)
FIG. 3. Pattern obtained with an FSH preparation after purification by means of zone electrophoresis on starch (borate buffer, pH 7.9 in a 40-cm. trough, 206 v., 72 hr.).
Biological
TABLE III Characterization in Hypophysectomized FSH Obtained from Zone Electrophoresis Organ
Preparation
Total dos.5’
lo. oitrats
Rats of Purified on Starch
weights
Female
uterus
Ovaries
Prostate
.I-
Saline 131aD” 131aD 131aD 131aD 131aD Saline
9.075 0.100 0.150 0.300 0 0.010 0.025 0.038 0.040 1.30 0
m&z.
+w.
m&-.
R2OB
*5 5 5 4 7 4 4 3 3 3 5
9.8
f
0.76
11.7 10.9 14.2 9.5 7.4 10.6 15.5 17.2 26.9 7.6
& f f f f f f f f f
1.1 0.5 2.3~ 0.7 0.5 O.gc 0.1 2.8 4.7 0.8
15.4 14.7 14.9 14.1 ,66.0 12.7
f * f f f f
mg.
0.8 1.2 0.6 2.3 11.0 0.8
6.3 f 4.2 f
0.3 0.5
13.9 f 7.9 f
1.1 0.5
a The total dose for the R-preparation (representing starting material) was estimated on a dry-weight basis; the dose for the purified preparations (D-fractions) was estimated on a nitrogen basis, assuming 16% N for the FSH protein. h Mean f standard error. c Total dose producing follicular development with beginning antrum formation (M.E.D.). d See Fig. 3. 143
144
RAACKE,
4.0
LOSTROH
5.0
AND LI
6.0PH7.0
8.0
9.0
-1.0
FIQ. 4. Plot of zone electrophoretic mobilities as a function of pH of several commonly encountered components in FSH preparations; the active component is D.
(D fraction in Fig. 2) is 4.6. This is very close to the previously published isoelectric point for FSH of 4.5 (6), a coincidence which might be explained by the fact that the mobility curves of all the commonly encountered contaminants in the FSH preparations investigated converge at 4.6. DISCUSSION
It was evident throughout the present investigation that the FSH activity was not associated with the main protein component in any of the preparations studied, as evidenced by the fact that the main component (segments 27-35 in Fig. 1, A zone in Fig. 2) was found to be comparatively inactive, at the same time that a highly active material (segment 22 in Fig. 1, D zone in Fig. 2) could be located. Although none of the preparations described in previous publications (5, 6) were available when the present study was undertaken, preparations obtained by the same method of isolation and possessing comparable activity show mainly one peak (Figs. 1 and 2). The previous erroneous identification of the main protein component with the activity can easily be understood
ZONE ELECTROPHORESIS ON STARCH OF FSH
145
on the basis of certain characteristics of the material as well as certain factors inherent in the previous methods of analysis. When examined in free solution by means of optical devices (moving-boundary electrophoresis and ultracentrifugation), these preparations tend to appear nearly homogeneous, not only because the A peak (Fig. 2) represents the greater bulk of the material, but also because the rest of the material (including the active substance) is not detectable as a separate component, but, rather, spreads out in such a way as to raise the base line more or less uniformly. Furthermore, because of the coincidence that all the components have an isoelectric point in the neighborhood of pH 4.5 (see Fig. 4), repeated fractionation with ammonium sulfate at this pH produces only an insignificant shift in the relative proportions of all the components; this was verified by the almost identical electrophoretic patterns given by all the fractions obtained from such fractional precipitation experiments. Because of this, the ratio of biological activity to total protein remains constant, within the limits of error of the assay, so that the illusion of homogeneity is again given. From an inspection of Figs. 1 and 2, it is evident that zone electrophoresis on starch accomplishes a very substantial purification of the starting material. On the basis of the yields obtained for the pooled fraction 131aD, approximately a tenfold purification could be expected, and the activity of the isolated material indicates that such a purification was actually attained. In normal rats, the activity of the purified fraction was 900 slope units as compared with SO-100 units for the starting material (Table II); and, in hypophysectomized animals, the M.E.D. for the former was 0.025 mg. as compared with 0.300 mg. for the latter (Table III). It should be pointed out again that, despite this considerable purification, even our best preparations of ovine FSH obtained by zone electrophoresis on starch are not yet pure. From the work of Steelman et al. (15, 16), the same appears to be true for FSH of porcine origin. All that can be said with any certainty at present is that the behavior of the active principle throughout has been consistent with that of an acid protein with an isoelectric point in the neighborhood of pH 4.5. ACKNOWLEDGMENTS This work was supported in part by grants from the National Institutes of Health, United States Public Health Service (G-4097). We wish to acknowledge the able technical assistance of Charles W. Jordan, Jr. and Elizabeth Hageman.
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RAACKE, LOSTROH AND LI SUMMARY
Preparations of follicle-stimulating hormone from sheep pituitary glands have been examined by means of zone electrophoresis on starch. It was found that the activity was not associated with the main protein component, and that the active component as well as the contaminating components had an isoelectric point at pH 4.6. Approximately a tenfold purification could be achieved by the electrophoretic procedure, REFERENCES 1. LI, C. H., in “Amino Acids and Proteins” (D. M. Greenberg, ed.), p. 487.
C. C Thomas, Springfield, Mass., 1951. 2. COLVIN, J. R., SMITH, D. B., AND COOK, W. H., Chem. Revs. 64, 687 (1954). 3. LI, C. H., Acta Endocrinol. 10, 255 (1952). 4. ACHER, R., AND FROMAGEOT,C., Ergeb. Physiol. biol. Chem. u. exptl. Pharmakol. 48, 287 (1955).
5. LI, C.H.,SIMPSON, M.E., AND EVANS, H.M.,Science 109, 445 (1949). 6. LI, C. H., AND PEDERSEN, K. O., J. Gen. Physiol. 36,629 (1952). 7. TISELIUS, A., AND FLODIN, O., Advances in Protein Chem. 8, 461 (1954). 8. KUNKEL, H. G., AND SLATER, R. J., Proc. Sot. Exptl. Biol. Med. 80, 42 (1952). 9. F~NSS-BECH, P., AND LI, C. H., J. BioZ. Chem. 207, 175 (1954). 10. LOWRY,O.H.,ROSEBROUGH,N. J., FARR, A.L., AND RANDALL, R.J.,J. Biol. Chem. 193, 265 (1951). 11. RAACKE, I. D., AND Lr, C. H., J. Biol. Chem. 216, 277 (1955). 17, 366 (1954). 12. RAACKE, I. D., LI, C. H., AND LOSTROH, A., Acta Endocrinol. 13. STEELMAN, S. L., AND POHLEY, F. M., Endocrinology 63, 604 (1953). 14. EVANS, H.M., SIMPSON,M.E., TOLKSDORF, S., AND JENSEN, H., Endocrinology 26, 529 (1939); SIMPSON, M. E., VAN WAGENEN, G., AND CARTER, F., Proc. Sot. Exptl. Biol. Med. 91, 6 (1956). 15. STEELMAN, S. L., LAMONT, W. A., AND BALTES, B. J., Endocrinology 66, 216 (1965). 16. STEELMAN, S. L., LAMONT, W. A., AND BALTES, B. J., Acta Endocrinol. 22, 186 (1966).