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The isolation of human pituitary follicle-stimulating hormone
218
P R E L I M I N A R Y NOTES
PN I33~)
The isolation of human pituitary follicle-stimulating hormone Several papers dealing with the purification of human pituitary F S H have been published 1-4. The physico-chemical characterization has, however, been very limited and no unequivocal criterion of homogeneity has been reported for any preparation. A procedure is presented here, developed by one of us (P. R.), giving a FSFI homogeneous ill both the ultracentrifuge and in the free zone-electrophoresis apparatus. The starting material was whole frozen human pituitaries collected at autopsy. Normally about 300 glands (about 200 g) were used in each preparation. All steps were performed at 0-4 U. The glands were initially treated as described earlier 5 in connection with the preparation of human growth hormone. After removal of the human growth hormone-containing material at 0.5 satd. (NH4)2S()4 solution, F S H was precipitated by a slow addition of solid (NH4) 2SO4 (175 g/1 solution). Continuous stirring for I h was followed by centrifugation for io rain at 1600o g. The precipitate was dissolved and immediately subiected to fractionation. About 2.8 g of crude F S H was obtained per IOO g of frozen pituitary tissue. The potency was 1-1. 5 times that of the ovine F S H preparation NIH-FSH-S~, when estimated by the ovarian augmentation method in immature female rats 6. (Lyophilization was avoided throughout the whole purification procedure and both the doses for biological assays and the yields were based on absorbancy measurements, assuming an absorbancy coefficient of io 3 at 280 m~.) Fractionation of the crude FSFI was achieved by ion-exchange column chromatography on DEAE-cellulose. The adsorbent (about 12oo ml for a normal preparation) was equilibrated with 0.02 M potassium phosphate (pH 7.0) and operated at a rate of about 25 ml/em2/h. Under these conditions the F S H was adsorbed; about 5o % of the applied amount of protein, including the essential part of the luteinizing hormone present, passed the column unretarded. One-step increase in the buffer concentration to 0.06 M eluted the follicle-stimulating activity, appearing in a welldefined peak in the chromatogram. During this step the flow rate of the column was decreased to about IO ml/cm2/h. The active fraction was concentrated by ultrafiltration 7 and submitted to filtration through a column (21o >~ 2.2 cm) packed with Sephadex G-Ioo equilibrated with o.i M potassium phosphate buffer (pH 7.o) also containing 0.3 M NaC1. Development of the chromatogram by this buffer at a rate of about 4 ml/cm2/h resulted in a concentration of the activity, being now associated with about io % of the material filtered through the column. Rechromatography of the active material on Sephadex under identical conditions gave a further elimination of inert protein. By the procedure outlined a follicle-stimulating fraction was prepared in a yield of about 3 mg per IOO g of pituitary tissue. Its biological activity in the augmentation assay was estimated to be 15o-2oo times the potency of the N I H - F S H - S 1 preparation. When investigated for sedimentation homogeneity in a Spinco model-E ultracentrifuge only one boundary could be observed (Fig. I). The corrected sedimentation constant (szo,w) is calculated to 2. 9 S. Investigations for electrophoretie homogeneity by analytical eleetrophoresis indicated, however, the presence of three components. Different preparative electrophoretic procedures have been investigated concerning their application to this separation problem and to the alnotmts of material available (5-1o nag). Electrophoresis in aerylamide gel with migration-elution of the Biochim. Biophy,~. ,4c/~,, 82 (t9(;4) 2t,~ 22o
PRELIMINARY NOTES
219
zones8 was found to give the best results under following conditions. The column used (28 × 2.1 cm) was equipped with both central (diameter 0.8 cm) and outer water-cooling. Attached to the bottom was a separate elution cell, operating according to the same principles as the cells described9,10 earlier. A constant counter flow through the cell was maintained by a peristaltic pump. The polymerization
Fig. i. Sedimentation patterns of FSH from the second gel filtration on Sephadex G-ioo after 25, 57, 89, 121, 153 and 185 min at 59780 rev./min and 20.0°. Buffer, sodium phosphate (pH 7.0, I 0.o2) containing also o.io M NaC1. In I the angle of the schlieren diaphragm was 7°0 in II 60° and in III-VI 50°. (5 % (w/v) acrylamide, o.13 % (w/v) N,N'-methylene-bisacrylamide and 0.07 % (w/v) ammonium persulfate) was performed in o.2 M Tris-I-IC1 buffer (pFI 8.7) also used in the electrode vessels. The length of the gel column was 25 cm. The elution cell was filled with grains of polyacrylamide gel prepared by pressing gel through a 6o-mesh screen. Prior to electrophoresis, material from the second gel filtration was concentrated by ultrafiltration and dialyzed against the 0.2 M buffer, diluted I:IO. The zone (about I ml), applied on the top of the gel column, contained 6 % sucrose and was composed of equal volumes of the dialysate and of an 0.2 % suspension of agarose n in the diluted buffer. The agarose suspension was added to prevent convection in the zone and the function of the sucrose was to make the zone heavier than the 0.2 M buffer layered above it. Initially the current was 3 ° mA but after the zone had migrated into the gel, the current was increased to 80 mA. The rate of elution was about 15 ml/h and 3-ml fractions were collected during about 50 h. The electropherogram, based on absorbancy measurements at 230 m~, indicated a satisfactory separation of the three components observed in analytical runs. Only one of the components, obtained in a yield of about I m g per IOO g of pituitary tissue, was found to be associated with the follicle-stimulating activity. Preliminary data indicate, however, no significant increase in specific activity. This may be the result of an instability of the highly purified FSH in the diluted solutions (about o.2/~g protein per ml) used in the biological assays. It may also reflect the difficulties in preparing the small biological doses (1-2 ~g per animal). Owing to the elution technique and to the small amounts of material applied, the absorbancy values at 280 m~ are very low. Irregularities in the base-line, which are difficult to correct, can therefore give rise to large errors in the calculated doses. Re-electrophoresis of the active component from the gel electrophoresis in a column n (50 × 0.5 cm) with an o.18 % agarose suspension as supporting medium gave no further resolution. The experiment was performed in o.I M Tris-HC1 buffer (pH 8.7) at a current of 7, 5 mA. Material from the central part (about 7° %) of the protein peak obtained in this run was tested for electrophoretic homogeneity by the Biochim. Biophys. Acta, 82 (1964) 218-22o
220
PRELIMINARY NOTES
m u c h more accurate m e t h o d of free zone electrophoresis 12. The recorder indicated a sharp symmetrical peak (Fig. 2), corresponding to a substance with a mobility calculated to be --6.1. lO -5 cm2/V.sec. z:r
.
.
.
.
I
:
~ o:
11.S cr:n
I
i
"O
g
O
Fig. 2. U l t r a v i o l e t r e c o r d i n g from t h e free zone e t e c t r o p h o r e s i s of t h e h i g h l y puri fi e d F S H . Buffer, o . i M T r i s - H C l (pH 8.7); i n n e r d i a m e t e r of t h e e l e c t r o p h o r e s i s tube, 3.o m m ; c u r r e n t , 4.2 m A ; t e m p . , a b o u t 1% T h e d i a g r a m s refer to o, a n d 56 rain of electrophoresis. The a r r o w s i n d i c a t e t h e p o s i t i o n of t h e s t a r t i n g zone.
The authors are indebted to Professor A. TISELIUS for his kind interest in this work. We also wish to express our gratitude to Dr. S. HJERT~,N for performing the free zone electrophoresis. Financial support from U.S. Public H e a l t h Service, National Institute of Health (Grant AM o6o6o) a n d from the Swedish Medical Research Council are gratefully acknowledged.
Institute of Biochemistry, University of Uppsala, Uppsala (Sweden) Department of Obstetrics and Gynecology, Akademiska Sjukhuset, University of Uppsala, Uppsala (Sweden)
P. R o o s
C. A. GEMZELL
1 ~k. SEGALOFF AND S. L. STEELMAN, Recent Progr. Hormone Res., 15 (1959) 127. 2 p. R o o s AND C, A. GEMZELL, Ciba Found. Colloq. Endocrinol., 13 (196o) 209. a C. H. LI, P. G. SQUIR~ AND U. GR6SCHEL, Arch. Biochem. Biophys,, 86 (196o) i i o . 4 W. R. BUTT, A. C. CROOKE AND F. J. CUNNINGHAM, Biochem. J., 81 (1961) 5965 p. R o o s , H. R. FEVOLD AND C. A. GEMZELL, Biochim. Biophys. Acta, 74 (1963) 525 • S. L. STEELMAI~ ANn F. M. POHLEV, Endocrinology, 53 (1953) 6o4. 7 B. VoN HOFSTEN AND S. O. FALKBRING, Anal, Biochem., I (196o) 436. 8 S. HJERTI~N, J. Chromatog., IZ (1963) 66. 9 j . POR.ATH, E. B. LINDNER AND S. ]ERSTEDT, Nature, 182 (1958) 744. 10 M. A. NAUGHTON AND K. W. TAYLOR, Biochem. J., 77 (196o) 46. 11 S. HJERT~N, J. Chromatog., in t h e press. 12 S. HJERTt~N, Protides Biol. Fluids, Proc. Colloq., 7 (1959) 28.
Received November 8th, 1963 Biochim. Biophys. Acta, 82 (I964) 218-22o
P R E L I M I N A R Y NOTES
PN I33~)
The isolation of human pituitary follicle-stimulating hormone Several papers dealing with the purification of human pituitary F S H have been published 1-4. The physico-chemical characterization has, however, been very limited and no unequivocal criterion of homogeneity has been reported for any preparation. A procedure is presented here, developed by one of us (P. R.), giving a FSFI homogeneous ill both the ultracentrifuge and in the free zone-electrophoresis apparatus. The starting material was whole frozen human pituitaries collected at autopsy. Normally about 300 glands (about 200 g) were used in each preparation. All steps were performed at 0-4 U. The glands were initially treated as described earlier 5 in connection with the preparation of human growth hormone. After removal of the human growth hormone-containing material at 0.5 satd. (NH4)2S()4 solution, F S H was precipitated by a slow addition of solid (NH4) 2SO4 (175 g/1 solution). Continuous stirring for I h was followed by centrifugation for io rain at 1600o g. The precipitate was dissolved and immediately subiected to fractionation. About 2.8 g of crude F S H was obtained per IOO g of frozen pituitary tissue. The potency was 1-1. 5 times that of the ovine F S H preparation NIH-FSH-S~, when estimated by the ovarian augmentation method in immature female rats 6. (Lyophilization was avoided throughout the whole purification procedure and both the doses for biological assays and the yields were based on absorbancy measurements, assuming an absorbancy coefficient of io 3 at 280 m~.) Fractionation of the crude FSFI was achieved by ion-exchange column chromatography on DEAE-cellulose. The adsorbent (about 12oo ml for a normal preparation) was equilibrated with 0.02 M potassium phosphate (pH 7.0) and operated at a rate of about 25 ml/em2/h. Under these conditions the F S H was adsorbed; about 5o % of the applied amount of protein, including the essential part of the luteinizing hormone present, passed the column unretarded. One-step increase in the buffer concentration to 0.06 M eluted the follicle-stimulating activity, appearing in a welldefined peak in the chromatogram. During this step the flow rate of the column was decreased to about IO ml/cm2/h. The active fraction was concentrated by ultrafiltration 7 and submitted to filtration through a column (21o >~ 2.2 cm) packed with Sephadex G-Ioo equilibrated with o.i M potassium phosphate buffer (pH 7.o) also containing 0.3 M NaC1. Development of the chromatogram by this buffer at a rate of about 4 ml/cm2/h resulted in a concentration of the activity, being now associated with about io % of the material filtered through the column. Rechromatography of the active material on Sephadex under identical conditions gave a further elimination of inert protein. By the procedure outlined a follicle-stimulating fraction was prepared in a yield of about 3 mg per IOO g of pituitary tissue. Its biological activity in the augmentation assay was estimated to be 15o-2oo times the potency of the N I H - F S H - S 1 preparation. When investigated for sedimentation homogeneity in a Spinco model-E ultracentrifuge only one boundary could be observed (Fig. I). The corrected sedimentation constant (szo,w) is calculated to 2. 9 S. Investigations for electrophoretie homogeneity by analytical eleetrophoresis indicated, however, the presence of three components. Different preparative electrophoretic procedures have been investigated concerning their application to this separation problem and to the alnotmts of material available (5-1o nag). Electrophoresis in aerylamide gel with migration-elution of the Biochim. Biophy,~. ,4c/~,, 82 (t9(;4) 2t,~ 22o
PRELIMINARY NOTES
219
zones8 was found to give the best results under following conditions. The column used (28 × 2.1 cm) was equipped with both central (diameter 0.8 cm) and outer water-cooling. Attached to the bottom was a separate elution cell, operating according to the same principles as the cells described9,10 earlier. A constant counter flow through the cell was maintained by a peristaltic pump. The polymerization
Fig. i. Sedimentation patterns of FSH from the second gel filtration on Sephadex G-ioo after 25, 57, 89, 121, 153 and 185 min at 59780 rev./min and 20.0°. Buffer, sodium phosphate (pH 7.0, I 0.o2) containing also o.io M NaC1. In I the angle of the schlieren diaphragm was 7°0 in II 60° and in III-VI 50°. (5 % (w/v) acrylamide, o.13 % (w/v) N,N'-methylene-bisacrylamide and 0.07 % (w/v) ammonium persulfate) was performed in o.2 M Tris-I-IC1 buffer (pFI 8.7) also used in the electrode vessels. The length of the gel column was 25 cm. The elution cell was filled with grains of polyacrylamide gel prepared by pressing gel through a 6o-mesh screen. Prior to electrophoresis, material from the second gel filtration was concentrated by ultrafiltration and dialyzed against the 0.2 M buffer, diluted I:IO. The zone (about I ml), applied on the top of the gel column, contained 6 % sucrose and was composed of equal volumes of the dialysate and of an 0.2 % suspension of agarose n in the diluted buffer. The agarose suspension was added to prevent convection in the zone and the function of the sucrose was to make the zone heavier than the 0.2 M buffer layered above it. Initially the current was 3 ° mA but after the zone had migrated into the gel, the current was increased to 80 mA. The rate of elution was about 15 ml/h and 3-ml fractions were collected during about 50 h. The electropherogram, based on absorbancy measurements at 230 m~, indicated a satisfactory separation of the three components observed in analytical runs. Only one of the components, obtained in a yield of about I m g per IOO g of pituitary tissue, was found to be associated with the follicle-stimulating activity. Preliminary data indicate, however, no significant increase in specific activity. This may be the result of an instability of the highly purified FSH in the diluted solutions (about o.2/~g protein per ml) used in the biological assays. It may also reflect the difficulties in preparing the small biological doses (1-2 ~g per animal). Owing to the elution technique and to the small amounts of material applied, the absorbancy values at 280 m~ are very low. Irregularities in the base-line, which are difficult to correct, can therefore give rise to large errors in the calculated doses. Re-electrophoresis of the active component from the gel electrophoresis in a column n (50 × 0.5 cm) with an o.18 % agarose suspension as supporting medium gave no further resolution. The experiment was performed in o.I M Tris-HC1 buffer (pH 8.7) at a current of 7, 5 mA. Material from the central part (about 7° %) of the protein peak obtained in this run was tested for electrophoretic homogeneity by the Biochim. Biophys. Acta, 82 (1964) 218-22o
220
PRELIMINARY NOTES
m u c h more accurate m e t h o d of free zone electrophoresis 12. The recorder indicated a sharp symmetrical peak (Fig. 2), corresponding to a substance with a mobility calculated to be --6.1. lO -5 cm2/V.sec. z:r
.
.
.
.
I
:
~ o:
11.S cr:n
I
i
"O
g
O
Fig. 2. U l t r a v i o l e t r e c o r d i n g from t h e free zone e t e c t r o p h o r e s i s of t h e h i g h l y puri fi e d F S H . Buffer, o . i M T r i s - H C l (pH 8.7); i n n e r d i a m e t e r of t h e e l e c t r o p h o r e s i s tube, 3.o m m ; c u r r e n t , 4.2 m A ; t e m p . , a b o u t 1% T h e d i a g r a m s refer to o, a n d 56 rain of electrophoresis. The a r r o w s i n d i c a t e t h e p o s i t i o n of t h e s t a r t i n g zone.
The authors are indebted to Professor A. TISELIUS for his kind interest in this work. We also wish to express our gratitude to Dr. S. HJERT~,N for performing the free zone electrophoresis. Financial support from U.S. Public H e a l t h Service, National Institute of Health (Grant AM o6o6o) a n d from the Swedish Medical Research Council are gratefully acknowledged.
Institute of Biochemistry, University of Uppsala, Uppsala (Sweden) Department of Obstetrics and Gynecology, Akademiska Sjukhuset, University of Uppsala, Uppsala (Sweden)
P. R o o s
C. A. GEMZELL
1 ~k. SEGALOFF AND S. L. STEELMAN, Recent Progr. Hormone Res., 15 (1959) 127. 2 p. R o o s AND C, A. GEMZELL, Ciba Found. Colloq. Endocrinol., 13 (196o) 209. a C. H. LI, P. G. SQUIR~ AND U. GR6SCHEL, Arch. Biochem. Biophys,, 86 (196o) i i o . 4 W. R. BUTT, A. C. CROOKE AND F. J. CUNNINGHAM, Biochem. J., 81 (1961) 5965 p. R o o s , H. R. FEVOLD AND C. A. GEMZELL, Biochim. Biophys. Acta, 74 (1963) 525 • S. L. STEELMAI~ ANn F. M. POHLEV, Endocrinology, 53 (1953) 6o4. 7 B. VoN HOFSTEN AND S. O. FALKBRING, Anal, Biochem., I (196o) 436. 8 S. HJERTI~N, J. Chromatog., IZ (1963) 66. 9 j . POR.ATH, E. B. LINDNER AND S. ]ERSTEDT, Nature, 182 (1958) 744. 10 M. A. NAUGHTON AND K. W. TAYLOR, Biochem. J., 77 (196o) 46. 11 S. HJERT~N, J. Chromatog., in t h e press. 12 S. HJERTt~N, Protides Biol. Fluids, Proc. Colloq., 7 (1959) 28.
Received November 8th, 1963 Biochim. Biophys. Acta, 82 (I964) 218-22o