Binding of Corticosteroids by Plasma Proteins. Iv. the Electrophoretic Demonstration of Corticosteroid Binding Globulin

BINDING OF CORTICOSTEROIDS BY PLASMA PROTEINS. IV. THE ELECTROPHORETIC DEMONSTRATION OF CORTICOSTEROID BINDING GLOBULIN

William H. Daughaday

J Clin Invest. 1958;37(4):519-523. https://doi.org/10.1172/JCI103633.

Research Article

Find the latest version: https://jci.me/103633/pdf BINDING OF CORTICOSTEROIDS BY PLASMA PROTEINS. IV. THE ELECTROPHORETIC DEMONSTRATION OF CORTICOSTEROID BINDING GLOBULIN 1, 2 By WILLIAM H. DAUGHADAY WITH THE TECHNICAL ASSISTANCE OF IDA KOZAK (From the Metabolism Division, Department of Medicine, Washington University School of Medicine, St. Louis, Mo.) (Submitted for publication September 30, 1957; accepted December 5, 1957) The usefulness of equilibrium paper electropho- corticosterone when tested under conditions of resis to detect the binding of corticosteroids by low hormone concentration. plasma proteins was described in an earlier paper The second corticosteroid .binding system is of this series (2). Plasma was dialyzed initially operative at high plasma concentrations of cortisol. against the electrophoretic buffer to which carbon Most of this binding can be attributed to albumin labeled corticosteroids had been added. Later this whose binding characteristics have been adequately buffer was used to saturate the electrophoretic described (5-7). Cortisol and corticosterone are paper. Thus, the concentration relationship be- bound much less firmly by albumin than the other tween the buffer steroid and the protein bound steroid hormones mentioned above. steroid was maintained throughout the develop- The failure to observe two corticosteroid bind- ment of the electrophoresis. Consequently, there ing zones in our equilibrium paper electrophoresis was little tendency for the bound steroid to trail experiments can be attributed to the fact that these the advancing protein components as occurs with experiments were carried out with amounts of customary electrophoresis of steroids (3). In corticosterone and cortisol far in excess of those the experiments carried out with this method us- needed to saturate the corticosteroid binding glob- ing barbital buffer, pH 8.8, both corticosterone- ulin. Binding, predominantly by albumin, could 4-C14 and cortisol-4-C14 appeared to migrate with be anticipated under these conditions. It was not the albumin peak of plasma. possible to carry out equilibrium paper electro- Subsequent experiments with carbon labeled phoresis at physiologic cortisol concentrations be- steroids using dialysis equilibrium suggested the cause the small amount of plasma which can be ap- possibility that there were two important corti- plied to the electrophoretic paper limits the amount costeroid binding proteins of human plasma (4). of radioactive steroid available for counting. In The first of these binding systems probably ac- the present paper, this difficulty was circumvented counts for nearly all the binding of cortisol at by using continuous flow paper electrophoresis of normal physiologic concentrations. Under such equilibrated plasma, which has permitted the dem- circumstances about 99 per cent of the cortisol in onstration of a corticosteroid binding globulin plasma is protein-bound. As the concentration that is distinct from human albumin. of hormone in the plasma rises, the degree of bind- ing falls rapidly at first and then very much more METHODS slowly. Like plasma, plasma Fraction IV-4 has Serum was collected from laboratory workers and also been demonstrated to have a high affinity for medical students, two to three hours after breakfast. Before subjecting this serum to continuous flow electro- corticosterone and cortisol. The affinity of this phoresis, it was dialyzed extensively against the electro- fraction for progesterone, testosterone, estrone, phoretic buffer in the presence of the radioactive steroid and estradiol is much less than for cortisol and under study. Ten ml. of serum in a Visking cellophane bag was dialyzed against 1,500 ml. of buffer. The amount 1 A preliminary report of these findings has been pub- of steroid added to the serum and buffer was estimated lished (1). to provide about 0.5 ,ug. of steroid bound to the serum 2 This investigation was supported by a research grant, and an equilibrium concentration in the buffer. To speed C-255, from the National Institute of Arthritis and Meta- the attainment of equilibrium, 0.5 ,ug. of the labeled steroid bolic Diseases of the National Institutes of Health, Pub- was added to the plasma directly. To the buffer was lic Health Service. added 2.3 Ag. of cortisol-4-CG or corticosterone-4-C". 519 520 WILLIAM H. DAUGHADAY

(9). The protein bound hexose was determined in sev- eral experiments by the method of Winzler (10). Radio- 120~ activity was determined in each tube by extracting 3 ml. so. of eluent with 25 ml. of chloroform in a large stoppered 60.1 tube. The extracts were washed with 2.5 ml. of 0.1 N sodium hydroxide and 2.5 ml. of water. Twenty ml. of 20- the final chloroform extracts was evaporated in 40 ml. 401 centrifuge tubes. The residues, of negligible mass, were 1 2 3 4 5 6 7 8 9 10 PI 12 13 14 15 16 17 18 19 20 21 22 transferred to small brass cups in alcohol. Radioactivity 600. was determined in a gas flow counter operating in the proportional range. I-400j400.1 The cortisol-4-C1' and the corticosterone-4-C' used in 3200X these experiments were obtained from the Endocrinology

100. Study Section of the National Institutes of Health. Both compounds had a specific activity of 1.467 milli- curies per millimole. The progesterone-4-C1" was ob- FIG. 1. CONTINUOUS FLOW ELECTROPHORESIS OF Hu- tained from Tracer Lab, Inc., and had a specific activity MAN SERUM CARRIED OUT WITH BARBITAL BUFFER, PH of 1.63 millicuries per millimole. 8.8, IONIC STRENGTH 0.045 IN THE PRESENCE OF CORTI- COSTERONE-4-C"4 The general reproducibility of the electrophoretic experiments was excellent, although slight changes Serum was applied to curtain above tube 14. The elu- in the rate of migration and the exactness of definition tion tubes are indicated by the Nos. 1 through 22 between occurred. The accuracy of counting was better than + 10 the two curves. per cent.

In the experiments with progesterone-4-C"4, 1.87 ,ug. of RESULTS this steroid was added to the buffer. Nonradioactive cortisol, 350 /Ag., was added in certain experiments with The binding of cortisol-4-C14 was first examined progesterone-4-C"4. electrophoretically in barbital buffer. When the Continuous flow electrophoresis was carried out us- electrophoresis was performed in a buffer with an ing the Karler-Misco apparatus at 40 C. Two electro- of phoretic buffers were used-barbituric acid-sodium bar- ionic strength 0.045, the peak of radioactivity biturate, pH 8.8, ionic strength 0.045; acetic acid-sodium of the bound steroid followed the main albumin acetate, pH 5.2, 0.02 M. A fractionation plate, similar peak (Figure 1). Only a slight asymmetry with to that described by Shetlar, Cahill, Stidworthy, and a shoulder of increased radioactivity in the albu- Shetlar (8), gave improved separation of the protein min containing tubes was apparent. The position fraction. The curtains were made from Schleicher and Schuell paper No. 588. The upper reservoirs of the ap- paratus, which provide the flow of buffer for the curtain, were filled with the buffer which had been dialyzed against the serum in the presence of the radioactive ster- so- were filled with stock buffer. oid. The electrode vessels *50- As soon as the curtain was wet with buffer, the current i 40. was turned on for three to five hours to create a steady state. In the barbital buffer the current flow was 13 milliamperes with 700 volts and 4 to 6 milliamperes with 20-I0 800 to 900 volts with the acetate buffer. 2 3 4 5 4 9 10 11 12 14 IS 14 Ito191 1 a T EI 6 7 13 Serum was applied to the paper through a 2 mm. wick cut from Schleicher and Schuell paper No. 470A. In a period of from 36 to 40 hours, 5 to 10 ml. of serum were fractionated. The eluent was collected in 22 small 300200- - test tubes. Each tube contained between 3 and 4 ml. of a ~~~~~~~~~~~~0, eluent at the end of a run. The curtain was removed and dried rapidly with a hair drier. Drying was completed and the proteins fixed to the paper by heating to 1000 C. FIG. 2. CONTINUOUS FLOW ELECTROPHORESIS OF HUMAN for 30 minutes in an oven. The positions of the pro- SERUM IN THE PRESENCE OF CORTICOSTERONE-4-C' tein bands were identified by staining with bromphenol Acetate buffer, pH 5.2, 0.02 M was used. Serum was blue. applied to curtain above tube 12. The dotted line on the The eluent tubes were made up to a total volume of lower graph indicates protein concentration measured by 5 ml. each. Aliquots were analyzed for protein using the Lowry, Rosebrough, Farr, and Randall procedure the method of Lowry, Rosebrough, Farr, and Randall (9). ELECTROPHORESIS OF CORTICOSTEROID BINDING GLOBULIN 521 When the electrophoresis of normal serum was carried out with cortisol-4-C14, a similar distri- bution of radioactivity-was observed (Figure 3). In this particular experiment there was a greater concentration of the anodally migrating protein components in the region of the cortisol binding than observed in the experiment with corticos- terone. The binding pattern with progesterone-4-C14 is of considerable interest (Figure 4) in that there is a clearly defined double peak of binding of radioactivity. One peak is associated with the anodally migrating component which binds corti- costeroids, but in addition there is significant bind- FIG. 3. CONTINUOUS FLOW ELECTROPHORESIS OF HUMAN ing in the region of the albumin peak. This find- THE OF CORTISoL-4-C14 SERUM IN PRESENCE ing is consistent with the interpretation of the Acetate buffer, pH 5.2, 0.02 M was used. Serum was applied to curtain above tube 12. The dotted line on the binding of progesterone-4-C14 in dialysis equi- lower graph indicates the protein concentration measured librium experiments with different quantities of by the Lowry, Rosebrough, Farr, and Randall procedure carrier nonradioactive progesterone (4). It was (9). suggested that the binding of progesterone was relatively less for corticosteroid binding globulin of the radioactivity in respect to the albumin peak and relatively greater for albumin as compared was influenced by minor experimental variations. to cortisol. To support this view, the ability of Lowering the ionic strength of the barbital buffer cortisol to displace progesterone-4-C14 from the increased the mobility of the bound radioactive corticosteroid binding globulin has been studied. steroid. With a buffer of ionic strength of 0.02, In the electrophoretic experiments, depicted in the bound steroid slightly preceded the albumin Figure 5, 350 ug. of nonradioactive cortisol was peak. added to the preliminary dialysis carried out with To gain further information concerning the 10 ml. of serum, 1,500 ml. of acetate buffer and binding of corticosterone, experiments have been conducted using 0.02 M acetate buffer, pH 5.2. Albumin migrates little at this pH and descends directly from the point of application above tube 12. In control experiments in which cortisol4-C04 I. was added directly to the curtain in the absence of serum the steroid descended somewhat ca- I thodally from the point of application with peak concentrations in tubes 14 and 15. The displace- ment of the noncharged steroid can be attributed to electroendosmotic movement of the buffer. When the experiment was carried out with serum I the distribution of radioactivity was very different from the control experiment (Figure 2). The greatest concentrations of the labeled cortisol were located in tubes 5, 6, and 7. These tubes are on FIG. 4. CONTINUOUS FLOW ELECTROPHORESIS OF HUMAN the anodal side of the albumin which was con- SERUM IN THE PRESENCE OF PROGESTERONE-4-C1' in tubes 10 and 11. The tubes con- Acetate buffer, pH 5.2, 0.02 M was used. Serum was centrated applied to curtain above tube 12. The dotted line on the taining the greatest amounts of the labeled steroid lower graph indicates protein concentration measured by were found to contain only small quantities of the Lowry, Rosebrough, Farr, and Randall procedure protein. (9). 522 WILLIAM H. DAUGHADAY sociated with M-2. The fastest minor protein peak in our experiments probably corresponds to M-1, and M-2 was not clearly separated from al- bumin. The presence of bound hexose and hex- osamine in these fractions has been confirmed by analysis. The anodal migration of corticosteroid binding globulin occupies a position intermediate between that reported for M-1 and M-2, and in certain experiments, such as in Figure 1, these tubes contain only minute amounts of protein. This observation cannot be construed as evidence that the corticosteroid binding material is other FIG. 5. CONTINUOUS FLOW ELECTROPHORESIS OF Hu- than protein. The ease with which corticosteroid MAN SERUM IN THE PRESENCE OF PROGESTERONE-4-C' binding globulin of plasma is saturated with small AND NONRADIOACTIVE CORTISOL. additions of corticosteroids indicates that only Acetate buffer, pH 5.2, 0.02 M. was used. Serum ap- plied above tube 12. minute amounts of the protein are normally pres- ent. The preliminary binding experiments which progesterone-4-C'4. Under these conditions, the have been done with plasma protein fractions in- binding of the progesterone-4-C14 by the anodally dicate that corticosteroid binding globulin may be contained in Fraction IV-4, a fraction known to be moving protein was virtually eliminated and the binding by the albumin peak greatly accentuated. rich in glycoproteins (2). The relatively low affinity of progesterone for Analyses of the bound hexose distribution are also included in this graph. the corticosteroid binding globulin has been ex- perimentally useful in permitting the demonstra- tion of binding of this protein and albumin. The DISCUSSION change in distribution of progesterone with the The application of the principle of equilibrium addition of cortisol is explained by the higher electrophoresis to continuous flow electrophoresis affinity of the corticosteroid binding globulin for has presented no particular problems. The ap- cortisol. paratus selected for use has been favorable for such studies because of the small capacity of the SUM MARY reservoirs feeding the buffer to the curtain. Other 1. The binding of cortisol-4-C14, corticosterone- designs of this type of instrument have larger buf- 4-C14, and progesterone-4-C14 by human serum fer reservoirs which make the initial dialysis of proteins has been studied by continuous flow elec- buffer with serum more troublesome. trophoresis. Careful predialysis of the serum, the The existence of corticosteroid binding globu- steroid, and the electrophoretic buffer has pre- lin with mobility characteristics distinct from albu- vented elution artifacts. min seems evident from the experimental results. 2. With barbital buffer, pH 8.8, the peak of In the customary barbital buffer system this pro- corticosterone binding migrates more slowly than tein has the mobility of an alpha globulin. albumin and is present in the alpha globulin re- Anodal mobility of the corticosteroid binding gion. In acetate buffer, pH 5.2, the corticosteroid globulin in acetate buffer, pH 5.2, is indicative of binding globulin moves anodally away from the a low isoelectric point. These features suggest albumin component. an acidic glycoprotein. At this pH, two anodally 3. Binding studies conducted with progester- migrating glycoproteins have been recognized. one-4-C14 have permitted the demonstration of The fastest component has been described by binding both by the anodally moving binding pro- Mehl, Golden, and Winzler (11) as M-1 and the tein and by albumin. The bound progesterone second component with a mobility only slightly could be displaced off the anodally moving com- faster than albumin has been called M-2. The ponent onto the albumin peak by adding cortisol thyroxine binding globulin is believed to be as- to the system. ELECTROPHORESIS OF CORTICOSTEROID BINDING GLOBULIN 523 4. These observations provide direct evidence Solubility determinations. J. biol. Chem. 1954, of the existence of a corticosteroid binding globu- 206, 411. lin which is separate from albumin and suggest its 6. Westphal, U. Steroid protein interactions. III. Spectrophotometric demonstration of interaction glycoprotein nature. between proteins and progesterone, desoxycorti- costerone and cortisol. Arch. Biochem. 1957, 66, 71. REFERENCES 7. Westphal, U., and Ashley, B. D. Spectrophotometric 1. Daughaday, W. H. Corticosteroid binding by a observations on interaction between proteins and plasma alpha globulin (abstract). J. clin. Invest. A'-3-ketosteroids. Fed. Proc. 1957, 16, 269. 1957, 36, 881. 8. Shetlar, M. R., Cahill, C., Stidworthy, G., and Shet- 2. Daughaday, W. H. Binding of corticosteroids by lar, C. L. Comparison of continuous and strip plasma proteins. II. Paper electrophoresis and paper electrophoresis technics for study of serum equilibrium paper electrophoresis. J. clin. Invest. glycoproteins. Proc. Soc. exp. Biol. (N. Y.) 1956, 1956, 35, 1434. 93, 44. 3. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Westphal, U., Firschein, H. E., and Pearce, E. M. Randall, R. Protein measurement Binding of hydrocortisone-4-C1' and J. with the progesterone- Folin phenol reagent. J. biol. Chem. 1951, 193, 4-C1 to serum albumin, demonstrated by paper 265. electrophoresis. Science 1955, 121, 601. 10. Winzler, R. J. Determination of serum glycopro- 4. Daughaday, W. H. Binding of corticosteroids by teins in Methods of Biochemical Analysis, D. plasma proteins. III. The binding of corticos- Glick, Ed. New York, Interscience Publishers, teroid and related hormones by human plasma and 1955, vol. 2, p. 279. plasma protein fractions as measured by equilibrium 11. Mehl, J. W., Golden, F., and Winzler, R. J. Muco- dialysis. J. clin. Invest. 1958, 37, 511. proteins of human plasma. IV. Electrophoretic 5. Eik-Nes, K., Schellman, J. A., Lumry, R., and Sam- demonstration of mucoproteins in serum at pH 4.5. uels, L. T. The binding of steroids to protein. I. Proc. Soc. exp. Biol. (N. Y.) 1949, 72, 110.