Transcortin: A Corticosteroid-binding Protein of Plasma. VII. Half-Life in Normal and Estrogen-treated Subjects Avery A. Sandberg, … , S. Nienhouse, W. R. Slaunwhite Jr. J Clin Invest. 1964;43(3):461-466. https://doi.org/10.1172/JCI104931. Research Article Find the latest version: https://jci.me/104931/pdf Journal of Clinical Investigation Vol. 43, No. 3, 1964 Transcortin: A Corticosteroid-binding Protein of Plasma. VII. Half-Life in Normal and Estrogen-treated Subjects * AVERY A. SANDBERG, M. WOODRUFF, H. ROSENTHAL, S. NIENHOUSE, AND W. R. SLAUNWHITE, JR. (From the Roswell Park Memorial Institute, Buffalo, N. Y.) In 1959 we described elevated plasma concen- The present study was undertaken to deter- trations of transcortin during pregnancy and after mine whether the elevated transcortin levels fol- the administration of estrogens (1, 2). Since lowing estrogen therapy are due 1) to increased then, a number of investigations utilizing a va- synthesis of the protein or 2) to decreased turn- riety of methodologies to ascertain the binding over and, hence, prolonged half-life of transcortin. of cortisol to transcortin have confirmed our ob- This study has been made feasible through the servations (3-8). The different techniques used isolation by Seal and Doe (9, 10) of a highly and the diverse arithmetic approaches employed purified and well-characterized preparation of all indicate that the plasma concentration of trans- transcortin, making possible the injection of la- cortin rises about threefold during pregnancy or beled transcortin free from any significant pro- after estrogen therapy. These findings (3-8) tein contaminants. Another important advance are in essential agreement with our original, less that encouraged us to undertake the study has sophisticated observations and calculations. An been the feasibility of utilizing labeled proteins important contribution has been the isolation by in the study of their half-lives and the demon- Seal and Doe (9, 10) of a highly purified plasma stration that the introduction of a small number transcortin preparation, making it possible to as- of iodine atoms into a protein appears not to certain the amino acid composition of the protein change its biological behavior (11-15). and many of its physical properties. They have shown that transcortin has one binding site for Materials and Methods cortisol and a molecular weight of approximately The transcortin used in this study was isolated ac- 45,000 (9, 10). More recently, these workers cording to the method of Seal and Doe (9, 10).1 The have demonstrated, by means of isolation studies, protein was iodinated according to the technique of that the plasma transcortin concentration is ap- Pressman and Eisen (16) as modified by Day, Planinsek, 100 ml in normal sub- Korngold, and Pressman (17). The iodination proce- proximately 2.5 mg per dures described below were standardized many times jects and 7.5 mg per 100 ml in estrogen-treated in our laboratory utilizing various proteins (17). The subjects (7). These and other values for nor- iodination of the transcortin was carried out on the morn- mal, estrogen-treated, and pregnant subjects (3-8) ing when the subjects were injected. Since we did not are higher than our original calculations (1). know the length of time during which the plasma would can have to be sampled, I"25 was chosen because its half-life Our low values be attributed to the crudeness is greater than that of 181. The results indicate that of the early techniques and the less critical theo- I1" could have served as well. retical physicochemical approaches utilized. The The iodide in 266 ,uc of ,KIJu (carrier free) and 80 methods used in our laboratory now obviate these A1l of 0.0025 M KI was oxidized by the addition of 0.1 shortcomings and yield values similar to those ob- ml of 1 N HCl and 0.05 ml of 0.02 M NaNO,. The tained other workers. pH was immediately made slightly alkaline by adding by 0.5 ml of a 1: 4 (vol/vol) mixture of 1 N NaOH and M * 0.167 borate buffer (pH 7.9) in 0.133 M NaCl. The Submitted for publication September 19, 1963; ac- alkaline iodine solution was added to 3.0 mg of trans- cepted November 14, 1963. cortin dissolved in 0.6 ml of water. The reaction mix- This study has been supported in part by grant A-1240 from the National Institute of Metabolic and 1The transcortin was kindly supplied by Dr. U.. S. Endocrine Disorders, U. S. Public Health Service. Seal. 461 462 SANDBERG, WOODRUFF, ROSENTHAL, NIENHOUSE, AND SLAUNWHITE ture was immediately passed through a 4- X 40-mm col- dogenous transcortin. Radioactive iodine and carbon umn of Amberlite IR-4B (OH) anion exchange resin were assayed simultaneously in a dual chainel liquid (analytical grade) to remove iodide ion. At this step, scintillation spectrometer in a dioxane-ethoxyethanol 22% of the I'2' reacted with transcortin. After passage solvent (20). The degree of correlation of the two through another column of IR-4B resin to ensure com- isotopes was taken as a measure of the identity of ex- plete removal of iodide, the iodination yield was 16%, ogenous I126-transcortin with the endogenous protein. which was equivalent to 0.4 atoms of iodine per mole The chromatography was performed at 4° as follows. of transcortin. This solution was diluted to 250 ml with Approximately 30 ml of plasma was dialyzed overnight physiological saline. against 10 vol of distilled water. The pH of the plasma Treatment of two patients with carcinoma of the was adjusted to 5.0, and the plasma was centrifuged. prostate with diethylstilbestrol (15 mg per day) was The supernatant liquid was applied to a 2- X 30-cm initiated 2 weeks before the injection of 1125-transcortin column of DEAEC equilibrated with buffer, which was and continued throughout the experimental period. 0.025 M in NaCl and 0.015 M in NaH.PO at pH 5.0. Three male laboratory personnel served as untreated After the plasma had percolated into the resin, 0.1 Ac of controls. Just before the injection of IY5-transcortin, 4-C4-cortisol(_0 ,uc per mg) in buffer was added. 30 ml of blood was drawn in heparin. The saline solu- Elution with the same buffer was commenced and con- tion of I'-transcortin (50 ml) was injected intrave- tinued until the optical density at 280 mA of the effluent nously over a period of 1 to 3 minutes. At periodic in- was between 0.2 and 0.6. At this point most of the al- tervals small (10-ml) samples of blood from an oppo- bumin and its associated yellow pigment had been eluted, site arm vein were withdrawn for assay of radioactivity. but the blue band due to ceruloplasmin was at the top of At 1 hour and at 9 days after injection, 50-ml samples the column. A gradient in ionic strength at a con- of blood were drawn, and chromatography of the plasma stant pH of 5.0 was started, using 800 ml of the initial from these samples was performed. buffer in a mixing chamber and dropping inito it a buffer Determination of traniscortin concentration. Plasma consisting of 1: 9 (vol/vol) 0.2 M NaH?PO: 0.09 M cortisol determinations were performed according to NaCl at pH 5.0. Fractions of 30 ml in volume were col- the method of Plager, Cushman, and Chase (18) on lected at a flow rate of less than 1 ml per minute until the preinjection sample of plasma. Equilibrium dialyses the blue band had passed out of the column. were performed at various concentrations of cortisol The protein concentration of each fraction was de- as previously described (1), both before and after heat- termined by comparison with an ovalbumin standard ing at 60° for 20 minutes (4). The protein concen- using the absorption band at 280 m,u. The amount of tration was determined with a Scatchard type plot (19), I125 and C4 in 1 ml of every other fraction was deter- plotting the ratio of the percentage of unbound versus mined in a dual channel liquid scintillation spectrometer. the concentration of bound cortisol. Our experience The high voltage and discriminator were set so that (1), as well as that of others (5, 10), has shown that 90% of the I125 counts appeared in the lower channel and the equilibrium constant of association does not change 42.5% in the upper channel. Under these conditions the with estrogen stimulation, that is, estrogen stimulation efficiency of counting (using both channels) of J125 was produces more transcortin rather than a different pro- 78%o; that of C4 was 86%. Binding of C14-cortisol was tein. Our data, after subtraction of albumin binding performed by equilibrium dialysis (1) on a suitably of cortisol, yield a value of 1 x 10'M' for the equi- diluted sample so that the percentage bound fell between librium constant of association. Using this value, and 40 and 60. The percentage bound per milligram of pro- a molecular weight of 45,000 for the corticosteroid- tein was then calculated. Unless all determinations are binding protein, the concentration of transcortin binding brought to the same end-point (not necessarily this one), sites was calculated for each plasma. Since Seal and comparisons are invalid (19). Doe (9, 10) have shown that there is only one binding site on transcortin for cortisol, this value is also the Results concentration of transcortin. Determination of the half-life of transcortint. From All the subjects were injected between 10 a.m. each specimen, 1 or 2 ml of whole blood was removed and 12 noon and (except normal subject R.E.) for counting.