Abstract
Abstract Human pregnancy serum was previously reported to exhibit a high binding affinity for testosterone; steroid binding was measured by a semimicrotechnique, based on the principle of equilibrium dialysis but utilizing Sephadex G-25 in a batchwise fashion. This property may be ascribed to a testosterone-binding component, presumably a protein, present in very low concentration in serum. The testosterone-binding component was readily separated from corticosteroid-binding globulin (both steroid-binding proteins are present at elevated levels in late pregnancy) by column chromatography of pooled pregnancy serum on microgranular diethylaminoethyl cellulose. Stepwise elution furnished four major fractions: γ-globulin, β-globulin, albumin, and α-globulin, in that sequence. The testosterone-binding component appeared in the β-globulin fraction, whereas the corticosteroid-binding globulin (measured by its cortisol- and progesterone-binding activities) appeared in the albumin fraction; the latter fraction also exhibited considerable testosterone-binding activity, attributable to albumin itself and, in part, to corticosteroid-binding globulin. A portion of the β-globulin fraction (after precipitation in 50% ammonium sulfate) was chromatographed on a column of Sephadex G-100; the testosterone-binding protein was thereby separated from the bulk of the protein (the former emerged last from the column). Considerable losses in testosterone-binding activity were, however, encountered at each stage of purification, and so the over-all increase in specific binding activity (i.e. testosterone-binding activity per g of total protein) was only about 4-fold. Other fractionation procedures, i.e. column chromatography on hydroxylapatite and on fibrous DEAE-cellulose, were not as efficacious, but nonetheless informative; the testosterone-binding component is readily precipitated from whole serum in 50% saturated ammonium sulfate. The testosterone-binding component is distinctly different from corticosteroid-binding globulin in its chromatographic behavior and steroid-binding properties. There is also an appreciable difference in molecular size according to gel filtration studies: the testosterone-binding component has a Stokes molecular radius of about 47 A compared to 36 A for corticosteroid-binding globulin; the same value was obtained for the latter protein from the sedimentation and diffusion data of other investigators.
Highlights
METHODSTritium Determination described (l), in a Packardsampaluetsomawtiecre Tcroi-uCnatrebd, liaqsuidprevsicoiunstillylation spectrometer, model 314 AX
Human pregnancy serum was previously reported to exhibit a high binding aflinity for testosterone; steroid binding was measured by a semimicrotechnique, based on the principle of equilibrium dialysis but utilizing Sephadex G-25 in a batchwise fashion
I.e. column chromatography on hydroxylapatite and on fibrous DEAE-cellulose, were not as efficacious, but informative; the testosteronebinding component is readily precipitated from whole serum in 50% saturated ammonium sulfate
Summary
Tritium Determination described (l), in a Packardsampaluetsomawtiecre Tcroi-uCnatrebd, liaqsuidprevsicoiunstillylation spectrometer, model 314 AX. At = 2 (1.5 x 10-q V where At is the total steroid-binding activity (in liters) of a given protein solution, the total volume of which (in milliliters) is V, and which affords an assay value of N/v as defined above. The ratio of the nP, values is 1.04; in other words, the estimated molar concentration of the testosterone-binding component in the standard solution will differ by only 4q/ when the assay is performed at l/& = 1.2 X lo and at 1.5 X lo M-‘. Ackers [15] has, in a fashion somewhat similar to that described below, obtained a good correspondence between the batchwise and column methods in estimating Kd values for certain standard proteins when Sephadex G-100 (or G-75) was used; such a correspondence did not hold, for the more loosely crosslinked G-200 gels.
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