Human (h) GH in plasma exists as a series of size isomers, which are in part explained by the presence of hGH oligomers. However, certain aspects of circulating large mol wt hGH, such as its high relative proportion compared to that in the pituitary, are poorly understood. To explore whether binding of hGH to plasma protein(s) could contribute to the phenomenon of large mol wt hGH, we incubated freshly prepared monomeric [125I]hGH or biosynthesized [3H]hGH with normal human plasma or serum at pH 7.4 for various time periods at 22 and 37 C. Plasma radioactive hGH patterns were then analyzed simultaneously with unincubated tracer hGH by Sephadex G-100 and G-200 chromatography. We found that part of the radioactivity was converted to a component with an apparent mol wt of 85,000, suggesting binding to a plasma protein(s). This phenomenon was inhibited in a dose-dependent fashion by unlabeled hGH. Saturation/Scatchard analysis indicated an association constant (Ka) of 2-3 X 10(8) M-1 and a maximum binding capacity of 20 ng hGH/ml plasma. Binding was rapid, reversible, and specific. A number of polypeptide hormones, including human placental lactogen, hPRL and rat GH, did not inhibit hGH binding. However, the 20K variant of hGH interacted weakly with the plasma binding component (Ka, 1.2 X 10(7) M-1; maximum binding capacity, 450 ng/ml). The binding component was heat labile and could be partially purified by gel permeation chromatography and affinity chromatography on a hGH-Sepharose column. Its estimated mol wt is 60,000-65,000, and it appears to bind one molecule of hGH to form a complex of 80,000-85,000 mol wt. The binding component is neither albumin nor an immunoglobulin. Under physiological conditions, a minimum of 15-18% of circulating hGH is presumably bound to this plasma component. We conclude that a specific, high affinity, low capacity binding protein for hGH with mol wt of 60,000-65,000 exists in normal and hypopituitary human plasma. hGH complexed with this protein forms part of big-big hGH. The biological significance of this binding protein remains to be investigated.

We recently described a specific binding protein for human GH (hGH) in human plasma, with which a substantial portion of circulating hGH is complexed. The biological function of the complexed fraction is unknown. To test the hypothesis that complexed hGH may have different in vivo kinetics than free hGH, we compared the MCRs, distribution volumes (Vd), and degradation rates of complexed and free [125I] hGH in the rat. A partially purified GH-binding protein preparation, generated by affinity chromatography on a hGH column, was used for this purpose. A mixture of hGH with binding protein (equivalent to the amount contained in 0.9 mL human plasma) was injected iv as a single dose. Parallel experiments were conducted with hGH in the absence of binding protein. Disappearance of total, immunoprecipitable, and trichloroacetic acid-precipitable radioactivity from rat plasma was followed, and MCR, Vd, and degradation rates were derived by standard mathematical techniques. The MCR was 6-fold slower for complexed than for free hGH (2.3 vs. 14 mL/min X kg), Vd was 4-fold smaller for complexed hGH than for free hGH (71 vs. 256 mL/kg), and initial degradation rate was 4.5-fold lower for complexed than for free hGH (13.2% vs. 59.9%/15 min). The Vd of complexed hGH was close to the intravascular volume, while the Vd for free hGH corresponded to the extracellular volume. We conclude that one function of the hGH-binding protein is relative confinement of hGH to the vascular compartment, thereby protecting it from degradation and prolonging its biological half-life.

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The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH.

Human GH (hGH) extracted from pituitary glands consists of several molecular forms. Monomeric pituitary forms include the single chain 22,000-dalton polypeptide (22K; hGH-B), a 20,000-dalton variant with a 15-amino acid deletion (20K), 3 proteolytically cleaved 2-chain forms (hGH-C, -D, and -E), 2 deamidated forms, an acetylated form (fast hGH), and other, only partially characterized forms. It is not known which of these forms is secreted, nor what the precise nature of circulating hGH is. To answer these questions, we have extracted hGH from human plasma obtained by plasmapheresis from normal volunteers after L-dopa stimulation of hGH secretion and from acromegalic patients. We extracted and concentrated hGH by immunoadsorbent chromatography and examined its chemical nature by polyacrylamide gel electrophoresis under native and denaturing (sodium dodecyl sulfate and urea), nonreducing and reducing (dithiothreitol) conditions as well as by isoelectric focusing. In all cases, the predominant form of hGH present in plasma was 22K, which accounted for approximately 85% of all immunoreactive hGH. In addition, we found evidence for the presence of 20K as a minor form (approximately 7% of all hGH) and of one or more acidic forms (N-acetylated, deamidated, or cleaved hGH; 5-10% of all hGH). Exact identification of the acidic form(s) was not possible. However, the highly bioactive cleaved forms hGH-D and -E were judged to be undetectable (less than 5% of all hGH). We conclude that 1) several monomeric molecular forms of hGH circulate in normal and acromegalic man; 2) the pattern of circulating hGH forms reflects in part their relative prevalence in the pituitary gland; 3) proteolytically cleaved 2-chain hGH forms with enhanced bioactivity are not detectable in blood; and 4) monomeric hGH circulating in acromegaly is qualitatively indistinguishable from normal hGH.