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Characterization of Corticotropin-Releasing Hormone Binding Protein in Human Plasma by Chemical Cross-Linking and its Binding during Pregnancy^*

Department of Medicine, Institute of Clinical Endocrinology, and the Maternal Division, Maternal and Perinatal Center (M.I.), Tokyo Women’s Medical College Tokyo 162 Japan

Address requests for reprints to: Toshihiro Suda, M.D., Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women’s Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan.

A human plasma CRH-binding protein (CRHBP) was identified and characterized by chemical cross-linking of ¹²⁵I-Tyr-hCRH to human plasma using disuccinimidyl suberate. The apparent mol wt of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 43,000. The mol wt was slightly lower in the nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the mol wt of ¹²⁵I-Tyr-CRH, the BP appeared to have a mol wt of approximately 38,000. Binding was specific since the appearance of the 43,000 dalton band was not affected by unlabeled ACTH, vasopressin, serum albumin, or -globulin, but was inhibited by unlabeled hCRH dose dependently. Pretreatment of plasma with 0.1 mol/L HC1, 0.01 mol/L NaOH, 10 mmol/L dithiothreitol, or trypsin before cross-linking abolished its ability to bind ¹²⁵I-Tyr-hCRH. Rat, rabbit, or goat plasma or human cerebrospinal fluid did not bind ¹²⁵I-Tyr-CRH. It is unlikely that CRH-BP is a CRH receptor, because the estimated mol wt of the CRH-BP is smaller than the reported size of CRH receptors, and the CRH-BP did not bind to ovine CRH. The binding of ¹²⁵I-Tyr-CRH to CRHBP decreased in the third trimester of pregnancy, when plasma CRH levels were markedly elevated. However, after dissociating endogenous CRH from the CRH-BP, the binding was almost the same as in nonpregnant subjects. In addition, CRH-BP inhibited CRH-induced ACTH secretion from cultured rat anterior pituitary cells. We conclude that most of the increased plasma CRH found in pregnant women is bound to CRH-BP, and so is inactive, therefore plasma ACTH levels do not increase to above the normal range.

^* This work was supported in part by Grant-in-Aid 62570520 for General Scientific Research from the Japanese Ministry of Education, Science and Culture and a Research grant for Intractable Disease from the Japanese Ministry of Health and Welfare.

Received March 31, 1988.

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