Observation and quantitation of metal-binding sites in the sex steroid-binding protein of human and rabbit sera using the luminescent probe terbium |
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Authors: | J B Alexander Ross Paul B Contino Mark F Lulka and Philip H Petra |
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Institution: | (1) Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York, New York, New York;(2) Departments of Obstetrics/Gynecology and Biochemistry, University of Washington, Seattle, Washington |
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Abstract: | The first direct evidence for specific metal-binding sites in pure human and pure rabbit sex steroid-binding protein (SBP) is obtained using the luminescent lanthanide terbium. Terbium, a probe for calcium sites in proteins, provided protection of the SBP steroid-binding activity in diluted human serum samples equivalent to that provided by calcium. Pure SBP, first treated with ethylenediaminetetraacetate, was dialyzed against buffer containing TbCl3. After gel filtration to remove nonspecifically bound terbium, the protein was denatured in urea. The amount of protein-bound terbium was determined by luminescence enhancement of the lanthanide using the chelator dipicolinate, yielding four metal-binding sites per mole of dimer protein from both species. |
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Keywords: | metal-binding site sex steroid-binding protein terbium |
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