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Separation of Thyroxine(T₄)-Binding Proteins of Human Serum in Polyacrylamide Gel at pH 7.4. I. Effect of pH on Distribution of Tracer Quantities of T₄¹

Gerontology Research Center (Baltimore), National Institute of Child Health and Human Development, National Institutes of Health, USPHS, U. S. Welfare, Bethesda, Maryland
Department of Health, Education and Welfare Bethesda, Maryland
Baltimore City Hospitals Baltimore, Maryland 21224

A polyacrylamide gel electrophoresis system buffered with piperazine-N,N'-bis-(2-ethane sulfonic acid) (PIPES) has been shown to be capable of resolving thyroxine-binding globulin (TBG), albumin and thyroxine-binding prealbumin (TBPA) at physiologic pH. In this system at tracer levels of thyroxine (T₄), TBG binds 50% more T₄ at pH 7.4 than at pH 9.0 (Tris-borate buffer). Inversely, binding of T₄ by TBPA was reduced at physiologic pH but high at alkaline pH. The pH-dependence of TBPA binding confirms previously reported electrophoretic studies in agarose. The pH-dependence of T₄ binding by the hormone-specific proteins explains in part disparities in binding data previously reported from various electrophoretic systems. Albumin binding of T₄ was not substantially affected by changes of pH. Consistent, highly individualized post-TBG zone migration of T₄ (range: 4–15%) was noted and was attributed to binding of hormone by proteins with relatively low affinities for thyroxine. Post-TBG zone binding of T₄ was greater at pH 7.4 than at pH 9.0. While large quantities of nonprotein-bound T₄ have been reported to accumulate in polyacrylamide gel systems, the presently employed pH 7.4 and pH 9.0 methods showed less than 2% of the tracer in the nonprotein-associated form. The in vivo role for TBG as the major serum reservoir for T₄ is supported by the binding data at pH 7.4. The mechanism of pHdependence of binding by the serum proteins is unclear, but may involve structural alterations of the carrier proteins, with corresponding changes in the number of available T₄-binding sites.

¹ Portions of this work have appeared in abstract form (Clin Res 17: 282, 1969).

Received September 27, 1969.