A Model for Ultracentrifugal Quantification of [ 35 S]Bromosulphthalein-Binding to Plasma Proteins in the Presence of Radioimpurities 1

Abstract

Summary

Solutions of [³⁵S]bromosul-phthalein ([³⁵S]BSP) in heparinized canine plasma, in the proportions established in vivo after injecting BSP intravenously to test liver function, were ultracentrifuged at 226,000 g for 24 hr at 5°. Protein-free supernatant was replaced by Krebs-Ringer buffer (pH 7.40), the protein sediment resuspended, and the mixture recentrifuged. That process was repeated several times, and the radioactivity of each resulting supernatant was measured. Since [³⁵S]BSP could not be adequately purified, supernatant radioactivities reflected both [³⁵S]BSP and radioim-purity. Therefore, a model was derived that (i) interpreted the rapid decrease in supernatant radioactivities of initial centrifugations and the gradual fall thereafter; and (ii) allowed us to determine picomoles of non-protein-bound [³⁵S]BSP. Results indicated that only 0.053% (SD 0.0013%) of BSP in our system was not protein-bound.