Feb 10, 1976

Affinity labeling of the primary bilirubin binding site of human serum albumin

The Journal of Biological Chemistry
C C KuenzleK J Wilson

Abstract

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.

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Mentioned in this Paper

Covalent Interaction
N-ethyl-5-phenylisoxazolium-3'-sulfonate
Bilirubin
ALB
Albumin Human, USP
Bilirubin, (4E,15E)-Isomer
Human Serum Albumin [EPC]
Serum Bilirubin Measurement
Plasma Protein Binding Capacity
Blood Bilirubin Measurement

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