Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition. Results of a novel ultrafiltration method

Richard A. Weisiger, J. Donald Ostrow, Ronald K. Koehler, Cecile C. Webster, Pasupati Mukerjee, Lorella Pascolo, Claudio Tiribelli

Research output: Contribution to journalArticle

Abstract

Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [14C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [14C]bilirubin was strongly dependent on assay conditions, falling from (5.09 ± 0.24) × 107 liters/mol at lower albumin concentrations (15 μM) to (0.54 ± 0.05) × 107 liters/mol at higher albumin concentrations (300 μM). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11-0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 ± 0.26) × 107 liters/mol to (0.65 ± 0.03) × 107 liters/mol). Affinities decreased by about half in the presence of chloride (50 mM). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.

Original languageEnglish
Pages (from-to)29953-29960
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number32
DOIs
Publication statusPublished - Aug 10 2001

ASJC Scopus subject areas

  • Biochemistry

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