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Updated: Jul 10, 2026
Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms
Published on: April 7, 2011
The cobalt-albumin binding assay: insights into its mode of action
David Bar-Or1, Leonard T Rael, Raphael Bar-Or
1Swedish Medical Center, Trauma Research Laboratory, Englewood, CO, USA; Swedish Medical Center, Emergency Department, Englewood, CO 80113, USA. dbaror@dmibio.com
Background:
We previously hypothesized that the N-terminus of human serum albumin (HSA) is altered during ischemic events, thus establishing the foundation for the cobalt-HSA binding assay phenomenon. In this investigation, we attempt to clarify the mode of action of the cobalt-HSA binding assay by direct observations of cobalt binding to HSA.
Methods:
High pressure liquid chromatography coupled to positive electrospray ionization mass spectrometry (HPLC/MS) was used to study cobalt binding to HSA in the plasma of patients with and without evidence of myocardial ischemia.
Results:
Strong binding of cobalt to the N-terminus of HSA occurs at pH>7.0. No differences in cobalt binding to the N-terminus of HSA are observed in ischemic versus non-ischemic patients' plasma despite differences in the cobalt-HSA binding assay. Plasma free cysteine/cystine ratio appears to play a role in the quantitative response of the cobalt-HSA binding assay.
Conclusions:
The main determinants of the cobalt-HSA binding assay mechanism of action include but are not limited to: the proportion of intact N-terminus of HSA, HSA concentration, plasma cysteine/cystine ratio, plasma pH, and the state of oxidation of cys34 of HSA. Assay improvements that consider and take these factors into account could lead to an improved cobalt-HSA binding assay with greater clinical utility.
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