Albutran Research and Production Enterprise, Minsk, Belarus
Background: Albumin is the major component of the blood transport system that provides the transport of many important metabolites, delivery of drugs, binding of toxins and their transfer to detoxifying organs. In that way albumin supports the functioning of all organs and body systems.
Recent studies showed that the structure and binding properties of albumin can be significantly altered in a variety of clinical conditions, and albumin may play an important role in the diagnosis and monitoring of patients with different disease states.
Objective: Development of the technique for evaluating the structural and functional changes of serum albumin.
Methods: Non-covalent spin labeling of albumin combined with electron paramagnetic resonance (EPR) spectroscopy is the basic technique used for evaluating the structural and functional changes that occur to albumin in different conditions. This technique is relatively simple and allows studying the proteins without altering the native protein structure, and utilizes commercially available fatty acid spin labels.
Results: The method makes use of the inherent tendency of albumin to non-covalently bind fatty acids. Binding on albumin of fatty acids previously labeled with nitroxide radical is the basis for this technique.
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Albumin has several specific binding sites for long chain fatty acids which provide extremely high binding constants. This makes problematic the evaluation of albumin binding parameters, because the potion of unbound fatty acid spin probe in serum samples is extremely low and varying due to stochastic factors such as concentrations of various metabolites, temperature, pH variations etc. Addition of a specified amount of ethanol to the mixture of serum probe and the fatty acid spin probe allows providing the controlled reducing of albumin binding constant and overcoming the influence of stochastic factors, and thereby significantly reducing variations in measured results.
Analysis of the EPR spectrum generated from the fatty acid spin probe bound to albumin in serum reveals several distinct spectral components. Two major spectral components are representative of the portion of fatty acids bound on albumin. Two other components are representative of unbound fatty acids present in the solution singularly or organized into clusters of fatty acid micelles.
Analysis of concentration ratio of the bound and unbound fatty acid probe allows evaluating the binding efficiency and capacity of albumin sites. Spectral line shape provides information on evaluation of the structural features of albumin sites, and the whole albumin globule.
Comparison of the changes that occur to the mobility, binding affinity, and distribution of the fatty acid spin label on albumin in normal healthy individuals with those changes observed in patients with disease states, such as sepsis, SIRS, cancer and others, has revealed the alterations which can be readily assessed with EPR analysis. The differences observed offer excellent discrimination between healthy and disease states.
Conclusion: The EPR test of serum albumin is the novel noninvasive method which allows evaluation in vitro of changes of serum albumin structure and function that can occur in pathological conditions and may play an important role in the diagnosis.