Use of cysteine as a spectroscopic probe for determination of heme-scavenging capacity of serum proteins and whole human serum

https://doi.org/10.1016/j.jpba.2019.05.013Get rights and content
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Highlights

  • Detection of free extracellular heme is challenging issue.

  • Cysteine can be used as an absorbance spectroscopy probe for distinguishing protein-bound from free heme.

  • Use of cysteine allow detection of heme bound to proteins with low affinity.

  • The strategy might be used for assessment of heme-binding capacity of plasma or serum of patients with hemolytic diseases.

Abstract

Heme serves as a prosthetic group of numerous proteins involved in the oxidative metabolism. As result of various pathological conditions associated with hemolysis or tissue damage, large quantities of hemoproteins and heme can be released extracellularly. Extracellular heme has pronounced pathogenic effects in hemolytic diseases, mediated by its pro-oxidative and pro-inflammatory activities. The pathogenic potential of heme is mostly expressed when the molecule is in protein unbound form. The pathological relevance of free heme deems it necessary to develop reliable approaches for its assessment. Here we developed a technique based on UV–vis absorbance spectroscopy, where cysteine was used as a spectroscopy probe to distinguish between heme-bound to plasma proteins or hemoglobin from free heme. This technique allowed estimation of the heme-binding capacity of human serum, of particular heme scavenging proteins (albumin, hemopexin) or of immunoglobulins. The main advantage of the proposed approach is that it can distinguish free heme from heme associated with proteins with a wide range of affinities. The described strategy can be used for evaluation of heme-binding capacity of human plasma or serum following intravascular hemolysis or for estimation of stoichiometry of interaction of heme with a given protein.

Keywords

Heme
Hemolysis
Heme-binding proteins
Human serum
Absorbance spectroscopy

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These authors contributed equally to the work.