Spontaneous and Ionizing Radiation-Induced Aggregation of Human Serum Albumin: Dityrosine as a Fluorescent Probe

Abstract

The use of spectroscopic techniques has shown that human serum albumin (HSA) undergoes reversible self-aggregation through protein–protein interactions. It ensures the subsequent overlapping of electron clouds along with the stiffening of the conformation of the interpenetrating network of amino acids of adjacent HSA molecules. The HSA oxidation process related to the transfer of one electron was investigated by pulse radiolysis and photochemical methods. It has been shown that the irradiation of HSA solutions under oxidative stress conditions results in the formation of stable protein aggregates. The HSA aggregates induced by ionizing radiation are characterized by specific fluorescence compared to the emission of non-irradiated solutions. We assume that HSA dimers are mainly responsible for the new emission. Dityrosine produced by the intermolecular recombination of protein tyrosine radicals as a result of radiolysis of an aqueous solution of the protein is the main cause of HSA aggregation by cross-linking. Analysis of the oxidation process of HSA confirmed that the reaction of mild oxidants (Br2•−, N3•, SO4•−) with albumin leads to the formation of covalent bonds between tyrosine residues. In the case of •OH radicals and partly, Cl2•−, species other than DT are formed. The light emission of this species is similar to the emission of self-associated HSA.

Influence of Ionizing Radiation on Spontaneously Formed Aggregates in Proteins or Enzymes Solutions

“…One of the methods of protein cross-linking is the formation of dityrosine bridges, which are formed as a result of the recombination of long-lived tyrosyl radicals (TyrO • ) generated under oxidative conditions. Our own study [ 4 ] and data presented in the literature [ 11 , 12 ] indicate that in aqueous solutions of tyrosine, TyrO • radicals are formed as one of the products of reaction with hydroxyl radicals. As a result of the reaction of • OH or radicals with HSA molecules, stable protein aggregates of low and high molecular weight are formed.…”

“…To better understand the mechanism of protein aggregation induced by • OH or radicals, we conducted pulse radiolysis experiments and spectroscopic measurements for solutions of peptide and proteins, including papain, ovalbumin, insulin, calf thymus histone, β-lactoglobulin or lysozyme. The HSA oxidation process related to electron transfer has been investigated by pulse radiolysis and photochemical methods and has recently been described by us [ 4 ]. The aim of our study was an attempt to answer the question of whether it would be possible to observe the formation of DT both by pulse radiolysis and spectroscopic techniques.…”

“…In the case of mild oxidants as azide radicals, HSA aggregates are characterized by classic DT fluorescence. Our measurements clearly revealed that penetrates to the HSA interior [ 4 ]. However, in the reaction of albumin with • OH radicals, essentially, we did not observe DT.…”

“…The formation of protein dimers is accompanied by the appearance of a new “blue” emission in the spectrum. Fortunately, the luminescence of light by radiation-induced protein aggregates differs from the emission of self-aggregates (mainly dimers) [ 4 ]. In most cases, as a result of the irradiation of protein solutions during oxidative stress, the formation of dimers/aggregates leads to an emission band with a maximum of about 400 nm.…”

“…During protein aggregation in neat protein solutions, conformational changes in their structure can be registered, and they consist mainly in increasing the content of the β-sheet structure. Hydrogen bond networks with β-sheet cross-structure and/or π–π stacking interactions in protein aggregates are the cause of specific emission in albumin solutions [ 4 ]. Radiation-induced aggregates show specific emission different from non-irradiated solutions (emission from self-aggregates).…”