engleski

Agitation induced reversible self-association of protein rHu-G-SCF

Granulocyte colony-stimulating factor (rHuG-CSF), or so-called filgrastim, is a member of the class of cytokines that stimulates the synthesis of granulocytes in the human body. Filgrastim is structurally composed of 175 amino acids and can be used as a drug for the treatment of neutropenia. While affected by different types of stressors, the monomeric protein form of filgrastim has a tendency to self-associate and produce dimers, trimers, tetramers, and other complex aggregates. A stressor such as heat can induce the formation of irreversible filgrastim aggregates, while agitation stimulates the reversible self-association mechanism. Produced irreversible forms can emerge due to the formation of covalent bonds of monomer protein structures, of which the most significant are disulfide bridges. Further on, reversible filgrastim aggregate forms occur due to the non-covalent bonds, i.e. hydrogen bonds, electrostatic, hydrophobic, and van der Waals interactions. Researchers have discovered that reversible self- associated proteins in the human body can promote the development of Alzheimer's, Parkinson's, Huntington's, and prion's diseases, as well as amyotrophic lateral sclerosis [1], however, the complexity of the filgrastim structure makes the understanding of aforementioned self-association processes extremely challenging. Therefore, to examine the mechanism of the reversible protein binding reaction, the filgrastim aqueous solution was agitated at a constant rate increasing the agitation time by 60-600 s. The analysis was performed immediately after agitation by high performance liquid chromatography (HPLC) and the obtained results were normalized by a mathematical model that derives a more precise quantitative analysis of chromatographic peaks in four steps. Such a mathematical model enabled the illustration of the Lumry-Eyring equation framework that explains the mechanism of an aggregation process of filgrastim.

protein self-association, agitation, filgrastim

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