ИСТИНА

Beta-2-microglobulin (β2M) is a low-molecular weight protein (11.8 kD, 99 amino acid residues) which is localized on the external membrane surface of the most eukaryotic cells. The normal concentration of β2M in plasma is about 1-3 mg / L, but in case of the chronic renal failure it significantly increases. Prolonged persistence of high β2M concentrations in patient’s blood leads to the formation and accumulation of amyloid fibrils on the basis of this protein (both full-length β2M, and its truncated forms - without the 6 and 10 N-terminal amino acid residues) which results in lesions of various tissues and organs. A comparative study of amyloid fibrils on the basis of various forms of β2M is the aim of the present work. Аbsorption spectra, intrinsic tryptophan fluorescence and fluorescence excitation spectra of the studied fibrils were recorded, as well as the their CD spectra in the far UV region, which showed the differences in the secondary structure of the fibrils. For morphological characterization and evaluation of the dimensions of amyloid fibrils their images were obtained by electron microscopy. To detect more subtle differences in the structure of the fibrils on the basis of various forms of β2M their interaction with a specific fluorescent probe thioflavin T (ThT) was studied. Investigation of the spectral characteristics of ThT solutions with amyloid fibrils led to conclusion that ThT most effectively interacts with fibrils based on β2M without 6 N-terminal amino acids. Using absorption spectroscopy of solutions obtained by equilibrium microdialysis parameters of ThT binding with these fibrils were determined. It is shown that all binding sites of ThT to the amyloid fibrils on the basis of β2M are identical (there is one mode of binding with a binding constant ~ 104 M-1). The existence of this binding mode is due to the incorporation of the dye in the groove formed by amino acid side chains of amyloid fibrils along their long axis perpendicular to the beta-sheets. It is interesting to compare these results with the data obtained by the study of ThT interaction with amyloid fibrils on the basis of other proteins and peptides. In particular, fibrils on the basis of insulin, lysozime and Abeta-peptide along with ThT binding mode similar to the mode, present in the β2M fibrils, have another one with higher binding constant, existence of which is caused by the interaction of ThT with the aggregates of these fibrils. It is proved by electron microscopy that amyloid fibrils on the basis of β2M do not form such aggregates. Based on these data, we can conclude that the amyloid fibrils on the basis of the full-length β2M and its truncated forms without the 6 and 10 N-terminal amino acid residues have different structures. The results of this work may be significant for the understanding of the fundamental mechanisms of amyloid fibril formation, as well as for the treatment of hemodialysis amyloidosis.