Electron microscopy of stable electrophoretic fractions of natural humic acids – the key to the understanding of their structural organization

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Abstract

Transmission electron microscopy (TEM) with contrast staining by uranyl acetate solution was used to study morphological differences between soil humic acids (HAs) and their A, B and C + D fractions obtained using coupling preparative low-pressure size exclusion chromatography and analytical polyacrylamide gel electrophoresis. The electrophoretic mobility of fractions varied in order C + D > B > A. The distribution of various morphological elements between fractions showed that large structures such as vesicle-like formations 70–150 nm long and 30–80 nm wide with clear edges were found exclusively in fraction A and occupied ~55% of the TEM image area. On the other hand, long fibrils with a length of 60–100 nm, a width of 4–6 nm and a thickness of 2–3 nm, as well as their bundles with a length of >150 nm and a diameter of 30–70 nm were identified only in the C + D fraction and occupied ~59 % area of TEM images. Smaller morphological elements such as point particles with a diameter of 2–3 nm, ring particles with a diameter of 4–6 nm, worm-shaped short particles with a length of 20–30 nm, and spheroids with a diameter of 10– 30 nm were observed in all samples, but in varying quantities. Significant morphological differences between the fractions can be explained by their composition, previously established by using a few physico-chemical methods. The ratio Car(165–108 ppm)/Calk(108–0 ppm), or aromaticity index, calculated from 13C-NMR, could be one of the indicators of the various morphological structures formation. The obtained TEM results clearly confirm the supramolecular organization of soil HAs.

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About the authors

O. E. Trubetskaya

Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS

Author for correspondence.
Email: olegi03@yahoo.com
Russian Federation, prosp. Nauki, 6, Pushchino, 142290

O. M. Selivanova

Institute of Protein Research, Russian Academy of Sciences

Email: olegi03@yahoo.com
Russian Federation, prosp. Nauki, 4, Pushchino, 142290

V. V. Rogachevsky

Institute of Cell Biophysics, Russian Academy of Sciences

Email: olegi03@yahoo.com
Russian Federation, prosp. Nauki, 3, Pushchino, 142290

O. A. Trubetskoj

Institute of Basic Biological Problems, Russian Academy of Sciences

Email: olegi03@yahoo.com
Russian Federation, prosp. Nauki, 2, Pushchino, 142290

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Supplementary files

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2. Fig. 1. Electrophoresis of chernozem humic acids and fractions A, B, C + D in 10% PAAG (a); fractionation of humic acids on a column with Sephadex G-75 (b); absorption spectra of humic acids and fractions at a concentration of preparations of 20 mg/l (c); 13C NMR spectra (d) and distribution of fluorescence intensity of preparations when illuminated with UV light of a wavelength of 312 nm (d).

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3. Fig. 2. TEM images of chernozem HA preparations and fractions A, B, C + D. Numbers 1–7 correspond to the ordinal numbers of the morphological forms presented in Table 2: point particles (1), ring particles (2), worm-like particles (3), spheroids (4), droplet vesicle-like formations (5), long fibrils (6), bundles of long fibrils (7). The aromaticity index (AI), i.e. Sarom/Salif, was calculated on the basis of 13C NMR spectra by the ratio of the areas above the chemical shift regions of aromatic (165–108 ppm) and aliphatic (108–0 ppm) carbon according to our previously published data [32].

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4. Fig. 3. TEM images of point (1) and ring (2) particles in various micrographs of chernozem HA preparations and fractions A, B, C + D; examples of worm-like particles (3), spheroids (4), long fibrils in two different projections (6), and bundles of long fibrils (7).

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