Abstract
Humic substances are considered to be composed of relatively small, heterogeneous molecules bound by weak linkages. The dissociation of acid-soluble constituents from soil humic acids (HAs) during the preparative polyacrylamide gel electrophoresis in the presence of concentrated urea has been previously demonstrated. Moreover, the dissociation of acid-soluble constituents has been attributed to the action of concentrated urea. The aim of this study was to investigate the effects of concentrated urea on the dissociation of acid-soluble constituents of soil HAs. Three types of soil HAs were solubilized in 0.1 M NaOH containing 7 M urea and precipitated after 16 h by acidifying the samples to pH 1.0. The acid-soluble constituents were separated from the dark-colored precipitates by concentrated urea treatment and accounted for 16–45 % of the total organic carbon in HAs. Approximately half of the acid-soluble constituents was recovered in the DAX-8-adsorbed fraction. The humification degree of the DAX-8-adsorbed fraction was considerably lower than that of the corresponding unfractionated HA. In contrast, the humification degree of the precipitated fraction increased due to the separation of acid-soluble constituents. The molecular sizes of the DAX-8-adsorbed and DAX-8-non-adsorbed fractions, estimated by high-performance size exclusion chromatography, were similar and smaller than the precipitated fraction. Three-dimensional excitation-emission matrix fluorescence spectroscopy revealed that the acid-soluble constituents exhibited fluorescence similar to that of fulvic acid (FA), added to which the DAX-8-non-adsorbed fraction exhibited protein-like fluorescence. Diffuse reflectance infrared Fourier transform spectroscopy showed that the DAX-8-adsorbed fraction contained proteinous moieties and the DAX-8-non-adsorbed fraction was rich in proteinous and polysaccharide moieties. The present findings suggest that soil HAs are formed by the molecular associations between dark-colored acid-insoluble constituents, FA-like acid-soluble constituents, protein-like constituents, and polysaccharides bound by weak linkages.
Highlights
Humic substances are considered to be composed of relatively small, heterogeneous molecules bound by weak linkages
The dissociation of humic acid (HA) constituents observed in this study can be attributed to the disruption of the hydrogen-bonding and hydrophobic interactions triggered by concentrated urea
Our previous study has demonstrated that acid-soluble constituents were separated from HAs during preparative polyacrylamide gel electrophoresis (PAGE) in the presence of concentrated urea [7]
Summary
Humic substances are considered to be composed of relatively small, heterogeneous molecules bound by weak linkages. The dissociation of acid-soluble constituents from soil humic acids (HAs) during the preparative polyacrylamide gel electrophoresis in the presence of concentrated urea has been previously demonstrated. The aim of this study was to investigate the effects of concentrated urea on the dissociation of acid-soluble constituents of soil HAs. Soil organic matter is a complex, heterogeneous mixture resulting from the decomposition of plants, animals, and microorganisms in the soil environment. Soil humic acids (HAs) are usually extracted from soil using an alkaline solution and precipitated by acidification; these are considered to be heterogeneous in composition. Acidification of the electrophoretic fractions resulted in the separation of acid-soluble constituents from the dark-colored precipitates. Our previous studies have demonstrated that acid-soluble HA constituents can be dissociated by electrophoresis in the presence of concentrated urea
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