Fractionation of soil humic acids by preparative polyacrylamide gel electrophoresis in the presence of concentrated urea

Abstract

To separate soil humic acids (HAs) into their constituents and characterize them, polyacrylamide gel electrophoresis (PAGE) was carried out in the presence of 7 M urea using a preparative electrophoresis system. Two types of soil HAs were fractionated into nine fractions by PAGE. The dark-colored constituents were recovered from the electrophoretic fractions by precipitation on acidification, and the brown-colored constituents dissolved in the acidic solution of fast-moving fractions were recovered by adsorption onto DAX-8 resin. High-performance size-exclusion chromatography (HPSEC) confirmed that the constituents of the HAs were separated based on their molecular size by PAGE. The dark-colored constituents exhibited higher degrees of humification than did the corresponding unfractionated HAs, except for the constituents remaining in the electrophoretic gels at the end of electrophoresis. Diffuse reflectance infrared spectroscopy revealed that the chemical properties of the dark-colored constituents changed regularly: the content of carboxyl groups decreased and the proportions of proteinous, aliphatic and polysaccharide moieties increased with increasing molecular size. The humification degrees of the constituents adsorbed onto DAX-8 resin were considerably lower than those of the corresponding unfractionated HAs. The chemical properties of the DAX-8-adsorbed constituents were different from those of the dark-colored constituents. Observation of electrophoretic fractions under blue light (470 nm) and HPSEC with fluorescence detection at an excitation wavelength of 460 nm and an emission wavelength of 520 nm showed that green fluorescent substances were largely concentrated in the smallest molecular size fractions and were partitioned into both the dark-colored precipitates and DAX-8-adsorbed fractions. The proportion of organic carbon recovered by precipitation and adsorption onto DAX-8 resin was 45-63%, indicating that substantial parts of the HA constituents were missing. The unrecovered constituents were considered to be acid-soluble, nearly colorless substances. The dissociation of the acid-soluble constituents from the acid-insoluble dark-colored constituents during the preparative PAGE of soil HAs was ascribed to disruption of hydrogen bonding and hydrophobic interactions caused by concentrated urea.

Separation of acid-soluble constituents of soil humic acids by dissolution in alkaline urea solution and precipitation with acid

“…These results indicated that the acid-soluble constituents of HA were characterized by a low degree of humification and that their dissociation from HA resulted in an increase in the humification degree of precipitated constituents. These findings were in agreement with our previous study, where preparative PAGE of HAs was carried out in the presence of concentrated urea [7]. The degree of humification of the precipitated fraction was significantly higher when treated with alkaline concentrated urea.…”

“…The DAX-8-non-adsorbed fraction (>1 kDa) showed a similar molecular size to that observed for the DAX-8-adsorbed fraction; however, the intensity of the peak was observed to be relatively lower. The molecular sizes of the acid-soluble constituents dissociated by concentrated urea treatment were similar to those dissociated by preparative PAGE in the presence of concentrated urea [7].…”

“…Our previous study has demonstrated that acid-soluble constituents were separated from HAs during preparative PAGE in the presence of concentrated urea [7]. This study further revealed that the dissociation of acid-soluble constituents from HAs occurred when the HAs were dissolved in alkaline concentrated urea solution, followed by the acidification of the solution.…”

“…The chromatograms were monitored by UV absorption using a photodiode array detector (MD-2018, JASCO Corp., Tokyo, Japan) and by fluorescence at excitation and emission wavelengths of 460 and 520 nm, respectively, using a fluorescence detector (FP-920, JASCO Corp., Tokyo, Japan). To estimate the molecular weight (MW), the column was calibrated using polyethylene glycols as the MW standards [7].…”

“…We further subjected the HAs to polyacrylamide gel electrophoresis (PAGE) in the presence of 7 M urea, using a preparative electrophoresis system [7]. Acidification of the electrophoretic fractions resulted in the separation of acid-soluble constituents from the dark-colored precipitates.…”