Aggregation and Disaggregation of Humic Supramolecular Assemblies by NMR Diffusion Ordered Spectroscopy (DOSY-NMR)

Abstract

Diffusion ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) was applied to a number of fulvic (FA) and humic (HA) acids of different origin. Spectral separation achieved by DOSY based on diffusion coefficients (D), and correlated to molecular sizes by calibration standards, showed that carbohydrates had the largest molecular size in FA, whereas alkyl or aromatic components were the most slowly diffusing moieties in HA. At increasing concentrations, these components had invariably lower D values in DOSY spectra for all humic samples,thereby indicating an aggregation into apparently larger associations, whose increased hydrodynamic radius was confirmed by viscosity measurements. When humic solutions were broughtfrom alkaline to acidic pH (3.6), components diffusivity detected by DOSY increased significantly, suggesting a decrease of aggregation and molecular size. A general comparison of HA and FA molecular sizes was achieved by multivariate statistical analysis. While a larger extent of aggregation and disaggregation was observed for HA than for FA, no aggregation was detected, under similar conditions, for a true macropolymeric standard. Such difference in diffusion between a polymeric molecule and humic samples, is in line with the supramolecular nature of humic matter. The possible formation of humic micelles was also investigated by both changes of diffusivity in DOSY spectra and shift of 1H NMR signals. Except for HA of peat and soil origin, revealing a self-assembling in micelle-like structures at the 4 mg mL(-1) concentration, no other humic sample showed evidence of critical micelle concentration (cmc) up to 20 mg mL(-1). These results indicated that DOSY-NMR spectroscopy is a useful technique to evaluate components of different molecular size in natural humic superstructures.

Electrospray and photoionization mass spectrometry for the characterization of organic matter in natural waters: a qualitative assessment

“…A new view has emerged, however, which reconciles observations of macromolecular size to MSbased observations of low molecular weight. The new view describes NOM and humic substances as a dynamic, noncovalent association of small, recognizable biological molecules in various stages of decomposition (Hedges et al 2000;Burdon 2001;Wrobel et al 2003;Sutton and Sposito 2005;Smejkalova and Piccolo 2008). A similar aggregation behavior has been observed in noncovalent oligomers in fossil fuels (Piccolo et al 2002;Mullins et al 2008).…”

Humic substances stimulate maize nitrogen assimilation and amino acid metabolism at physiological and molecular level

“…be explained with the tendency to increase the size of supramolecular aggregation of humic molecules with concentration [74] and the concomitant enhanced capacity to provide specific bioactive molecules to targeted root cell membranes [6]. Among all enzymes, only the AS showed a different trend of regulation in response to HS, possibly because of a competition of its own substrate with the biosynthesis of amino acids of the Asp family [75], thus favouring the accumulation of a large amount of Asn in seedling leaves treated with the greatest HS concentration.…”

Physiological responses to humic substances as plant growth promoter

“…It is thus conceivable that molecules released from humic superstructures may then access cell membranes and induce different physiological responses. The assembling and disassembling behavior of HA molecular domains and their dynamic release of different molecular constituents [38] may then explain why humic compounds had been previously found to possess hormone-like activities similar to those of auxins, gibberellins, cytokinins and polyamines [39]. The ultimate physiological response would involve a large level of cell activity and tissue differentiation, which result in root growth.…”