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Determination of DTNB-reactive sulphydryl groups in soil humic acids: their enrichment in humic acids from volcanic acid soils



Determination of DTNB-reactive sulphydryl groups in soil humic acids: their enrichment in humic acids from volcanic acid soils



European Journal of Soil Science 45(2): 183-191



Thirteen acid soils were collected from typical volcanic regions in Japan (S content: 0.9-4.1, mean = 2.2 g/kg; pH (H2O): 2.81=3.93, mean = 3.33), as well as nine reference soils (S: 0.6-1.7, mean = 1.1 g/kg; pH (H2O): 4.10-4.74, mean = 4.47). Humic acids were extracted from the soils separately with 0.1M NaOH and precipitated by acidification (pH = 2, HCl). After purification, the humic acids were subjected to colorimetric analysis using a DTNB reagent for the active sulfhydryl (-SH) group. Since humic acids have significant absorption at 412 nm, the coloured compound (5-mercapto-2-nitrobenzoic acid) was separated from the humic acids by ultrafiltration or solvent extraction prior to the colorimetric measurement. Humic acids also caused discoloration of the coloured compound when they coexisted in the reaction solutions. Thus, the reproducible determination of -SH was accomplished by employing a standard addition technique (-SH standard: cysteine). Although -SH contents obtained by the ultrafiltration method were considerably greater than those by the solvent-extraction method, probably due to the denaturation of humic acids by the higher buffer concentration used in the ultrafiltration method, they correlated well with each other. The humic acids from acid soils contained apparently greater concentrations of -SH (120-510, mean = 270 mg S/kg by the ultrafiltration method; 8-110, mean = 39 mg S/kg by the solvent-extraction method) compared to those from reference soils [20-260, mean = 90 mg S/kg by the former; not detectable (<5)-34, mean = 11 mg S/kg by the latter]. This -SH enrichment in the humic acids from acid soils may result from the degradation and subsequent humification of S-rich debris of plants and micro-organisms and/or direct incorporation of volcanic acid gas (H2S) into the humic acids.

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