Re-examining the glomalin-purity of glomalin-related soil protein fractions through immunochemical,lectin-affinity and soil labelling experiments

Due to analytical similarities with the mycorrhizal glycoprotein glomalin, ubiquitous citrate and heat-extractable soil protein fractions have been assumed to be predominantly glomalin-stabilised within soil. Often termed glomalin-related soil protein (GRSP), little however is actually known of the “glomalin-purity” of these soil fractions. We undertook western and lectin blots and crossed immuno/lectin affinity electrophoresis (CIE/CLAE) analysis of “easily extractible” GRSP fractions, as well as liquid chromatography-tandem mass spectrometry (LC–MS/MS) of “total” GRSP fractions. To further test whether soil saprobes contribute to GRSP production, we amended soil with ¹⁴C-sucrose and examined whether ¹⁴C could be traced in the GRSP pool over a 500-day incubation period.While only four of six bands on SDS–PAGE profiles of easily extracted GRSP reacted with anti-glomalin MAb32B11 and the lectin Con A under our blotting conditions, CIE/CLAE indicated the presence of a single protein moiety in the easily extractible GRSP pool. LC–MS/MS analysis of total GRSP pooled from various soils also showed that although traces of protein tentatively assignable to soil bacteria were present in GRSP, their concentrations were low. Additionally, specific activity of total GRSP in ¹⁴C-labelled soil was relatively depleted compared to the bulk soil and soil microbial biomass. This suggests that little GRSP of heterotrophic origin was laid down over the incubation period, although the potential presence of a pre-existing ¹⁴C-free GRSP background, as well as of low microbial dynamics in the absence of any further substrate inputs to the soil warrant caution with this inference.