Glomalin‐related soil protein in French temperate forest soils: interference in the Bradford assay caused by co‐extracted humic substances

Abstract

Thermostable soil protein, known as glomalin, is an important component of soil carbon stocks. Thought to originate from endomycorrhizal fungi, Glomales, this operationally-defined fraction of soil organic matter contains proteins of diverse origin as well as non-protein material, including humic substances. Accumulation results from the balance between production/release and subsequent degradation. Quantification of the protein is subject to uncertainty because of the co-extraction of other components that interfere with the Bradford assay. We studied 10 topsoils from French temperate forests, taken from the national forest monitoring network (Renecofor). Two fractions were extracted, easily extractable (EE) at neutral pH and total extractable (T) at pH 8. Protein was quantified with the colorimetric Bradford method, either by direct calibration using bovine serum albumin (BSA) or by extrapolation of the standard addition plot of BSA. Solubilized organic matter was characterized by using absorbance at 465 and 665 nm and by three-dimensional fluorescence excitation-emission spectroscopy. Neither soil properties nor forest cover influenced glomalin-related soil protein (GRSP) content. Direct assay gave the GRSPEE to be about 1 g kg−1 soil, and GRSPT in the range 3–10 g kg−1, accounting for about 2% of soil organic carbon and about 15% of soil nitrogen. Standard addition plots indicated a two to sixfold under-estimation of protein in total extracts, caused by negative interference with the Bradford assay. The GRSPEE was correlated significantly with both estimates of GRSPT. Under-estimation of GRSPT by direct assay was not related to the E4:E6 ratio but was correlated significantly with the intensity of absorbance at either 460 or 660 nm and with one of the fluorescence peaks. We conclude that GRSPEE is not necessarily more recent than GRSPT and that both fractions may be probes of protein content, but that absolute contents may be under-estimated because of co-extracted humic substances

Towards meaningful quantification of glomalin‐related soil protein (GRSP) taking account of interference in the Coomassie Blue (Bradford) assay

“…This indicates that interaction between added BSA and soil components, assumed to be polyphenols, had more effect on the spectrum at wavelength 595 nm than at 465 nm. The much greater estimate of GRSP with the standard addition accords with a previous study of forest soil where we reported a two‐ to sixfold increase in estimated GRSP with the standard addition method (Jorge‐Araújo et al, ). Furthermore, there was no tendency for the estimated GRSP to level off at the greatest dilution for the direct assay.…”

“…Attempts to do this have been largely unsuccessful, with very small yields; however, yields are rarely given in published reports. Jorge‐Araújo et al () postulated that irreversible, possibly co‐valent, bonds are formed between soil proteins and polyphenols during the high‐pressure, high‐temperature extraction. If this is the case, soil protein and added BSA might not experience the same interference.…”

“…Nevertheless, quantification is limited by the short working range of this method, the nonlinear response and interference, both positive and negative, from co-extracted compounds. These effects result in dilution effects that complicate the exact quantification of the protein component (Jorge-Araújo, Quiquampoix, Matumoto-Pintro, & Staunton, 2015;Redmile-Gordon, Armenise, White, Hirsch, & Goulding, 2013;Reyna & Wall, 2014).…”

Glomalin amount and compositional variation, and their associations with soil properties in farmland, northeastern China

“…) and were also reported by previous reports ( Rosier et al, ; He et al, ). About 15% of soil N were from GRSP contribution ( Jorge‐Araújo et al, ), and GRSP‐N relations were reported as soil [N (g kg −1 ) = 0.004GRSP+0.1069, R 2 = 0.558] ( Fokom et al, ). In this paper, stepwise regression and SEM analysis showed non‐direct effects of soil N on GRSP amounts in soil (Tab.…”

Is glomalin an appropriate indicator of forest soil reactive nitrogen status?

“…Moreover, easy extractable GRSP (EE‐GRSP) and total GRSP (T‐GRSP) fraction can be distinguished, depending on the chosen extraction procedures. The concept of glomalin has been faced with criticism due to the uncertainty about the chemical characterization, co‐extraction of non‐protein material and the doubts regarding the origin of this soil organic fraction ( Jorge‐Araújo et al, ). However, glomalin can be still viewed as an operationally‐defined part of more persistent soil organic matter with evidenced relationships to soil quality and mycorrhiza ( Wu et al, ; Vasconcellos et al, ).…”