Polyphenolic compounds interfere with quantification of protein in soil extracts using the Bradford method

doi:10.1016/j.soilbio.2006.08.012

Soil Biology and Biochemistry

Volume 39, Issue 2, February 2007, Pages 691-694

https://doi.org/10.1016/j.soilbio.2006.08.012 Get rights and content

Abstract

The Bradford protein quantification assay is based on an absorbance shift in Coomassie brilliant blue G-250 (CBB). Samples extracted for glomalin, a protein produced by arbuscular mycorrhizal (AM) fungi, are quantified using the Bradford assay. CBB is known to react with polyphenolic substances, and co-extraction of glomalin and humic substances is known to occur. The effects of increasing concentrations polyphenolic compounds were measured. The addition of any amount of polyphenolic compounds increased the Bradford reactive fraction (BRF) of soil extract. Caution is required when interpreting BRF data, as comparison of BRF data from different studies or different field sites is problematic. The BRF may represent recalcitrant organic material in soil, though its relationship to AM fungi remains unclear.

Section snippets

Acknowledgements

Thanks to Dr. David Nehl for his editing and analysis advice. Thank you to the New South Wales National Parks and Wildlife Service and State Forests of New South Wales for permission to collect samples at the BNR and JC field sites. This work has been supported by the Cotton Catchment Communities CRC, Australia.

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Protein accumulation and distribution in floodplain soils and river foam

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Cited by (73)

Potential interaction mechanisms between PAHs and glomalin related-soil protein (GRSP)
2023, Chemosphere
Glomalin-related soil protein (GRSP) has gained widespread attention because of its benefits to carbon sequestration, improving soil quality and fixing heavy metals. However, studies on how GRSP affects the environmental fate of organic contaminants are scarce. In this study, different types of GRSPs were isolated from forest soils and characterized to study the binding of GRSPs and PAHs under different environmental conditions. The results indicated that GRSPs contain abundant functional groups (such as –NH, –COOH, and CO) and material composition, like humic acid, proteins, and lipids. For the tested GRSPs, EE-GRSP has lower DOC, SUVA₂₆₀ and SUVA₂₈₀ values, as well as higher E2/E3 values, indicating that EE-GRSP has lower hydrophobicity and molecular weight. These properties can lead to strong interactions between GRSP and PAHs, especially with benzopyrene, which has a high K_ow and K_sw and a large molecular size, with binding constants ranging from 16,119 to 163,697 L·kg⁻¹. Furthermore, low pH (pH = 3) and temperature (15 °C) could increase GRSP's aggregation, enhance the GRSP binding ability with PAHs, whose binding constants were 11,595 and 5067.3 L·kg⁻¹. Therefore, the binding between GRSP and PAHs may lead to changes in the fate of PAHs in the soil and affect the environmental risk of PAHs. The results presented here will deepen our understanding of the environmental function of GRSPs and provide a theoretical basis to further elucidate the mechanisms of GRSPs and organic pollutants.
Contributions of mycorrhizal fungi to soil aggregate formation during subalpine forest succession
2023, Catena
Although soil structure is essential for land sustainability, resilience, and fertility, the biological factors of aggregate formation and dynamics, especially the role of mycorrhizal fungi, are still unclear. Soils from a secondary succession, grasslands → shrublands → secondary forests → primary forests in subalpine regions, were sampled to compare the contribution of mycorrhizal fungal communities to the aggregate structure. Four aggregate size classes (>2000 and 250–2000 µm, large and small macroaggregates, respectively; 53–250 µm microaggregates; and < 53 µm silt and clay fraction) were related to microbial community structure, root biomass, and morphology, mycorrhizal infection rate, and external mycorrhizal hyphae. The proportion of large macroaggregates increased by 10 %, while small macroaggregates decreased by 9 % during vegetation succession, increasing the mean weight diameter by 17 %. The Shannon–Wiener and Chaos indexes of arbuscular mycorrhizal fungi (AMF) in grassland soil were three times and in shrubland, they were 24 times higher than in the primary spruce forests. The Simpson index of AMF had a positive linear relationship with soil structure. The mycorrhizal hyphal density was maximal in primary forests and could explain the aggregate size distribution before the arboreous layer developed. In contrast, dissolved organic carbon (DOC) and the diameter of the mycorrhizal hyphal contributed to the distribution of aggregates during later succession stages. Thus, biological aggregation mechanisms in the studied subalpine region followed ecosystem succession, with the mycorrhizal fungal communities being the crucial factor affecting the soil structure mainly during grassland and shrubland succession (AMF stage). In contrast, during the ECM stage, the contribution of DOC to aggregate formation became more critical.
Accumulation and vertical distribution of glomalin-related soil protein in French temperate forest soils as a function of tree type, climate and soil properties
2023, Catena
Soil organic matter (SOM) is of critical importance to the functioning of ecosystems because of its effects on soil nutritional quality, microbial activity and soil structure. SOM is made up of a variety of compounds ranging from chemically reactive and mobile forms to stable, recalcitrant forms. An operationally defined fraction of soil organic matter known as glomalin-related soil protein (GRSP) is thought to be of fungal origin, very stable and responsible for increased physical stability of soil. To date, the origin of this fraction of SOM and the factors controlling its accumulation in soils are not well understood. The aim of this study was to contribute to the understanding of the origin of this fraction and the factors which contribute to its accumulation in the soil. This study was carried out on archived soils from 102 sites of the long-term monitoring network for forest ecosystems in France (RENECOFOR), from depths up to 1 m. The effects of tree type, climate and soil properties on the accumulation and vertical distribution of GRSP were assessed. GRSP was greater under conifer forests than broadleaf forests. GRSP in the top soil layer was positively correlated with both SOC and total nitrogen, and inversely related with pH, with the strongest effect observed at very acid (pH ≤ 4.0). Both GRSP and SOC were found to follow exponential declines with depth. The contrast in vertical distribution of GRSP and SOC supports the hypothesis that GRSP is a distinct fraction of SOM. Climate and soil properties had significant influence on the vertical distribution of GRSP. The accumulation of GRSP, and the enrichment of SOC in GRSP, at acid pH could indicate a fungal origin of GRSP.
Quantification of protein by acid hydrolysis reveals higher than expected concentrations in red wines: Implications for wine tannin concentration and colloidal stability
2022, Food Chemistry
Protein is reportedly negligible in most red wines, due to its loss following co-precipitation with phenolic substances. A method for protein quantification in red wine was developed which overcame analytical interference from phenolic substances, based on ethanol precipitation, followed by acid-hydrolysis and amino acid quantification. Protein concentration was surveyed in a range of red wines produced from V. vinifera and interspecific (Vitis spp) hybrids, revealing higher than expected concentrations, ranging from 23 mg/L ± 2.57 to 380 mg/L ± 16. The results showed that tannin extracted from grapes remains soluble in wine in the presence of protein even at high protein (>100 mg/L) and tannin (>500 mg/L) concentrations. As a further consequence of this, the particle size and concentration of colloids within high- and low-protein wines were similar, independent of protein or tannin concentration. Higher wine tannin concentration was also correlated with increased heat stability of wine protein.
Estimates of protein in coastal marsh soils: A case study of the utility of the Bradford assay for quantifying soil protein
2022, Geoderma
The fraction of soil protein extracted by autoclaving soil (AESP) has emerged as a potential indicator of available organic nitrogen (N), aggregate stability, and soil carbon (C) storage potential. Estimates of AESP are often based on colorimetric assays—primarily the Bradford assay—yet the results of these assays can be confounded by interference from polyphenolic contaminants of soil protein extracts. The extent to which polyphenolic interference in the Bradford assay may contribute to over- or underestimation of AESP content and whether a correction can be made for the interference has remained unclear. Here, we present a case study of how Bradford-based estimates of AESP compared to maximum and minimum estimates of AESP across two chronosequences of marsh soils that ranged widely in soil organic matter concentration (20–580 mg·g⁻¹). Our maximum estimates were based on the amount of N in the dialyzed extracts; our minimum estimates were based on the proportion of the extracted N that was accounted for by hydrolysable amino acids. We observed that the Bradford-based estimates usually followed trends that were qualitatively similar to those of the maximum and minimum estimates across different marshes. However, the Bradford-based estimates were almost always greater than the maxima (by up to 450%) and never below the minima. This extent of overestimation was positively correlated with the C:N ratio of the extracts, but the relationships between the overestimates and C:N ratios differed between the two chronosequences. Contrary to what has been previously suggested, the dilution of extracts only exacerbated the extent of overestimation in the Bradford assay. Instead of a continued reliance on the Bradford assay for AESP measurements, we propose that the N content of semi-purified soil extracts serve as a surrogate metric for AESP, primarily because it may be more amenable to inter-study comparisons while still being feasible for most soil laboratories.
Glycoproteins of arbuscular mycorrhiza for soil carbon sequestration: Review of mechanisms and controls
2022, Science of the Total Environment
Citation Excerpt :
The covalent bonds generated between proteins and polyphenolic compounds during extraction make GRSP extraction and quantification difficult and partly uncertain (Jorge-Araújo et al., 2015). The GRSP measured through the Bradford protein is discussed in terms of fungal origin and the age of the EE and T-GRSP pools (Staunton et al., 2020; Whiffen et al., 2007). GRSP are operationally defined based on the extraction method and therefore recent studies have refined the terms such as autoclaved citrate extractable (ACE) protein and citrate extractable soil proteins (CESP) as more appropriate to designate GRSP (Holátko et al., 2021; Hurisso et al., 2018) (Table 1).
Glycoproteins, e.g., glomalin related soil proteins (GRSP), are sticky organic substances produced by arbuscular mycorrhizal fungi (AMF). This review summarizes the information on i) the biochemical nature, physical state and origin of GRSP, ii) GRSP decomposition and residence time in soil, iii) GRSP functions, in particular the physical, chemical, and biochemical roles for soil aggregation and carbon (C) sequestration, and finally iv) how land use and agricultural management affect GRSP production and subsequently, organic C sequestration. GRSP augment soil quality by increasing water holding capacity, nutrient storage and availability, microbial and enzymatic activities, and microbial production of extracellular polysaccharides. After release into the soil, GRSP become prone to microbial decomposition due to stabilization with organic matter and sesquioxides, and thereby increasing the residence time between 6 and 42 years.
Temperate soils contain 2-15 mg GRSP g^-1, whereas arid and semiarid grasslands amount for 0.87-1.7 mg g^-1, and GRSP are lower in desert soils. GRSP content is highest in acidic soils as compared to neutral and calcareous soils. Conservation tillage, organic fertilizers and AMF inhabiting crops (e.g. maize, sorghum, soybean, and wheat) increase GRSP production and transform C into stable forms, thereby sustaining soil health and reducing CO₂ emissions. Crop rotations with non-mycorrhizal species (e.g. rapeseed) and fallow soils reduce AMF growth and consequently, the GRSP production. The GRSP production increases under nutrient and water deficiency, soil warming and elevated CO₂. In the context of global climate change, increased C sequestration through GRSP induced aggregate formation and organic matter stabilization prolong the mean residence time of soil C. Protecting soils against degradation under intensive land use, stable aggregate formation, and prolonging the residence time of C calls for strategies that maximize GRSP production and functions based on reduced tillage, AMF-relevant crop rotations and organic farming.

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Soil Biology and Biochemistry

Abstract

Section snippets

Acknowledgements

References (26)

Enhancement of copper and cadmium adsorption on kaolin by the presence of humic acids

Chemosphere

Relating foliar dehydration tolerance of mycorrhizal Phaseolus vulgaris to soil and root colonization by hyphae

Journal of Plant Physiology

A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding

Analytical Biochemistry

Survival of inocula and native AM fungi species associated with shrubs in a degraded Mediterranean ecosystem

Soil Biology & Biochemistry

Mechanism of dye response and interference in the Bradford protein assay

Analytical Biochemistry

Color reaction of hydrolyzable tannins with Bradford reagent, Coomassie brilliant blue

Phytochemistry

Some aspects of complex interactions involving soil mesofauna: analysis of the results from a Scottish woodland

Ecological Modelling

Some aspects of interrelations between fungi and other biota in forest soil

Mycological Research

Microbiota accompanying different arbuscular mycorrhizal fungal isolates influence soil aggregation

Pedobiologia

Changes in soil enzyme activity, fertility, aggregation and C sequestration mediated by conservation tillage practices and water regime in a maize field

Applied Soil Ecology

What is humic acid? A perspective of the past forty years

Does the presence of glomalin relate to reduced water infiltration through hydrophobicity?

Canadian Journal of Soil Science

Protein accumulation and distribution in floodplain soils and river foam

Ecology Letters

Cited by (73)

Potential interaction mechanisms between PAHs and glomalin related-soil protein (GRSP)

Contributions of mycorrhizal fungi to soil aggregate formation during subalpine forest succession

Accumulation and vertical distribution of glomalin-related soil protein in French temperate forest soils as a function of tree type, climate and soil properties

Quantification of protein by acid hydrolysis reveals higher than expected concentrations in red wines: Implications for wine tannin concentration and colloidal stability

Estimates of protein in coastal marsh soils: A case study of the utility of the Bradford assay for quantifying soil protein

Glycoproteins of arbuscular mycorrhiza for soil carbon sequestration: Review of mechanisms and controls

Short communicationPolyphenolic compounds interfere with quantification of protein in soil extracts using the Bradford method

Abstract

Section snippets

Acknowledgements

Chemosphere

Journal of Plant Physiology

Analytical Biochemistry

Soil Biology & Biochemistry

Analytical Biochemistry

Phytochemistry

Ecological Modelling

Mycological Research

Pedobiologia

Applied Soil Ecology

Does the presence of glomalin relate to reduced water infiltration through hydrophobicity?

Canadian Journal of Soil Science

Protein accumulation and distribution in floodplain soils and river foam

Ecology Letters

Short communication
Polyphenolic compounds interfere with quantification of protein in soil extracts using the Bradford method