Glomalin extraction and measurement

We investigated extraction from soil of glomalin, a glycoprotein produced by arbuscular mycorrhizal fungi, and we examined its measurement. The most commonly used protocols for extracting glomalin require autoclaving of soil in citrate solution, followed by centrifugation to separate the supernatant, and then measurement by either Bradford protein assay or enzyme-linked immunosorbent assay (ELISA). We found that lengthening the time of autoclaving increased easily extractable glomalin extraction. Delay of centrifugation after autoclaving, however, diminished Bradford-reactive substances in the supernatant, suggesting that extracted substances might be reversibly immobilized on soil particles. Surprisingly, increasing the volume of extraction solution did not accelerate extraction of "total glomalin", but instead, substantially increased the amount extracted. Multiple autoclave cycles nevertheless denature glomalin, which may not be as heat-resistant as thought. Repeated 1-h autoclaving of supernatant diminished both its Bradford-reactive substances (7.3% h^-1) and immunoreactive protein (22% during the first hour and 9.5% h^-1 of the remainder thereafter), although a large initial volume of extractant could reduce the loss of immunoreactive protein. Proteins and polyphenols that survive the extraction process are measured non-specifically by the Bradford assay. When we added other glycoproteins to dry soils, we recovered a maximum 34% bovine serum albumin and 22% bovine mucin, primarily in the first two, 1-h extraction cycles. These added proteins may adhere to soil organic matter and thereby be protected from denaturation. In addressing the endpoint of glomalin extraction, we found that the Michaelis-Menten equation closely fits cumulative glomalin per extraction cycle such that its asymptote provides an objective estimate of total extractable glomalin for a given set of extraction conditions. Additionally, the equation provides a curvature parameter that reflects the soil-specific efficiency of an extraction protocol. Although the soils that we investigated with 7.6% or more soil organic matter had the most asymptotic total glomalin, they were extracted the least efficiently.