Fluorescence Methods

Fluorogenic substrates are used to assay extracellular enzymes in aquatic and terrestrial environments. These substrates contain an artificial fluorescent molecule and one or more natural molecules (e.g., glucose, amino acids) and they are linked by a specific binding (e.g., peptide binding, ester binding). The substrates used are conjugates of the highly fluorescent compounds 4-methylumbelliferone

(MUB) and 7-amino-4-methyl coumarin (AMC). Marx et al. (2001) used this method to measure the activities of enzymes involved in C cycling ((3-d-glucosidase, (3-d-galactosidase, (3-cellobiase, (3-xylosidase), N cycling (leucine aminopeptidase, alanine aminopeptidase, lysine-alanine aminopeptidase), P cycling (acid phosphatase), and S cycling (arylsulfatase). Fluorogenic model substrates are not toxic and they are supplied to soil suspensions in high or increasing quantities to measure the maximum velocity of hydrolysis (Vmax). Fluorescence is observed after enzymatic splitting of the complex molecules (Fig. 3.5). The increasing interest of soil microbiologists in the use of fluorogenic substrates to measure soil enzyme activities is mainly because of their high sensitivity. A comparative study between a fluorimetric and a standard colorimetric enzyme assay based on p-nitrophenyl substrates generated similar values for the maximum rate of phosphatase and ( -glucosidase (Vmax), but the affinity for their respective substrates (as indicated by Km values, Michaelis-Menten constant) was up to two orders of magnitude greater for the 4-methylumbelliferyl substrates compared to the p-nitrophenyl substrates (Marx et al., 2001). This high sensitivity of fluorimetric enzyme assays provides an

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