The Use of p-Nitrophenol and 4-Methylumbelliferone in Soil Enzyme Assay.

p-Nitrophenyl and 4-methylumbelliferyl substrates are two classes of most commonly used chromogenic and fluorogenic substrates used for assaying soil enzyme activities.  It is well established that p-nitrophenol is relatively stable and can be quantitatively detected in soil suspensions with little interference by soil components.  Method employing 4-methylumbelliferone can provide greater sensitivity; however significant quenching of signal by soil components can occur. With the increasing interest in the use of high throughput microplate format assays for soil enzymes that utilize only small quantity of soil for each analysis, highly sensitive detection methods are needed. Therefore, this study was conducted to directly compare the use of these two classes of substrates for soil enzyme assays and provide thorough evaluations on variables that are pertinent to the assay and its subsequent data interpretation.  Following comparison of fluorometric and spectrophotometric assays for determining activities of N-acetyl-β-D-glucosaminidase, β-D-glucopyranosidase, aryl sulfatase, and alkaline phosphatase in eight soils of a range of properties, we found that the detected enzyme activities by these two methods could be considerably different in some soils for some enzymes but not in others.  Of the enzymes evaluated, the biggest difference was shown for activities of alkaline monophosphoesterase, which could be magnitude difference between the two methods.