Simultaneous Analysis of Multiple Enzymesin Environmental Samples Using Methylumbeiliferyl Substrates and HPLC C. Freeman* and G. B. Nevison Abstract Enzymes are considered to represent valuable biological indicators of ecosystemhealth. This study evaluates the potential for simultane- ous measurement of the activity of multiple enzymes in a single sample. The rate of degradation of a suite of artificial methylumbelliferyl substrates was monitored in a wetlandsoil samplefollowing separation of the individual components using high pressure liquid chromatogra- phy (HPLC). The methodallowed effective separation and quantita- tive monitoringof the hydrolysis of substrates for the enzymes sul- fatase, ~-glucosidase, xylosidase, and esterase. The proposed method appeared simple to perform, and offered considerable time-savings over conventional techniques. Tc HESIGNIFICANCE of enzymes, as indicators of the ycling of nutrients and energy and thus ecosystem health, is reflected in the multitude of enzymological studies in the literature (Tabatabai, 1982; Chrost, 1990; Wetzel, 1991; Sinsabaugh, 1994). Enzymes have, for ex- ample, been found of substantial value as indicators of microbial activity in coal strip mine spoils (Hersman et al., 1979), in the assessment of metal toxicities (Boular- bah et al., 1996), and for the identification of degraded soils (Dick, 1997). However, measurements of enzyme activities in peat-accumulating wetlands were all but nonexistent before the 1990s. This can be attributed to methodologicallimitations, in that the enzyme activities in peat (particularly at depth) are extremely low, and measurements are hampered by an abundance of in- terfering dissolved organic materials (Freeman et al., 1995). The following approach was developed following recent advances in the measurement of peatland enzyme activities. However, its use is not restricted to peatlands: Peat was selected primarily because it represents the most problematic matrix in which to carry out enzymic analyses, and therefore represents the most rigorous test of the new approach. The technique has evolved, following the application of fluorogenic assays to wetland samples. That method involves measuring the rate of release of fluorescent methylumbelliferone product following enzymic hydro- lysis of a non fluorescent methylumbelliferyl (MUF) model substrate (Freeman et al., 1995). Adoption fluorescence-based assays gave greater sensitivity than the earlier colorimetric assays (Tabatabai, 1982). But, even the improved method suffered from limitations caused by interfering compounds such as phenolics. These materials cause quench, a particularly problem- atic symptom of which is the absorbtion of radiation at School of Biological Sciences, Univ. of Wales, Bangor, Gwynedd, LL57 2UW, UK. Received 10 July 1998. *Corresponding author (c.freeman@bangor.ac.uk) Published in J. Environ. Qual. 28:1378-1380 (1999). both the excitation and emission wavelengths used in the fluorimetric analysis. The phenomenon is highly variable between samples (15-95%), and the only con- ventional means of addressing the problem involves la- bor intensive manual quench correction procedures (Freemanet al., 1995). The problems of quench have now been overcome through the use of high pressure liquid chromatography (HPLC) to separate the fluorescent products of interest from the interfering agents (Freeman, 1997). Further- more, it has since been found that the elimination of those interferences allows detection of product forma- tion using inexpensive UV detection, rather than its expensive fluorimetric counterpart (Kang et al., 1997), and without excessive loss of sensitivity. The work de- scribed in this paper has arisen as a direct consequence of observations made using the UV-HPLC approach. In those studies, it became apparent that the system was capable of resolving two distinct peaks; both the free-MUF and the model MUF-substrate from which that free-MUF was released. Thus, enzymic hydrolysis could be followed either as (i) the rate of free-MUF formation, or (ii) the rate of model substrate degrada- tion. Furthermore, it was noted that each different model compound eluted from the column with a differ- ent retention time. The latter observation allows us to propose a radical new approach to the measurement of enzyme activities. Instead of measuring each enzyme assay individually, it is possible to add a completesuite of enzyme-substrates in a single multiple enzyme assay. Using HPLC, it is then possible to monitor the degrada- tion of each component in the mixture of substrates. Materials and Methods The enzyme systems considered included [3-glucosidase, es- terase, and xylosidase, which participate in organic matter decomposition and hence C cycling. These were studied by following the rate of hydrolysisof the model substrates methyl- umbelliferyl (MUF)-[3-D-glucoside, MUF-acetate, and MUF- ~3-D-xyloside, respectively. Standard curvesfor each substrate can be seen in Fig. 1. It has also been recognized that in wetland soils, organic matter decomposition can be effected through a pathwaythat involves both C and S cycles, the fermentation-sulfate reduction pathway (Howes et al., 1984). As such, the enzyme sulfatase was also studied, and was as- sayed by following the hydrolysis of MUF-sulfate. The experimentswere carried out at 11°C, using peat from Cerrig-yr-Wyn, Plynlimon, mid-Wales (UK Nat. Grid Ref. SN 820 866) in an area dominated by Sphagnum and Juncus. A mixed substrate solution was preparedto contain 100txM each of methylumbelliferyl sulfate (MUF-S), methylumbelliferyl- Abbreviations: HPLC, high pressure liquid chromatography; MUF, methylumbelliferyl; MUF-S, methylumbelliferyl sulfate; MUF-G, methylumbelliferyl-[3-D-glucoside; MUF-X, methylumbelliferyl-[3- D-xyloside. 1378 Published July, 1999