CHAPTER 2
 EXPERIMENTAL CONSIDERATIONS IN DETERMINING OPTIMUM ENZYME
 ASSAY CONDITIONS IN WETLAND SYSTEMS

 Introduction

 Fluorogenic substrates have been widely adopted in investigations of enzyme

activities in a variety of systems such as lakes, grasslands, wetlands, streams,

groundwater and oceans (Chr6st, 1989; Freeman et al., 1995; Miettinen et al., 1996;

Sinsabaugh and Foreman, 2001; Debosz et al., 1999; Mayr et al., 1999, Shackle et al.,

2000; Wittmann et al., 2000; Burns and Ryder, 2001; Newman et al., 2003; Saiya-Cork et

al., 2002; Wittmann et al., 2004). These compounds have become popular due to their

production of fluorescent compounds, which exhibit less interference by highly colored

phenolic compounds than colorimetric substrates (Sinsabaugh et al., 1991; Freeman et al.,

1995). Methylumbelliferyl (MUF) and amidomethylcoumarin (AMC) substrates are

among the most widely adopted fluorogenic substrates.

 Fundamentally different methodological approaches have been adopted in the

literature. The most recent approaches include the use of microtiter plates (Marx et al.,

2001). Standardized approaches use substrate saturating conditions, standard

temperature, and an assay pH that maximizes the fluorescence of the fluorochrome.

Enzymes have differing optimum pH levels which have influenced the use of different

pH conditions between enzymes (Parham and Deng, 2000). The environmental approach

utilizes substrate concentrations similar to the local environment with assay temperature

and pH approximating field conditions. A compromise between these approaches is to