Determining Enzyme Activity by Radial Diffusion

Abstract

B ESIDES BEING OF INTEREST IN THEMSELVES, enzymes are useful markers of such diverse biological processes as differentiation, physiology, metabolism, genetics, and evolution. Enzymes are assayed to monitor food processing and are important in clinical diagnosis. The amount of enzyme present is standardly estimated by the amount of enzyme activity, usually by some spectrophotometric method. However, there are times when the investigator only needs to determine (a) whether the enzyme is present at all or (b) a rough approximation of the amount of enzyme activity. In these situations the assay may be conveniently performed using the radial diffusion method. Basically the radial diffusion assay is performed by placing the sample containing the enzyme in direct contact with the substrate dissolved or suspended in agar gel. As the enzyme diffuses into the gel, the reaction takes place. At the end of a set time the gel is monitored for some estimate of enzyme activity, such as a clear area that indicates the loss of substrate. The more enzyme present in the sample, the more it diffuses from the point of application, and therefore the larger the cleared zone. The clear area can be used (a) to establish that the enzyme was indeed present in the sample and (b) to estimate the amount of enzyme present. A comparable procedure is routinely performed in assaying antibiotic concentration (or sensitivity) and in quantitative immunochemistry. Theoretically the diffusion method can be used to determine any enzyme activity, provided that the reaction can be monitored visibly and that the substrate or product has limited diffusion through the agar gel. In practice the method has been limited to assays of hydrolytic enzymes. For example, amylase activity can be determined by add ing the sample to a starch -agar plate. After a specific amount of time the presence of starch in the plate is visualized by flooding the surface with an iodine solution. Clear, unstained zones indicate the absence of starch and therefore the presence of the starch-digesting enzyme. This method has been carefully analyzed (e.g., Mestecky et al. 1969; Mottonen 1970), and will be discussed in detail below. The diffusion method has also been used to detect microbial cellulases, proteases, and pectinases (Booth 1971). In many of these instances, as in the determination of elastase (Sbarra et al. 1960), the substrate-agar is relatively opaque since the substrate is more a suspension than a solution. Therefore the clear zone can be observed without staining. Alternately the substrate may be observed more clearly after acid precipitation or after the addition of a stain that is specific for the substrate. Other enzymes assayed by this method are muramidase (ysozyme), alkaline and acid phosphatase, deoxyribonuclease and ribonuclease (Jarvis and Lawrence 1969; Schill and Schumacher 1972). Commercial plates are now available for most of these enzyme assays.