Expression and Measurement of Enzyme Activity

The presence or absence of an enzyme is typically determined by observing the rate of the reaction(s) it catalyzes. Quantitative enzyme assays are designed to measure either the total amount of a particular enzyme (or class of enzymes) in units of moles or, more commonly, the catalytic activity associated with a particular enzyme. The two types of assays differ in that those in the latter category measure only active enzyme. The assays contained in this section are concerned primarily with the measurement of catalytic activity, or active enzyme. The assays are based on kinetic experiments, as activities are calculated from measured reaction rates under defined conditions. The basic Premise for these assays is that the amount of enzyme in a reaction mi xture can be determined from the rate at which the enzyme-catalyzed reaction occurs. 

The goal of most enzyme assays is to quantitatively measure the amount of enzyme activity (catalytic activity) present in a sample. Thus, assay results are typically reported in activity units. A unit of activity may be defined in 

It is also common for enzyme activities to be reported in units based on changes in reaction mixture properties that are themselves a function of the extent of the enzymatic reaction. These units are often difficult to interpret in 

The upshot of the above discussion is that a variety of enzyme activity units may be encountered. This makes it essential that all units, whatever their bases, be clearly defined. 

Enzyme assays are typically done under relevant conditions, be they physiological conditions, food-storage conditions, or conditions corresponding to maximal activity. This implies that consideration must be given to reaction mixture parameters such as pH, temperature, ionic strength, buffer composition, and other components not involved in the reaction. It is prudent to assume that changes in any of these parameters may affect enzyme activity. Analysts will often run assays under apparent optimum conditions (maximal activity), such as optimum pH, because these conditions tend to coincide with maximum assay sensitivity. It should be apparent from this discussion that assays using different reaction conditions may have only limited comparative value. 

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Cl.] Expression and Measurement of Enzyme Activity C1.2 Detecting Enzyme Activity A Case Study of Polygalacturonase... 

Extraction and measurements of enzyme activity was achieved as described previously (1). 5-ALAD activity was expressed in nanomole of PBG formed per second and per cotyledon. 

Many different measurements of enzymes are undertaken to acquire different types of information. For example, the presence of genes for an enzyme has been used to infer whether organisms are capable of performing particular functions, the expression of those genes or the appearance of the enzyme protein is used to indicate if and under what conditions the gene is functioning, while assay of the activity of the enzyme has been used to infer rates of particular processes (see Section 2). In fact, virtually all measurements classified as molecular and most rate measurements of uptake are, in fact, measurements of enzymes. Because strictly molecular methods (i.e., the capacity to perform a reaction see Chapter 30 by Zehr and Jenkins, this volume) and N uptake (i.e., the net result of enzymatically mediated processes see Chapter 6 by Mulholland and Lomas, this volume) are discussed elsewhere in this book in this chapter, we will focus primarily on measurements of enzyme activities. 

The spectrophotometer measures and displays the increase in absorbance at 410 nm as a function of time (AA/At). Whether the output from the instrument is in the form of a strip chart or is collected by a computer, the reaction velocities are usually expressed in terms of change in concentration per unit time, or converted to specified units of enzyme activity. The International Unit (U) for enzyme activity is defined as the amount of enzyme that transforms 1 pmol substrate to product in 1 min under specified assay conditions. The SI unit for activity is the katal, which is defined as the amount of enzyme that transforms 1 mol substrate per second under specified conditions. Thus 1 U = 16.7 nkatal. To convert slopes AA/At values) to velocities (v), the following equation is used ... 

In most studies of DPO activity, the main objective is usually a simple comparison of the potential of a particular tissue to undergo enzyme-catalyzed browning, for example, a comparison of the potential for enzymic browning of different apple or mushroom cultivars. Related to this are comparative studies of different inhibitors and processing regimes to control enzymic browning. In these circumstances, it is usually sufficient to provide comparative measurements rather than absolute values of enzyme activity, in which case results can be expressed in arbitrary units such as AmV/min for 02 electrode assays or AA/min for spectrophotometric assays. If more precise units are required, the 02 electrode results should be expressed as Anmol 02/min/(j.g protein. 

Photometric measurements performed in clinical laboratories use advanced chemical and biochemical methods and diverse instrumentation. Most analyses performed in clinical laboratories are based on spectrophotometric methods using photometric systems, such as absorption photometers, atomic absorption spectrophotometers and flame photometers. Typically, the result is expressed as mass concentration of analyte in solution (mg/dl), molar concentration (mmol/1), or catalytic concentration of enzyme activities in solution (U/l) [6],... 

Cellulase activity of the samples was determined as filter paper activity (FPA) expressed in filter paper units (FPU) using Mandels procedure (15), and (3-glucosidase activity was assayed using 4-nitrophenyl-(3-D-glucopyranoside substrate according to Berghem and Petterson s (16) method. All samples were analyzed in triplicate and the mean values were calculated. The relative standard deviation of enzyme activity measurements was always below 5%. 

Enzymatic assays can be applied in the marine environment to provide indirect information on dissolved compounds that are available to fuel bacterial production. Approaches that have been commonly appHed include measuring hydrolytic enzyme activities in seawater and monitoring degradation rates of model compounds. Protein hydrolysis in seawater is rapid as expressed by model protein studies (e.g., Nunn et al., 2003 Pantoja and Lee, 1999). This rapid and selective removal of dissolved proteins explains the relatively minor contribution from proteins to the accumulating DOM reservoir even though proteins are by far the most abundant intracellular biochemical. In an elegant study, Nunn and coworkers (2003) used matrix assisted laser desorption/ionization (MALDI) time of flight (TOP) mass... 

The base unit katal (symbol kat), mol/sec, is the catalytic amount of any catalyst, including enzymes, that catalyzes a reaction rate of 1 mol per second in an assay system." The kind of quantity measured is identified as catalytic amount. There is a constant relationship between the international unit (I pmol/min) and the katal (1 mol/sec) to convert, a y ue. in international units to nmol/sec, the value is multiplied by 16.67. Note, however, that dependence on reaction conditions applies to. SI units in the same way as to international units therefore data reported in the same units but obtained under different conditions may not be comparable. Replacement of the international unit for reporting enzyme activity is likely to be slow even units that antedated the international unit are sometimes used in clinical laboratory practice. (See Chapters 8 and 21 for further details on the expression of enzyme activity.)... 

To standardize how enzyme activities are expressed, the Enzyme Commission of the lUB proposed that the unit of enzyme activity be defined as the quantity of enzyme that catalyzes the reaction of 1 pmol of substrate per minute and that this unit be termed the international unit (U). Catalytic concentration is to be expressed in terms of U/L or kU/L, whichever gives the more convenient numerical value. In this chapter, the symbol U is used to denote the international unit. In those instances in which there is some uncertainty about the exact nature of the substrate or when there is difficulty in calculating the number of micromoles reacting (as with macromolecules such as starch, protein, and complex lipids), the unit is to be expressed in terms of the chemical group or residue measured in following the reaction (e.g., glucose units, or amino acid units formed). 

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