Units for Expressing Enzyme Activity

When enzymes are measured by their catalytic activities, the results of such determinations are expressed in terms of the concentration of the number of activity units present in a 

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). 

The international unit itself may eventually be replaced by the SI unit termed the katal, the SI derived unit for catalytic activity (see Chapter 1). It is defined as moles per second. The name katal had been used for this unit for decades, but did not become an official SI derived unit until 1999 with Resolution 12 of the 21st CGPM, on the recommendation of the International Federation of Cfinical chemistry and Laboratory Medicine. Both the International Union of Pure and Applied Chemistry and the lUB now recommend that enzyme activity be expressed in moles per second and that the enzyme concentration be expressed in terms of katals per liter (kat/L). Thus, lU = lO mol/fiOs = 16.7 X 10 mol/s, or l.Onkat/L - 0.06U/L. The formal adoption of the katal is hoped to discourage the use of a non-SI unit called unit, symbol U, defined as micromoles per minute. Units are more commonly used than the katal in practice at present, but their definition lacks coherence with the SI system. 

The amount of substrate transformed into products during an enzyme-catalyzed reaction can be measured with any appropriate analytical method, such as spectrophotometry, fluorometry, or chemiluminescence. For example, if an enzyme reaction is accompanied by a change in the absorbance characteristics of some component of the assay system, in either the visible or ultraviolet spectrum, it can be photometrically observed while it is proceeding. Self-indicating reactions of this type are particularly valuable as 

To measure enzyme activity reliably, all the factors that affect the reaction rate-other than tlie concentration of active enzyme—must be optimized and rigidly controlled. Furthermore, because the reaction velocity is at or near its maximum under optimal conditions, a larger analytical signal is obtained that can be more accurately and precisely measured than a smaller signal obtained under suboptimal conditions. Much effort has therefore been devoted to determining optimal conditions for measuring the activities of enzymes of clinical importance. 

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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.)... 

Lineweaver and Ballou49 have proposed a pectinesterase unit ( PE. u. ) for expressing PM activity. One such unit is equivalent to 1/930 PMU under the same experimental conditions or the quantity of enzyme that, at 30° and optimum pH, will catalyze the hydrolysis of pectin at an initial rate of one milliequivalent ester bonds per minute in a standard substrate (0.5% citrus pectin containing 8-11% methoxyl) and 0.15 M sodium chloride. The use of the latter unit is unfortunate since the values obtained for the activity in ordinary plant materials are obtained in the third decimal place and because the experimental conditions are so... 

There are no recognized enzyme standards or reference materials. The accepted basis for measurement is the rate of reduction of the substrate, commonly nicotinamide adenine dinucleotide (NAD) for many reactions. The reduced form, NADH, absorbs at 340 nm, and the rate of change of this absorbance is measured in an enzyme-activity assay. The absorptivity a in Beer s law) is known for NADH from this and the rate of change of absorbance per unit time, the activity of the enzyme can be calculated in micromoles of substrate converted per minute. This is referred to as an International Unit (lU), expressing the activity as lU/liter. Therefore, an accurate, absolute absorbance scale must be established in each case in order to make a valid assay. 

It is not always possible to express enzyme activity in lU this is the case of enzymes catalyzing reactions that are not chemically well defined, as it occurs with de-polymerizing enzymes, whose substrates have a varying and often undefined molecular weight and whose products are usually a mixture of different chemical compounds. In that case, units of activity can be defined in terms of mass rather than moles. These enzymes are usually specific for certain types of bonds rather than for a particular chemical structure, so in such cases it is advisable to express activity in terms of equivalents of bonds broken. 

For purposes of dosage, the specific activity of an enzyme is usually expressed as International Units (lU) rather than in terms of weight. However, unit measurements do not provide information on the absolute purity of a given product. Moreover, purity is not as critical an attribute for oral enzymes, as opposed to those adrninistered parenteraHy, inasmuch as the gastrointestinal tract is capable of disposing of most inert contaminants. 

In many situations, the actual molar amount of the enzyme is not known. However, its amount can be expressed in terms of the activity observed. The International Commission on Enzymes defines One International Unit of enzyme as the amount that catalyzes the formation of one micromole of product in one minute. (Because enzymes are very sensitive to factors such as pH, temperature, and ionic strength, the conditions of assay must be specified.) Another definition for units of enzyme activity is the katal. One katal is that amount of enzyme catalyzing the conversion of one mole of substrate to product in one second. Thus, one katal equals 6X10 international units. 

Enzyme Reference Serums. Several companies sell lyophilized or stabilized reference serums for the calibration of instruments and for quality control. The label values given for the enzymatic activity of these serums should never be taken at face value, as at times they may be quite erroneous (19,33). Also, these values should only be used for the assay with which they were standardized, as interconversion of activity from one method to another for the same enzyme may often lead to marked errors. For instance, it is not recommended that alkaline phosphatase expressed in Bodansky units be multiplied by a factor to convert it to the units of the Ring-Armstrong method, or any other method for that matter. 

Pectinesterase activity expressed as a unit corresponding to the microequivalent of ester bonds of pectin molecule, which were hydrolyzed during 1 min. at 45 °C and pH 5.0 under the conditions, which were optimum for these enzymes. Endopolygalacturonase and exopolygalacturonase activities were determined using a technique determined by Ufshitz [8]. Activity of pectintranseliminase was determined by procedure (lOJ. 

Velocity can be expressed in a number of different units. The most common is micromolar per minute (p,M/min) however, because the velocity depends on the amount of enzyme used in the assay, the velocity is often normalized for the amount of enzyme present by expressing the activity in units of micromoles per minute per milligram of enzyme [ jjnol/(min mg)]. This is called a specific activity. You may be wondering (or not) where the volume went—after all, product concentration is measured in molar units (M mol/L). Well, it s really still there, but it... 

The catalytic action of an enzyme, its activity, is measured by determining the increase in the reaction rate under precisely defined conditions—i.e., the difference between the turnover (violet) of the catalyzed reaction (orange) and uncatalyzed reaction (yellow) in a specific time interval. Normally, reaction rates are expressed as the change in concentration per unit of time (mol 1 s see p. 22). Since the catalytic activity of an enzyme is independent of the volume, the unit used for enzymes is usually turnover per unit time, expressed in katal (kat, mol s ). However, the international unit U is still more commonly used (pmol turnover min 1 U = 16.7 nkat). 

The principal limitation of these data is the lack of definition of the individual forms for the CYP2C subfamily. Analysis of this subfamily has remained problematic due to high cross-reactivities of all of the distinct forms with most antibody preparations. In addition, Western blot analysis does not distinguish between active and inactive forms of the protein. Furthermore, distinct enzymes may have different affinities for coenzymes necessary for catalytic activity, which will serve to unlink abundance of the protein and its catalytic activity. Therefore the assumptions must be made that the ratios of active to inactive protein are similar for all forms and that all forms have similar affinities for coenzymes. These assumptions may not be justified. However, even with these limitations, the study of Shimada et al. (1994) contributes greatly to our understanding of relative enzyme abundance in human liver. In addition, the relative abundance data, coupled with the absolute P450 content (per unit protein) and the turnover numbers for enzyme-specific substrates (per unit protein), can provide an estimate of the turnover number for individual enzymes in the human liver membrane environment. This provides an important benchmark for evaluation of turnover number data from cDNA-expressed enzymes. 

Since a principal P450 is defined by its catalytic activity, and activity per unit enzyme is variable for the same enzyme/substrate when the enzyme is expressed in different heterologous systems, it seems more appropriate to establish relative contributions based on enzyme activities in the incubations of cDNA-expressed enzymes and human tissue preparations. 

By using specific catalytic activities, the differences in V ax due to any factor are automatically compensated for in the calculation. Because this method establishes relative contribution, there is no a priori need to use the same units for both components of the RAF calculation as long as the same units are used within the cDNA-expressed enzyme data set and the human tissue data set. Moreover, knowledge of the P450 content in either the cDNA expression system or in human liver microsomes is not necessary in order to make an interpretation. 

One difficulty in framing this discussion is a lack of commonality in units for the expression systems. For example, the same substrate may not have been examined in all systems or activity may be expressed per mg total cell lysate protein, per mg cytosol-free cell membrane protein, per mg microsomal protein or per million cells. In this section, activity levels will be compared in the units originally reported. The following values, as determined in the human lymphoblast system, may be used to compare among the alternative methods of enzyme preparation cytosol-free membranes provide about a 2-fold enrichment in activity, microsomes provide 5-fold enrichment in activity and there are about 7 million cells per mg total protein. These ratios may differ somewhat for other mammalian cell systems but they are unlikely to be off by more than 2-fold. 

The actual molar concentration of an enzyme in a cell-free extract or purified preparation is seldom known. Only if the enzyme is available in a pure crystalline form, carefully weighed, and dissolved in a solvent can the actual molar concentration be accurately known. It is, however, possible to develop a precise and accurate assay for enzyme activity. Consequently, the amount of a specific enzyme present in solution is most often expressed in units of activity. Three units are in common use, the international unit (IU), the katal, and specific activity. The International Union of Biochemistry Commission on Enzymes has recommended the use of a standard unit, the international unit, or just unit, of enzyme activity. One IU of enzyme corresponds to the amount that catalyzes the transformation of 1 p,mole of substrate to product per minute under specified conditions of pH, temperature, ionic strength, and substrate concentration. If a solution containing... 

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. 

Biochemical characteristics of the "Nerve ending fraction" and of the "Synaptosomal fraction", obtained from calf brain. Ganglio-sides are expressed as nmoles bound N-acetylneuraminic acid enzyme activities in milli International Units ( 1 nmole transformed substrate min at 37°C - 30° for NADH-, and NADPH-Cyt C reductase and LDH). The data shown, referred to 1 g starting fresh tissue, are the mean values of 6 experiments the S. E. was in all cases lower than + 10 % of the mean values. 

Either 24 hour urine collection or timed urine samples collected at the same time each day is recommended, with the activity expressed per unit of time (Price 1982, Plummer et al. 1986). If the assessment is to be repeated with time, the samples should be collected over the same time period on each day because there is pronounced diurnal variation in excretion rate of some enzymes (Maruhn et al. 1977, Price 1982, Gossett et al. 1987). For spot urine samples or those where accurate timed collection is not possible, normalization of activity per unit of creatinine can be done and this has been shown to be reasonably well correlated to 24 hour enzyme activity (Vanderlinde 1981, Grauer et al. 1995). Diet and age-matched controls must be included if enzyme activity is to be normalized to creatinine, to control for the effects of these variables on creatinine excretion (Plummer et al. 1986, Casadevall et al. 1995). 

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