Enzyme activity, international unit

The specific activity of the enzyme in international units per milligram is... 

Calculation One unit of enzyme activity (FIP Unit) is defined as that quantity of a standard lipase preparation (Fungi Lipase-International FIP Standard) that liberates the equivalent of 1 (imol of fatty acid per minute from the Substrate Emulsion under the described assay conditions. The specific activity is expressed in international FIP units per milligram of the Sample Preparation. 

Besides specific activity, two other quantities are used for the characterization of enzymes the International Unit [U] (defined as the amount of enzyme which... 

Recently, an assay using chromophoric substrate (Chromozym TH) was developed [7], When Chromozym TH is added, the absorbance at 405nm will increase, and the linear section from 15 to 30 sec is used to calculate the activity (international unit, one unit is defined as the specific activity required to convert 1 pM substrate/min/mg of enzyme), Fig. (7). The increasing absorbance is resulted from the p-nitroanilide released off the substrate during hydrolyzation. As compared with the fibrin plate method, this chromogenic assay is more rapid, convenient and reproducible. The specificity of the assay, however, is lower than that of fibrin-plate method because the chromogenic substrate could be hydrolyzed by some other proteases, for instance thrombin. 

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. 

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

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. 

In each of the assays of potency the amount of the immunoglobulin and the amount of a corresponding standard preparation that are required to neutralize the infectivity or other biological activity of a defined amount of virus or to neutralize a defined amount of a bacterial toxin are determined. The two determined amounts and the assigned unitage of the standard preparation are then used to calculate the potency of the immunoglobulin in International Units (lU). ELISA, enzyme-linked immunosorbent assay. 

In addition to KM and vmax, the turnover number (molar activity) and the specific activity are two important parameters in enzyme catalyzed reactions. The turnover number indicates the number of substrate molecules converted per unit time by a single enzyme molecule. The specific activity is given in units and one international unit (i.u.) is the amount of enzyme that consumes or forms 1 pmol of substrate or 1 pmol of product per minute under standard conditions. 

This corresponds to the recommendations given in 1959 by a joint committee of the Clinical Chemistry Commission of IUPAC (International Union of Pure and Applied Chemistry) and the Enzyme Commission of IUB (International Union of Biochemistry). Thus, one unit of enzyme activity should be defined as that amount of enzyme which catalyzes the conversion of one micromole of substrate per minute under defined conditions (W9). 

Because of the difficulties in measuring the amount of enzyme in the conventional units of mass or molar concentration, the accepted unit of enzyme activity is defined in terms of reaction rate. The International Unit (IU) is defined as that amount of enzyme which will result in the conversion of 1 /nmol of substrate to product in 1 minute under specified conditions. The SI unit of activity, which is becoming more acceptable, is the katal and is defined as that amount of enzyme which will result in the conversion of 1 mol of substrate to product in 1 second. A convenient sub-unit is the nanokatal, which is equal to 0.06 International Units. 

International Unit (IU) Ratal (kat), the true SI unit of enzyme activity Specific activity that amount of enzyme protein which brings about the conversion of 1 (Imol of substrate to product per minute under stated conditions that amount of enzyme protein which brings about the conversion of 1 mole of substrate to product per second under stated conditions. enzyme activity (as IU or kat) per mg of total protein. This is a useful measure of the purity of an enzyme preparation... 

Analyses. Cellulase enzyme activities were determined as described earlier (21). Enzyme activities were expressed as international units (lU), i.e., mole glucose produced per minute. Cotton hydrolyzing activity was expressed as mg of glucose released in 24 hours per ml of enzyme. 

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 intrinsic variable expressed usually as international units (U) of enzyme activity per milligram protein... 

The specific enzyme activity is defined as the amount of converted substrate and formed product, respectively, per time unit and amount of enzyme at defined pH, temperature, and buffer composition. The specific activity is given as arbitrary units (e.g., units/mg/min of units/O.D./min the international unit lU is defined as the conversion of 1 pmol substrate and forming of 1 pmol product, respectively, per minute) or as SI unit kat/mg (Mol/s/kg). 

By international agreement, 1.0 unit of enzyme activity is defined as the amount of enzyme causing transformation of 1.0 gmol of substrate per minute at 25 °C under optimal conditions of measurement. The term activity refers to the total units of enzyme in a solution. The specific activity is the number of enzyme units per milligram of total protein (Fig. 3-23). The specific activity is a measure of enzyme purity it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure (Table 3-5). 

Turnover numbers can be measured only when the concentration of the enzyme is known. Partly for this reason the activity of an enzyme is usually given as specific activity, the units of activity per milligram of protein. One international unit is the amount of enzyme that produces 1 prnol of product per minute under standard (usually optimal) conditions. The International Union of Biochemistry17 has recommended a larger unit, the katal (kat), the amount of enzyme that converts one mol s 1 of substrate to product. 

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 ... 

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

Calculation of the Endocellulase Activity from the Intrinsic Viscosity Values. The enzymic degradation of polymeric substrates can occur at different bonds in the same substrate molecule, and the enzymic activity has to be defined here as the initial number of moles of glyco-sidic bonds split per second (53). This definition corresponds to the definition of the katal, symbolyzed kat. This unit is defined as the catalytic amount of any catalyst (including any enzyme) that catalyzes a reaction rate of one mole per second in an assay system (54), and it is recommended by the International Union of Pure and Applied Chemistry (55) for the quantitative evaluation of catalytic activities. 

According to the International Union of Biochemistry, one enzyme international unit (IU) was defined as the enzyme strength which can catalyze 1 jumole of substrate per minute. In describing the activity of the cellulase, one IU is equivalent to the strength to release 1 /rmoles of glucose per minute, because the molecular weight of the substrate, cellulose polymer, is not well defined. 

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. 

The value of the (overall) enzyme activity is usually provided in International Units or Units ... 

The esterification activity of Lipozyme IM-20 was measured according to the method described by Langone and Sant Anna (9), which determines the consumption rate of fatty acid at 60°C in a reaction system containing glycerol, lauric acid, and a given amount of the commercial enzyme preparation. One international unit of esterification activity is the quantity of enzyme that consumes 1 pmol of lauric acid/min under the reaction conditions. The enzyme used has an esterification activity of 20 IU/g. 

The Jhtemationai Union of Biochemistry (TUB) was officially founded in 1953 by an initiative of the British Biochemical Society. At ibis time enzyme standardization was in a chaotic slate, owing to the multiplicity of arbitrarily defined units of enzyme activity and the ilt-dtfned nomenclature. In 1955, the 1UB International Commission on Enzymes was created. This led to an improved enzyme nomoidature, which has been used since 1961, and the definition of the International Unit (LU.) of enzyme activity. 

A new international unit has been recommended. The katal (kat) is defined as the amount of enzyme activity that transforms 1 mole of substrate per second. Activities can also be given in millikatals (mkat), microkatals (fxkat), nanokatals (nkat), etc. Both specific activity and turnover number can also be expressed in these units. 

The routine unit of enzyme activity has been the international unit (I.U.), namely xmoles P formed (or S consumed) per minute. The specific activity of an enzyme preparation is the number of xmoles P formed (or S consumed) per minute per milligram of protein (clearly this will be very low in a crude cell extract and have a maximal value for a pure preparation of the enzyme). If the molecular mass is known, the specific activity of a pure enzyme measured in saturating (Fmax conditions) can be used to calculate the turnover number (or molecular activity ) of an enzyme, namely the number of P molecules formed (or S molecules transformed) per molecule of enzyme per second (units sec- ). If we recall that the maximal velocity (Fmax) equals k+2 (sec " ) [ET], we can see that the molecular activity equals k+2 (sec -1), that is, fal (sec-1). The katal is the S.I. unit of enzyme activity (moles substrate transformed sec -I) from whence come the corresponding units for specific activity (katal kilogram-1) and molar activity (katal per mole of enzyme). 

A partially purified Bacillus thurlnglensis var. israelensls (Bti) 6-endotoxin was used to Immunize rabbits. The antisera obtained have an improved specificity towards the mosquito larvacidal activity of the toxin, as opposed to antiserum raised when the whole crystal was used as immunogen. Using a two step/indirect ELISA (enzyme linked immunosorbent assay) procedure developed in our laboratory, fourteen experimental formulations were tested, and the results were compared with bioassays. An average of 69.1 international units 20% c.v. was found to associate with each ug of toxin detected by the ELISA. Our data indicate that when toxin specific antisera are available, Immunoassays can be used to predict the biological activity of Bti samples with reasonable accuracy. 

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

In most preparations the actual molar concentration of enzyme is unknown. Consequently, the amount of enzyme present can be expressed only in terms of its activity. In order to standardize the reporting of enzyme activities, the Commission on Enzymes of the International Union of Biochemistry has defined a standard unit ... 

A new unit of enzyme activity, the katal, has been proposed. One katal is that amount of enzyme that catalyzes the conversion of one mole of substrate per second. Thus, one International Unit 1/60/ikatal 16.67 nkatal. One katal = 6 X lO Units. Specific activity can be expressed as katals/kg protein or p.katals/mg protein. The molar activity of an enzyme is the katals/mole protein with units of sec . 

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